Patent Publication Number: US-2022235333-A1

Title: Modified Phenylalanine Dehydrogenase

Description:
This application is a Continuation of, and claims priority under 35 U.S.C. § 120 to, International Application No. PCT/JP2020/038341, filed Oct. 9, 2020, and claims priority therethrough under 35 U.S.C. § 119 to Japanese Patent Application No. 2019-187753, filed Oct. 11, 2019, the entireties of which are incorporated by reference herein. Also, the Sequence Listing filed electronically herewith is hereby incorporated by reference (File name: 2022-04-06T_US-636_Seq_List; File size: 75 KB; Date recorded: Apr. 6, 2022). 
    
    
     GENERAL FIELD 
     The present invention relates to a modified phenylalanine dehydrogenase, a method for analyzing phenylalanine using the same, and more. 
     BACKGROUND ART 
     The amino acid phenylalanine is a biomarker stored in large amounts in blood and/or urine of patients having phenylketonuria, which is a hereditary phenylalanine metabolic disorder disease, and measurement of phenylalanine is very important for clinical diagnosis. A diet low in phenylalanine is required for phenylketonuria patients, and measurement of a phenylalanine content in food is also important. As a method for measuring phenylalanine, an enzymatic method of measurement using a phenylalanine dehydrogenase derived from  Thermoactinomyces intermedius  (Patent Literature 1, for example) is known. 
     As a method for measuring phenylalanine, an enzymatic method of measurement using a phenylalanine dehydrogenase derived from  Thermoactinomyces intermedius  (Patent Literature 1, for example) is known. In addition, analyses of amino acid residues taking part in the activity and the like of phenylalanine dehydrogenases have been performed (Non Patent Literatures 1 to 4). 
     BACKGROUND ART REFERENCES 
     Patent Literature 
     Patent Literature 1: Japanese Examined Patent Application Publication No. H07-114713 
     Non Patent Literature 1: J Biochem. 1994 December; 116(6): 1370-1376 
     Non Patent Literature 2: Biochemistry 2000, 39, 31, 9174-9187 
     Non Patent Literature 3: Journal of Biotechnology Volume 142, Issue 2, 15 Jun. 2009, Pages 127-134 
     Non Patent Literature 4: PNAS 2016 113 (47) E7383-E7389 
     SUMMARY 
     In the phenylalanine measurement using the modified enzyme described herein, the characteristics of the enzyme related to the ability to measure phenylalanine such as, for example, substrate specificity, solubility, and enzyme activity, are desirably excellent. However, wild-type, or non-modified, phenylalanine dehydrogenase is reactive with tyrosine, for example, and thus has low substrate specificity and low accuracy of measurement of phenylalanine, which is problematic. In addition, wild-type phenylalanine dehydrogenase is poorly soluble in water and is thus in liquid suspension, which causes segregation of the enzyme by flocculation or the like. Therefore, the influence of this segregation on the accuracy of measurement of phenylalanine is of concern. Given these circumstances, for practical implementation of a more precise phenylalanine measurement, an enzyme having higher substrate specificity, solubility, and/or enzyme activity is in demand. 
     An aspect of the present invention is to provide a modified phenylalanine dehydrogenase suitable for practical implementation of more precise phenylalanine measurement. 
     The subject matter as described herein is a method of measuring a phenylalanine concentration by using an enzyme with improved characteristics related to measurement of phenylalanine, for example, substrate specificity, solubility, and enzyme activity, resulting in development of a phenylalanine dehydrogenase with these improved characteristics. 
     It is an aspect of the present invention to provide a modified phenylalanine dehydrogenase, comprising a mutation of at least one amino acid residue in a motif selected from the group consisting of: (1) GPALGGXRM (SEQ ID NO:3); (2) GRFXTGTDMGT (SEQ ID NO:4); (3) DF; (4) GXANN (SEQ ID NO:5); (5) RH; (6) VNXGGLIQV (SEQ ID NO:6), and (7) combinations thereof, wherein X indicates any amino acid, and wherein said modified phenylalanine dehydrogenase comprises at least one motif selected from the motifs (1) to (6); wherein said modified phenylalanine dehydrogenase has at least one characteristic selected from the group consisting of a substrate specificity, solubility, and phenylalanine dehydrogenase activity higher than a non-modified phenylalanine dehydrogenase. 
     It is a further aspect of the present invention to provide the modified phenylalanine dehydrogenase as described above, in which the mutation is a substitution selected from the group consisting of: (a) substitution of leucine in GPALGGXRM (SEQ ID NO:3); (b) substitution of threonine, the seventh amino acid from the left, in GRFXTGTDMGT (SEQ ID NO:4); (c) substitution of phenylalanine in DF; (d) substitution of asparagine, the fourth amino acid from the left, in GXANN (SEQ ID NO:5); (e) substitution of arginine in RH; (f) substitution of asparagine in VNXGGLIQV (SEQ ID NO:6); (g) substitution of leucine in VNXGGLIQV (SEQ ID NO:6); (h) substitution of glutamine in VNXGGLIQV (SEQ ID NO:6); (i) substitution of valine, the ninth amino acid from the left, in VNXGGLIQV (SEQ ID NO:6), and (j) combinations thereof. 
     It is a further aspect of the present invention to provide the modified phenylalanine dehydrogenase as described above, in which the mutation is a substitution selected from the group consisting of: (a) substitution of leucine in GPALGGXRM (SEQ ID NO:3) with tryptophane, phenylalanine, tyrosine, or methionine; (b) substitution of threonine, seventh amino acid from the left, in GRFXTGTDMGT (SEQ ID NO:4) with serine; (c) substitution of phenylalanine in DF with leucine or isoleucine; (d) substitution of asparagine, fourth amino acid from the left in GXANN (SEQ ID NO:5) with glycine, glutamine, threonine, lysine, proline, or serine; (e) substitution of arginine in RH with aspartic acid or glutamic acid; (f) substitution of asparagine in VNXGGLIQV (SEQ ID NO:6) with valine, aspartic acid, methionine, glutamine, proline, isoleucine, histidine, alanine, threonine, glycine, or cysteine; (g) substitution of leucine in VNXGGLIQV (SEQ ID NO:6) with phenylalanine, glutamine, histidine, asparagine, isoleucine, aspartic acid, glycine, glutamic acid, threonine, or serine; (h) substitution of glutamine in VNXGGLIQV (SEQ ID NO:6) with aspartic acid, glutamic acid, lysine, asparagine, serine, or arginine; and substitution of valine, ninth amino acid residue from the left, in VNXGGLIQV (SEQ ID NO:6) with tyrosine, tryptophane, glutamic acid, asparagine, threonine, isoleucine, lysine, glycine, serine, leucine, methionine, glutamine, phenylalanine, cysteine, or arginine, and (j) combinations thereof. 
     It is a further aspect of the present invention to provide the modified phenylalanine dehydrogenase as described above, in which the phenylalanine dehydrogenase comprises motifs (1) to (6) in numerical order. 
     It is a further aspect of the present invention to provide the modified phenylalanine dehydrogenase according to as described above, in which the phenylalanine dehydrogenase is derived from the genus  Thermoactinomyces.    
     It is a further aspect of the present invention to provide the modified phenylalanine dehydrogenase according to as described above, in which the phenylalanine dehydrogenase comprises: (A) the amino acid sequence of SEQ ID NO:1; (B) an amino acid sequence comprising a substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO:1; or (C) an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:1. 
     It is a further aspect of the present invention to provide a modified phenylalanine dehydrogenase, comprising a mutation of an amino acid residue corresponding to an amino acid residue selected from the group consisting of: R2, R10, Y11, C19, L41, G42, G43, C44, A50, S51, M66, C70, F77, K90, Y112, T115, D116, F124, R129, L137, K139, S140, K144, T147, K173, C200, C210, K216, K220, Q222, N227, R228, C234, C240, R255, C256, L257, N264, R271, Q277, K278, R279, S280, C282, N290, G293, L294, Q296, V297, R326, K328, N329, N331, C335, R340, K347, K348, and combinations thereof; wherein said phenylalanine dehydrogenase comprises: (A) the amino acid sequence of SEQ ID NO:1, (B) an amino acid sequence comprising substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO:1, or (C) an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:1, wherein said modified phenylalanine dehydrogenase has phenylalanine dehydrogenase activity, and wherein said phenylalanine dehydrogenase has an improved characteristic selected from the group consisting of substrate specificity, solubility, phenylalanine dehydrogenase activity, and combinations thereof. 
     It is a further aspect of the present invention to provide the modified phenylalanine dehydrogenase as described above, which comprises a substitution of an amino acid residue corresponding to an amino acid residue selected from group consisting of: R2D, R2E, R10D, R10E, Y11E, Y11D, C19A, C19S, L41W, L41F, L41Y, L41M, G42A, G43A, C44A, C44S, A50D, A50E, S51D, S51E, M66I, M66L, M66V, C70A, C70S, F77L, F77I, F77R, K90E, Y112L, T115S, D116E, F124L, F124I, R129K, L137V, K139E, S140A, K144G, T147A, T147S, T147N, K173E, K173D, C200A, C200S, C210S, C210A, K216D, K216E, K220D, K220E, Q222E, Q222D, N227D, N227E, R228E, R228D, C234A, C234S, C240S, C240A, R255E, R255D, C256A, C256S, L257K, N264G, N264Q, N264T, N264K, N264P, N264S, R271D, R271E, Q277D, K278D, K278E, R279D, R279E, S280D, C282S, C282A, N290V, N290D, N290M, N290Q, N290P, N290I, N290H, N290A, N290T, N290G, N290C, G293A, L294F, L294Q, L294H, L294N, L294I, L294D, L294G, L294E, L294T, L294S, Q296D, Q296E, Q296K, Q296N, Q296S, Q296R, V297Y, V297W, V297E, V297N, V297T, V297I, V297K, V297G, V297S, V297L, V297M, V297Q, V297F, V297C, V297R, R326E, K328E, K328D, N329D, N331E, N331D, C335A, C335S, R340D, R340E, K347D, K348E, and combinations thereof. 
     It is a further aspect of the present invention to provide method for analyzing phenylalanine, the method comprising measuring phenylalanine contained in a test sample using the modified phenylalanine dehydrogenase as described above. 
     It is a further aspect of the present invention to provide the method as described above, comprising mixing the test sample with nicotinamide adenine dinucleotide (NAD + ) and detecting NADH produced from NAD +  by the action of the modified phenylalanine dehydrogenase. 
     It is a further aspect of the present invention to provide a method for producing phenylpyruvate, the method comprising producing phenylpyruvate from phenylalanine using the modified phenylalanine dehydrogenase as described above. 
     It is a further aspect of the present invention to provide a polynucleotide encoding the modified phenylalanine dehydrogenase as described above. 
     It is a further aspect of the present invention to provide an expression vector comprising the polynucleotide as described above. 
     It is a further aspect of the present invention to provide a transformant comprising an expression unit of a polynucleotide encoding the modified phenylalanine dehydrogenase as described above. 
     It is a further aspect of the present invention to provide a method for producing a modified phenylalanine dehydrogenase, the method comprising producing a modified phenylalanine dehydrogenase comprising a mutation of at least one amino acid residue so as to improve a characteristic selected from the group consisting of substrate specificity, solubility, phenylalanine dehydrogenase activity, and combinations thereof, using the transformant as described above. 
     It is a further aspect of the present invention to provide a kit for analyzing phenylalanine, the kit comprising the modified phenylalanine dehydrogenase as described above. 
     It is a further aspect of the present invention to provide the kit for analyzing phenylalanine as described above further comprising at least one of a buffer solution or a buffer salt for reaction and nicotinamide adenine dinucleotide (NAD + ). 
     It is a further aspect of the present invention to provide an enzyme sensor for analyzing phenylalanine, the enzyme sensor comprising (a) an electrode for detection and (b) the modified phenylalanine dehydrogenase according to [1] immobilized or disposed on the electrode for detection. 
     The modified phenylalanine dehydrogenase as described herein has improved substrate specificity and is thus useful for quick, high-precision, and highly sensitive measurement of phenylalanine and/or production of phenylpyruvate. The modified phenylalanine dehydrogenase has improved solubility, thus does not cause segregation of enzyme by flocculation or the like, and is useful for uniform and high-precision measurement of phenylalanine. Furthermore, the modified phenylalanine dehydrogenase has improved phenylalanine dehydrogenase activity and is thus useful for quick and highly sensitive measurement of phenylalanine and/or production of phenylpyruvate. The modified phenylalanine dehydrogenase is useful as a liquid reagent in particular. The method of analysis is useful for diagnosis of diseases such as phenylketonuria and measurement of a phenylalanine content in food, for example. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a diagram of alignment of amino acid sequences near motif (1) (GPALGGXRM (SEQ ID NO:3)) of wild-type phenylalanine dehydrogenases derived from respective species. The arrows indicate representative mutation sites (L41, G42, and G43) for designing a modified phenylalanine dehydrogenase. 
         FIG. 2  is a diagram of alignment of amino acid sequences near motif (2) (GRFXTGTDMGT (SEQ ID NO:4)) and motif (3) (DF) of wild-type phenylalanine dehydrogenases derived from respective species. The arrows indicate representative mutation sites (T115 and F124). 
         FIG. 3  is a diagram of alignment of amino acid sequences near motif (4) (GXANN (SEQ ID NO:5)), motif (5) (RH), and motif (6) (VNXGGLIQV (SEQ ID NO:6)) of wild-type phenylalanine dehydrogenases derived from respective species. The arrows indicate representative mutation sites (N264, R271, N290, G293, L294, Q296, and V297) for designing a modified phenylalanine dehydrogenase. 
     
    
    
     DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS 
     A modified phenylalanine dehydrogenase is provided herein. The modified phenylalanine dehydrogenase can include a mutation of at least one amino acid residue so as to improve one or more characteristics related to measurement of phenylalanine such as substrate specificity, solubility, and phenylalanine dehydrogenase activity (hereinafter, also referred to simply as “enzyme activity” or “activity”). 
     Examples of the mutation of the amino acid residue include substitution, deletion, addition, and insertion; substitution is a particular example. 
     The amino acid residue to be mutated can be L-alanine (A), L-asparagine (N), L-cysteine (C), L-glutamine (Q), L-isoleucine (I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophane (W), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), L-lysine (K), or glycine (G) as a natural L-α-amino acid. When the mutation is substitution, addition, or insertion, the amino acid to be substituted, added, or inserted is the same as the amino acid residue to be mutated described above. In the following, L and α may be omitted in the expression of the amino acid. 
     A phenylalanine dehydrogenase (may be expressed as PheDH) is an oxidoreductase catalyzing the following reaction (EC 1.4.1.20): 
       L-phenylalanine+NAD + +H 2 O←→phenylpyruvate+NH 4   + +NADH.
 
     An enzyme derived from, that is, native to, any organism (for example, microorganisms such as bacteria, actinomycetes, and fungi, insects, fishes, animals, and plants) can be used as the phenylalanine dehydrogenase from which the modified phenylalanine dehydrogenase is derived; examples thereof include phenylalanine dehydrogenases derived from bacteria belonging to the genus  Thermoactinomyces  ( Thermoactinomyces intermedius  and  Thermoactinomyces  sp., for example), bacteria belonging to the genus  Lihuaxuella  ( Lihuaxuella thermophile,  for example), bacteria belonging to the genus  Baia  ( Baia soyae,  for example), bacteria belonging to the genus  Caldalkalibacillus  ( Caldalkalibacillus thermarum , for example), bacteria belonging to the genus  Bacillus  ( Bacillus badius, Bacillus  sp.,  Bacillus halodurans,  and  Lysinibacillus sphaericus  (also called  Bacillus sphaericus ), for example), bacteria belonging to the genus  Fictibacillus  ( Fictibacillus nanhaiensis,  for example), bacteria belonging to the genus  Lysinibacillus  ( Lysinibacillus sphaericus  (also called  Bacillus sphaericus ), for example), bacteria belonging to the genus  Sporosarcina  ( Sporosarcina ureae,  for example), bacteria belonging to the genus  Rhodococcus  ( Rhodococcus  sp., for example), and bacteria belonging to genera related thereto. Among the bacteria, bacteria belonging to the genus  Thermoactinomyces  are a particular example, and  Thermoactinomyces intermedius  is an even more particular example. Table 1 below lists examples of the wild-type phenylalanine dehydrogenase: 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Examples of wild-type phenylalanine dehydrogenase 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Identity (%) 
               
               
                   
                   
                   
                 by 
               
               
                   
                   
                   
                 GENETYX 
               
               
                   
                   
                 GenBank 
                 (registered 
               
               
                 Species origin 
                 UniProt ID 
                 Accession No. 
                 trademark)  ※1   
               
               
                   
               
               
                 
                   Thermoactinomyces  
                 
                 P22823 
                 P22823.1 
                 — 
               
               
                 
                   intermedius 
                 
                   
                   
                   
               
               
                 
                   Lihuaxuella thermophile 
                 
                 A0A1H8C 
                 SEM88729.1 
                 73 
               
               
                   
                 1K2 
                   
                   
               
               
                 
                   Baia soyae 
                 
                 — 
                 TCP62478.1 
                 57 
               
               
                   Thermoactinomyces sp.  DSM 
                 A0A1K2A 
                 SFX81991.1 
                 57 
               
               
                 45891 
                 6F4 
                   
                   
               
               
                 
                   Caldalkalibacillus thermarum 
                 
                 F5L9G2 
                 F5L9G2.1 
                 57 
               
               
                 TA2.A1 
                   
                   
                   
               
               
                 
                   Bacillus badius 
                 
                 Q59224 
                 Q59224.1 
                 55 
               
               
                 
                   Fictibacillus nanhaiensis 
                 
                 A0A1B1P 
                 ANT47406.1 
                 54 
               
               
                   
                 GG4 
                   
                   
               
               
                   Bacillus sp.  UTB2301 
                 Q9AJQ0 
                 BAB40584.1 
                 54 
               
               
                 
                   Lysinibacillus sphaericus  
                 
                 P23307 
                 P23307.1 
                 53 
               
               
                 
                   (Bacillus 
                   sphaericus) 
                 
                   
                   
                   
               
               
                 
                   Sporosarcina ureae 
                 
                 P97014 
                 P97014.1 
                 52 
               
               
                 
                   Bacillus halodurans  
                 
                 Q9KG94 
                 BAB03937.1 
                 51 
               
               
                 
                   Rhodococcus sp. 
                 
                 Q59771 
                 Q59771.2 
                 31 
               
               
                   
               
               
                   ※1  The identity of the amino acid sequence with respect to the wild-type phenylalanine dehydrogenase derived from  Thermoactinomyces  intermedius was calculated on “Gaps are NOT taken into account.”  
               
            
           
         
       
     
     Examples of the wild-type phenylalanine dehydrogenase include wild-type phenylalanine dehydrogenases having at least one, for example, one, two, three, four, five, or six of the following motifs labelled (1) to (6). The wild-type phenylalanine dehydrogenase may include a plurality of (for example, two, three, four, five, or six) of the motifs (1) to (6) in this order. The term “order” refers to the direction from the N-terminus toward the C-terminus in an amino acid sequence. The wild-type phenylalanine may include all of the motifs (1) to (6), and all the motifs (1) to (6) may be in this order. In the following, each of the motifs is indicated by a one-letter expression amino acid sequence, in which X indicates any amino acid, such as, any of the 20 kinds of amino acids that form proteins, that is, alanine (A), asparagine (N), cysteine (C), glutamine (Q), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophane (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), lysine (K), and glycine (G)). 
     
       
         
           
               
               
            
               
                   
                 motif (1): 
               
               
                   
                 (SEQ ID NO: 3) 
               
               
                   
                 a GPALGGXRM motif; 
               
               
                   
                   
               
               
                   
                 motif (2): 
               
               
                   
                 (SEQ ID NO: 4) 
               
               
                   
                 a GRFXTGTDMGT motif; 
               
               
                   
                   
               
               
                   
                 motif (3): 
               
               
                   
                 a DF motif; 
               
               
                   
                   
               
               
                   
                 motif (4): 
               
               
                   
                 (SEQ ID NO: 5) 
               
               
                   
                 a GXANN motif; 
               
               
                   
                   
               
               
                   
                 motif (5): 
               
               
                   
                 an RH motif; 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 motif (6): 
               
               
                   
                 (SEQ ID NO: 6) 
               
               
                   
                 a VNXGGLIQV motif. 
               
            
           
         
       
     
     Motifs (1), (2), (4), and (6) include the following motifs (1a), (2a), (4a), and (6a) and (6b), respectively, which are motifs indicated by shorter amino acid sequences. Consequently, the wild-type phenylalanine dehydrogenase may, for example, be a wild-type phenylalanine dehydrogenase that includes at least one (for example, one, two, three, four, five, six, or seven) of the motifs (1a), (2a), (3), (4a), (5), (6a), and (6b), and may also include a plurality of (for example, two, three, four, five, six, or seven) the motifs (1a), (2a), (3), (4a), (5), (6a), and (6b) in this order, and may include the motifs (1a), (2a), (3), (4a), (5), (6a), and (6b), and may include all of the motifs (1a), (2a), (3), (4a), (5), (6a), and (6b) in this order. 
     
       
         
           
               
               
            
               
                   
                 motif (1a): 
               
               
                   
                 (SEQ ID NO: 7) 
               
               
                   
                 a GPALGG motif in motif (1); 
               
               
                   
                   
               
               
                   
                 motif (2a): 
               
               
                   
                 (SEQ ID NO: 8) 
               
               
                   
                 a TGTDMGT motif in motif (2); 
               
               
                   
                   
               
               
                   
                 motif (4a): 
               
               
                   
                 an ANN motif in motif (4); 
               
               
                   
                   
               
               
                   
                 motif (6a): 
               
               
                   
                 a VN motif in motif (6); 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 motif (6b): 
               
               
                   
                 (SEQ ID NO: 9) 
               
               
                   
                 a GGLIQV motif in motif (6). 
               
            
           
         
       
     
     The modified phenylalanine dehydrogenase may include a mutation of at least one amino acid residue in at least one of the motifs (1) to (6), for example, at least one amino acid residue in at least one of the motifs (1a), (2a), (3), (4a), (5), (6a), and (6b)) in a phenylalanine dehydrogenase described above, having phenylalanine dehydrogenase activity, and showing at least one of substrate specificity, solubility, and phenylalanine dehydrogenase activity, which is higher than a wild-type phenylalanine dehydrogenase. 
     The mutation that imparts improved characteristics to the phenylalanine dehydrogenase related to an ability to measure phenylalanine is a substitution of leucine, the fifth amino acid glycine, or the sixth amino acid glycine (as counted from the left) in motif (1) (GPALGGXRM (SEQ ID NO:3)) of the amino acid sequence of the wild-type phenylalanine dehydrogenase. Motif (1) has nine consecutive amino acid residues of GPALGGXRM (SEQ ID NO:3) (X indicates any amino acid residue). The amino acid residue to be substituted can also be identified as leucine, the fifth amino acid glycine, or the sixth amino acid glycine in motif (1a) (GPALGG (SEQ ID NO:7)), indicated by the shorter amino acid sequence in motif (1). Although the position of motif (1) or (1a) in the amino acid sequence of the wild-type phenylalanine dehydrogenase can vary depending on the species origin of the enzyme, a person skilled in the art can determine the position of motif (1) or (1a) in the amino acid sequence of the wild-type phenylalanine dehydrogenase as appropriate and can thus identify the position of the leucine or the glycine (the fifth or sixth one) to be substituted. Normally, in the amino acid sequence of the phenylalanine dehydrogenase, motif (1) is located at positions 38 to 46, motif (1a) is located at positions 38 to 43, the leucine to be substituted is located at position 41, the glycine (the fifth one) to be substituted is located at position 42, and the glycine (the sixth one) to be substituted is located at position 43 (refer to, for example, Table 2 and  FIG. 1 ). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Sequences of conservation regions of motif (1) (GPALGGXRM (SEQ ID NO: 3) 
               
               
                 motif) and motif (1a) (GPALGG (SEQ ID NO: 7) motif) in wild-type phenylalanine 
               
               
                 dehydrogenases derived from respective species 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Conservation region 
               
               
                   
                   
                   
                   
                 Mutation 
                 (underline indicates 
               
               
                   
                   
                 GenBank 
                 Identity 
                 site 
                 representative 
               
               
                   
                 Species origin 
                 Accession No. 
                 (%) 
                 conservation 
                 mutation sites) 
               
               
                   
               
               
                  1 
                 
                   Thermoactinomyces 
                 
                 P22823.1 
                 —○ 
                 ○ 
                   
               
               
                   
                 
                   intermedius 
                 
                   
                   
                   
                   
               
               
                   
               
               
                  2 
                 
                   Lihuaxuella 
                 
                 SEM88729.1 
                 73 
                 ○ 
                 GPA   LGG   CRMIPY 
               
               
                   
                 
                   thermophile 
                 
                   
                   
                   
                 (SEQ ID NO: 17) 
               
               
                   
               
               
                  3 
                 
                   Baia soyae 
                 
                 TCP62478.1 
                 57 
                 ○ 
                 GPA   LGG   CRMIPY 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 27) 
               
               
                   
               
               
                  4 
                   Thermoactinomyces  sp. 
                 SFX81991.1 
                 57 
                 ○ 
                 GPA   LGG   CRMIPY 
               
               
                   
                 DSM 45891 
                   
                   
                   
                 (SEQ ID NO: 36) 
               
               
                   
               
               
                  5 
                 
                   Caldalkalibacillus 
                 
                 F5L9G2.1 
                 57 
                 ○ 
                 GPA   LGG   CRM 
               
               
                   
                   thermarum  TA2.A1 
                   
                   
                   
                 (SEQ ID NO: 45) 
               
               
                   
               
               
                  6 
                 
                   Bacillus badius 
                 
                 Q59224.1 
                 55 
                 ○ 
                 GPA   LGG   CRM 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 55) 
               
               
                   
               
               
                  7 
                 
                   Fictibacillus 
                 
                 ANT47406.1 
                 54 
                 ○ 
                 GPA   LGG   CRM 
               
               
                   
                 
                   nanhaiensis 
                 
                   
                   
                   
                 (SEQ ID NO: 63) 
               
               
                   
               
               
                  8 
                   Bacillus  sp. UTB2301 
                 BAB40584.1 
                 54 
                 ○ 
                 GPA   LGG   CRM 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 71) 
               
               
                   
               
               
                  9 
                 
                   Lysinibacillus 
                 
                 P23307.1 
                 53 
                 ○ 
                 GPA   LGG     
               
               
                   
                   sphaericus  ( Bacillus   
                   
                   
                   
                 (SEQ ID NO: 80) 
               
               
                   
                   sphaericus ) 
                   
                   
                   
                   
               
               
                   
               
               
                 10 
                 
                   Sporosarcina ureae 
                 
                 P97014.1 
                 52 
                 ○ 
                 GPA   LGG   CRM 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 89) 
               
               
                   
               
               
                 11 
                 
                   Bacillus halodurans 
                 
                 BAB03937.1 
                 51 
                 ○ 
                 GPA   LGG   CRM 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 98) 
               
               
                   
               
               
                 12 
                   Rhodococcus  sp. 
                 Q59771.2 
                 31 
                 x 
               
               
                   
               
            
           
         
       
     
     The mutation that imparts improved characteristics to the phenylalanine dehydrogenase related to an ability to measure phenylalanine is a substitution of the seventh amino acid threonine (as counted from the left) in motif (2) (GRFXTGTDMGT (SEQ ID NO:4)) of the amino acid sequence of the wild-type phenylalanine dehydrogenase. Motif (2) has 11 consecutive amino acid residues of GRFXTGTDMGT (SEQ ID NO:4) (X indicates any amino acid residue). The amino acid residue to be substituted can also be identified as the third amino acid threonine in motif (2a) (TGTDMGT (SEQ ID NO:8)) indicated by the shorter amino acid sequence in motif (2). Although the position of motif (2) or (2a) in the amino acid sequence of the wild-type phenylalanine dehydrogenase can vary depending on the species origin of the enzyme, a person skilled in the art can determine the position of motif (2) or (2a) in the amino acid sequence of the wild-type phenylalanine dehydrogenase as appropriate and can thus identify the position of the threonine to be substituted. Normally, in the amino acid sequence of the phenylalanine dehydrogenase, motif (2) is located at positions 109 to 119, motif (2a) is located at positions 113 to 119, and the threonine to be substituted is located at position 115 (for example, refer to Table 3 and  FIG. 2 ). 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Sequences of conservation regions of motif (2) (GRFXTGTDMGT (SEQ ID NO: 4) 
               
               
                 motif) and motif (2a) (TGTDMGT (SEQ ID NO: 8) motif) in wild-type phenylalanine 
               
               
                 dehydrogenases derived from respective species 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Conservation region 
               
               
                   
                   
                 GenBank 
                   
                 Mutation 
                 (underline indicates 
               
               
                   
                   
                 Accession  
                 Identity 
                 site con- 
                 representative 
               
               
                   
                 Species origin 
                 No. 
                 (%) 
                 servation 
                 mutation sites) 
               
               
                   
               
               
                  1 
                 
                   Thermoactinomyces 
                 
                 P22823.1 
                 — 
                 ○ 
                   
               
               
                   
                 
                   intermedius 
                 
                   
                   
                   
                   
               
               
                   
               
               
                  2 
                 
                   Lihuaxuella 
                 
                 SEM88729.1 
                 73 
                 ○ 
                 TG   T   DMGTNPEDFV 
               
               
                   
                 
                   thermophile 
                 
                   
                   
                   
                 (SEQ ID NO: 18) 
               
               
                   
               
               
                  3 
                 
                   Baia soyae 
                 
                 TCP62478.1 
                 57 
                 ○ 
                 TG   T   DMGT 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 28) 
               
               
                   
               
               
                  4 
                 
                   Thermoactinomyces 
                 
                 SFX81991.1 
                 57 
                 ○ 
                 GRFYTG   T   DMGT 
               
               
                   
                 sp. DSM 45891 
                   
                   
                   
                 (SEQ ID NO: 37) 
               
               
                   
               
               
                  5 
                 
                   Caldalkalibacillus 
                 
                 F5L9G2.1 
                 57 
                 ○ 
                 LNGRFYTG   T   DMGT 
               
               
                   
                 
                   thermarum 
                 
                   
                   
                   
                 (SEQ ID NO: 46) 
               
               
                   
                 TA2.A1 
                   
                   
                   
                   
               
               
                   
               
               
                  6 
                 
                   Bacillus badius 
                 
                 Q59224.1 
                 55 
                 ○ 
                 GRFYTG   T   DMGTN 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 56) 
               
               
                   
               
               
                  7 
                 
                   Fictibacillus 
                 
                 ANT47406.1 
                 54 
                 ○ 
                 LNGRFYTG   T   DMGT 
               
               
                   
                 
                   nanhaiensis 
                 
                   
                   
                   
                 (SEQ ID NO: 64) 
               
               
                   
               
               
                  8 
                   Bacillus  sp. 
                 BAB40584.1 
                 54 
                 ○ 
                 LNGRFYTG   T   DMGT 
               
               
                   
                 UTB2301 
                   
                   
                   
                 (SEQ ID NO: 72) 
               
               
                   
               
               
                  9 
                 
                   Lysinibacillus 
                 
                 P23307.1 
                 53 
                 ○ 
                 LNGRFYTG   T   DMGT 
               
               
                   
                 
                   sphaericus 
                 
                   
                   
                   
                 (SEQ ID NO: 81) 
               
               
                   
                 ( Bacillus   
                   
                   
                   
                   
               
               
                   
                   sphaericus ) 
                   
                   
                   
                   
               
               
                   
               
               
                 10 
                 
                   Sporosarcina 
                 
                 P97014.1 
                 52 
                 ○ 
                 LNGRFYTG   T   DMGT 
               
               
                   
                 
                   ureae 
                 
                   
                   
                   
                 (SEQ ID NO: 90) 
               
               
                   
               
               
                 11 
                 
                   Bacillus 
                 
                 BAB03937.1 
                 51 
                 ○ 
                 LNGRFYTG   T   DMGT 
               
               
                   
                 
                   halodurans 
                 
                   
                   
                   
                 (SEQ ID NO: 99) 
               
               
                   
               
               
                 12 
                   Rhodococcus  sp. 
                 Q59771.2 
                 31 
                 ○ 
               
               
                   
               
            
           
         
       
     
     The mutation that imparts improved characteristics to the phenylalanine dehydrogenase related to an ability to measure phenylalanine is a substitution of phenylalanine in motif (3) (DF) of the amino acid sequence of the wild-type phenylalanine dehydrogenase. Motif (3) has two consecutive amino acid residues of DF. Although the position of motif (3) in the amino acid sequence of the wild-type phenylalanine dehydrogenase can vary depending on the species origin of the enzyme, a person skilled in the art can determine the position of motif (3) in the amino acid sequence of the wild-type phenylalanine dehydrogenase as appropriate and can thus identify the position of the phenylalanine to be substituted. Normally, in the amino acid sequence of the phenylalanine dehydrogenase, motif (3) is located at positions 123 to 124, and the phenylalanine to be substituted is located at position 124 (for example, refer to Table 4 and  FIG. 2 ). 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Sequences of conservation regions of motif (3) (DF motif) in wild-type 
               
               
                 phenylalanine dehydrogenases derived from respective species 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Conservation region 
               
               
                   
                   
                 GenBank 
                   
                 Mutation 
                 (underline indicates 
               
               
                   
                   
                 Accession  
                 Identity 
                 site con- 
                 representative 
               
               
                   
                 Species origin 
                 No. 
                 (%) 
                 servation 
                 mutation sites) 
               
               
                   
               
               
                  1 
                 
                   Thermoactinomyces 
                 
                 P22823.1 
                 — 
                 ○ 
                   
               
               
                   
                 
                   intermedius 
                 
                   
                   
                   
                   
               
               
                   
               
               
                  2 
                 
                   Lihuaxuella 
                 
                 SEM88729.1 
                 73 
                 ○ 
                 TGTDMGTNPED   F   V 
               
               
                   
                 
                   thermophile 
                 
                   
                   
                   
                 (SEQ ID NO: 19) 
               
               
                   
               
               
                  3 
                 
                   Baia soyae 
                 
                 TCP62478.1 
                 57 
                 ○ 
                 PED   F     
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 29) 
               
               
                   
               
               
                  4 
                 
                   Thermoactinomyces 
                 
                 SFX81991.1 
                 57 
                 ○ 
                 PED   F     
               
               
                   
                 sp. DSM 45891 
                   
                   
                   
                 (SEQ ID NO: 38) 
               
               
                   
               
               
                  5 
                 
                   Caldalkalibacillus 
                 
                 F5L9G2.1 
                 57 
                 ○ 
                 PED   F   V 
               
               
                   
                   thermarum  TA2.A1 
                   
                   
                   
                 (SEQ ID NO: 47) 
               
               
                   
               
               
                  6 
                 
                   Bacillus badius 
                 
                 Q59224.1 
                 55 
                 ○ 
                 ED   F     
               
               
                   
               
               
                  7 
                 
                   Fictibacillus 
                 
                 ANT47406.1 
                 54 
                 ○ 
                 D   F     
               
               
                   
                 
                   nanhaiensis 
                 
                   
                   
                   
                   
               
               
                   
               
               
                  8 
                   Bacillus  sp. 
                 BAB40584.1 
                 54 
                 ○ 
                 PED   F   VHAA 
               
               
                   
                 UTB2301 
                   
                   
                   
                 (SEQ ID NO: 73) 
               
               
                   
               
               
                  9 
                 
                   Lysinibacillus 
                 
                 P23307.1 
                 53 
                 ○ 
                 D   F   VHA 
               
               
                   
                 
                   sphaericus 
                 
                   
                   
                   
                 (SEQ ID NO: 82) 
               
               
                   
                 ( Bacillus   
                   
                   
                   
                   
               
               
                   
                   sphaericus ) 
                   
                   
                   
                   
               
               
                   
               
               
                 10 
                 
                   Sporosarcina ureae 
                 
                 P97014.1 
                 52 
                 ○ 
                 ED   F   VHA 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 91) 
               
               
                   
               
               
                 11 
                 
                   Bacillus halodurans 
                 
                 BAB03937.1 
                 51 
                 ○ 
                 ED   F   VHA 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 100) 
               
               
                   
               
               
                 12 
                   Rhodococcus  sp. 
                 Q59771.2 
                 31 
                 x 
               
               
                   
               
            
           
         
       
     
     The mutation that imparts improved characteristics to the phenylalanine dehydrogenase related to an ability to measure phenylalanine is a substitution of asparagine as the fourth amino acid residue in motif (4) (GXANN (SEQ ID NO:5)) of the amino acid sequence of the wild-type phenylalanine dehydrogenase. Motif (4) has five consecutive amino acid residues of GXANN (SEQ ID NO:5) (X indicates any amino acid residue). The amino acid residue to be substituted can also be identified as asparagine as the second amino acid residue in motif (4a) (the ANN motif) indicated by the shorter amino acid sequence in motif (4). Although the position of motif (4) or (4a) in the amino acid sequence of the wild-type phenylalanine dehydrogenase can vary depending on the species origin of the enzyme, a person skilled in the art can determine the position of motif (4) or (4a) in the amino acid sequence of the wild-type phenylalanine dehydrogenase as appropriate and can thus identify the position of the asparagine to be substituted. Normally, in the amino acid sequence of the phenylalanine dehydrogenase, motif (4) is located at positions 261 to 265, motif (4a) is located at positions 263 to 265, and the asparagine to be substituted is located at position 264 (for example, refer to Table 5 and  FIG. 3 ). 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Sequences of conservation regions of motif (4) (GXANN (SEQ ID NO: 5) motif) and 
               
               
                 motif (4a) (ANN motif) in wild-type phenylalanine dehydrogenases derived from 
               
               
                 respective species 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Conservation region 
               
               
                   
                   
                 GenBank 
                   
                 Mutation 
                 (underline indicates 
               
               
                   
                   
                 Accession  
                 Identity 
                 site con- 
                 representative 
               
               
                   
                 Species origin 
                 No. 
                 (%) 
                 servation 
                 mutation sites) 
               
               
                   
               
               
                  1 
                 
                   Thermoactinomyces 
                 
                 P22823.1 
                 - 
                 ○ 
                   
               
               
                   
                 
                   intermedius 
                 
                   
                   
                   
                   
               
               
                   
               
               
                  2 
                 
                   Lihuaxuella 
                 
                 SEM88729.1 
                 73 
                 ○ 
                 AIVGSA   N   NQLVED 
               
               
                   
                 
                   thermophile 
                 
                   
                   
                   
                 RHG 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 20) 
               
               
                   
               
               
                  3 
                 
                   Baia soyae 
                 
                 TCP62478.1 
                 57 
                 ○ 
                 GSA   N   NQL 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 30) 
               
               
                   
               
               
                  4 
                   Thermoactinomyces  sp. 
                 SFX81991.1 
                 57 
                 ○ 
                 GSA   N   NQL 
               
               
                   
                 DSM 45891 
                   
                   
                   
                 (SEQ ID NO: 39) 
               
               
                   
               
               
                  5 
                 
                   Caldalkalibacillus 
                 
                 F5L9G2.1 
                 57 
                 ○ 
                 A   N   NQL 
               
               
                   
                   thermarum  TA2.A1 
                   
                   
                   
                 (SEQ ID NO: 48) 
               
               
                   
               
               
                  6 
                 
                   Bacillus badius 
                 
                 Q59224.1 
                 55 
                 ○ 
                 GSA   N   NQL 
               
               
                   
                   
                   
                   
                   
                 (   57) 
               
               
                   
               
               
                  7 
                 
                   Fictibacillus 
                 
                 ANT47406.1 
                 54 
                 ○ 
                 AIVGSA   N   NQL 
               
               
                   
                 
                   nanhaiensis 
                 
                   
                   
                   
                 (SEQ ID NO: 65) 
               
               
                   
               
               
                  8 
                   Bacillus  sp. UTB2301 
                 BAB40584.1 
                 54 
                 ○ 
                 GSA   N   NQL 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 74) 
               
               
                   
               
               
                  9 
                 
                   Lysinibacillus 
                 
                 P23307.1 
                 53 
                 ○ 
                 VGSA   N   NQL 
               
               
                   
                   sphaericus  ( Bacillus   
                   
                   
                   
                 (SEQ ID NO: 83) 
               
               
                   
                   sphaericus ) 
                   
                   
                   
                   
               
               
                   
               
               
                 10 
                 
                   Sporosarcina ureae 
                 
                 P97014.1 
                 52 
                 ○ 
                 GSA   N   NQL 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 92) 
               
               
                   
               
               
                 11 
                 
                   Bacillus halodurans 
                 
                 BAB03937.1 
                 51 
                 ○ 
                 A   N   NQL 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 101) 
               
               
                   
               
               
                 12 
                   Rhodococcus  sp. 
                 Q59771.2 
                 31 
                 ○ 
                 A   N   N 
               
               
                   
               
            
           
         
       
     
     The mutation that imparts improved characteristics to the phenylalanine dehydrogenase related to an ability to measure phenylalanine is a substitution of arginine in motif (5) (RH) of the amino acid sequence of the wild-type phenylalanine dehydrogenase. Motif (5) has two consecutive amino acid residues of RH. Although the position of motif (5) in the amino acid sequence of the wild-type phenylalanine dehydrogenase can vary depending on the derivation of the enzyme, a person skilled in the art can determine the position of motif (5) in the amino acid sequence of the wild-type phenylalanine dehydrogenase as appropriate and can thus identify the position of the arginine to be substituted. Normally, in the amino acid sequence of the phenylalanine dehydrogenase, motif (5) is located at positions 271 to 272, and the arginine to be substituted is located at position 271 (for example, refer to Table 6 and  FIG. 3 ). 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Sequences of conservation regions of motif (5) (RH motif) in wild-type 
               
               
                 phenylalanine dehydrogenases derived from respective species 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Conservation region 
               
               
                   
                   
                 GenBank 
                   
                 Mutation 
                 (underline indicates 
               
               
                   
                   
                 Accession  
                 Identity 
                 site con- 
                 representative 
               
               
                   
                 Species origin 
                 No. 
                 (%) 
                 servation 
                 mutation sites) 
               
               
                   
               
               
                  1 
                 
                   Thermoactinomyces 
                 
                 P22823.1 
                 - 
                 ○ 
                   
               
               
                   
                 
                   intermedius 
                 
                   
                   
                   
                   
               
               
                   
               
               
                  2 
                 
                   Lihuaxuella 
                 
                 SEM88729.1 
                 73 
                 ○ 
                 AIVGSANNQLVED   R     
               
               
                   
                 
                   thermophile 
                 
                   
                   
                   
                 HG 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 21) 
               
               
                   
               
               
                  3 
                 
                   Baia soyae 
                 
                 TCP62478.1 
                 57 
                 x 
                   
               
               
                   
               
               
                  4 
                 
                   Thermoactinomyces 
                 
                 SFX81991.1 
                 57 
                 x 
                   
               
               
                   
                 sp. DSM 45891 
                   
                   
                   
                   
               
               
                   
               
               
                  5 
                 
                   Caldalkalibacillus 
                 
                 F5L9G2.1 
                 57 
                 ○ 
                 ED   R   HG 
               
               
                   
                   thermarum  TA2.A1 
                   
                   
                   
                 (SEQ ID NO: 49) 
               
               
                   
               
               
                  6 
                 
                   Bacillus badius 
                 
                 Q59224.1 
                 55 
                 x 
                   
               
               
                   
               
               
                  7 
                 
                   Fictibacillus 
                 
                 ANT47406.1 
                 54 
                 x 
                   
               
               
                   
                 
                   nanhaiensis 
                 
                   
                   
                   
                   
               
               
                   
               
               
                  8 
                   Bacillus  sp. 
                 BAB40584.1 
                 54 
                 ○ 
                     R   HG 
               
               
                   
                 UTB2301 
                   
                   
                   
                   
               
               
                   
               
               
                  9 
                 
                   Lysinibacillus 
                 
                 P23307.1 
                 53 
                 ○ 
                     R   H 
               
               
                   
                   sphaericus  ( Bacillus   
                   
                   
                   
                   
               
               
                   
                   sphaericus ) 
                   
                   
                   
                   
               
               
                   
               
               
                 10 
                 
                   Sporosarcina ureae 
                 
                 P97014.1 
                 52 
                 ○ 
                     R   HG 
               
               
                   
               
               
                 11 
                 
                   Bacillus halodurans 
                 
                 BAB03937.1 
                 51 
                 ○ 
                     R   HG 
               
               
                   
               
               
                 12 
                   Rhodococcus  sp. 
                 Q59771.2 
                 31 
                 x 
               
               
                   
               
            
           
         
       
     
     The mutation that imparts improved characteristics to the phenylalanine dehydrogenase related to an ability to measure phenylalanine is a substitution of asparagine, the fifth amino acid glycine, leucine, glutamine, or the ninth amino acid valine in motif (6) (the VNXGGLIQV (SEQ ID NO:6) motif) of the amino acid sequence of the wild-type phenylalanine dehydrogenase. Motif (6) has nine consecutive amino acid residues of VNXGGLIQV (SEQ ID NO:6) (X indicates any amino acid residue). The amino acid residue to be substituted can also be identified as asparagine in motif (6a) (VN) indicated by the shorter amino acid sequence in motif (6) or the second amino acid glycine, leucine, glutamine, or valine in motif (6b) (GGLIQV (SEQ ID NO:9)) indicated by the shorter amino acid sequence in motif (6). Although the position of motif (6), (6a), or (6b) in the amino acid sequence of the wild-type phenylalanine dehydrogenase can vary depending on the derivation of the enzyme, a person skilled in the art can determine the position of motif (6), (6a), or (6b) in the amino acid sequence of the wild-type phenylalanine dehydrogenase as appropriate and can thus identify the position of the asparagine, the glycine, the leucine, the glutamine, or the valine to be substituted. Normally, in the amino acid sequence of the phenylalanine dehydrogenase, motif (6) is located at positions 289 to 297, motif (6a) is located at positions 289 to 290, motif (6b) is located at positions 292 to 297, the asparagine to be substituted is located at position 290, the glycine to be substituted is located at position 293, the leucine to be substituted is located at position 294, the glutamine to be substituted is located at position 296, and the valine to be substituted is located at position 297, (for example, refer to Table 7 and  FIG. 3 ). 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Sequences of conservation regions of motif (6) (VNXGGLIQV (SEQ ID NO: 6) 
               
               
                 motif), motif (6a) (VN motif), and motif (6b) (GGLIQV (SEQ ID NO: 9) motif) 
               
               
                 in wild-type phenylalanine dehydrogenases derived from respective species 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Conservation region 
               
               
                   
                   
                 GenBank 
                   
                 Mutation 
                 (underline indicates 
               
               
                   
                   
                 Accession  
                 Identity 
                 site con- 
                 representative 
               
               
                   
                 Species origin 
                 No. 
                 (%) 
                 servation 
                 mutation sites) 
               
               
                   
               
               
                  1 
                 
                   Thermoactinomyces 
                 
                 P22823.1 
                 — 
                 ○ 
                   
               
               
                   
                 
                   intermedius 
                 
                   
                   
                   
                   
               
               
                   
               
               
                  2 
                 
                   Lihuaxuella 
                 
                 SEM88729.1 
                 73 
                 ○ 
                 YAPDYLV   N   AG   GL   I   Q     
               
               
                   
                 
                   thermophile 
                 
                   
                   
                   
                     V   ADELEGF 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 22) 
               
               
                   
               
               
                  3 
                 
                   Baia soyae 
                 
                 TCP62478.1 
                 57 
                 ○ 
                 YAPDYLV   N   AG   GL   I   Q     
               
               
                   
                   
                   
                   
                   
                     V   A 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 31) 
               
               
                   
               
               
                  4 
                 
                   Thermoactinomyces 
                 
                 SFX81991.1 
                 57 
                 ○ 
                 YAPDYLV   N   AG   GL   IQ 
               
               
                   
                 sp. DSM 45891 
                   
                   
                   
                     V   A 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 40) 
               
               
                   
               
               
                  5 
                 
                   Caldalkalibacillus 
                 
                 F5L9G2.1 
                 57 
                 ○ 
                 V   N   AG   GL   I   QV     
               
               
                   
                   thermarum  TA2.A1 
                   
                   
                   
                 (SEQ ID NO: 50) 
               
               
                   
               
               
                  6 
                 
                   Bacillus badius 
                 
                 Q59224.1 
                 55 
                 ○ 
                     VN   G   GL   I   QV   ADEL 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 58) 
               
               
                   
               
               
                  7 
                 
                   Fictibacillus 
                 
                 ANT47406.1 
                 54 
                 ○ 
                 V   N   AG   GL   I   QV   ADEL 
               
               
                   
                 
                   nanhaiensis 
                 
                   
                   
                   
                 (SEQ ID NO: 66) 
               
               
                   
               
               
                  8 
                   Bacillus  sp. 
                 BAB40584.1 
                 54 
                 ○ 
                 V   N   AG   GL   I   QV   ADEL 
               
               
                   
                 UTB2301 
                   
                   
                   
                 (SEQ ID NO: 75) 
               
               
                   
               
               
                  9 
                 
                   Lysinibacillus 
                 
                 P23307.1 
                 53 
                 ○ 
                 V   N   AG   GL   I   QV   ADEL 
               
               
                   
                   sphaericus  ( Bacillus   
                   
                   
                   
                 (SEQ ID NO: 84) 
               
               
                   
                   sphaericus ) 
                   
                   
                   
                   
               
               
                   
               
               
                 10 
                 
                   Sporosarcina ureae 
                 
                 P97014.1 
                 52 
                 ○ 
                 V   N   G   GL   I   QV   ADEL 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 93) 
               
               
                   
               
               
                 11 
                 
                   Bacillus halodurans 
                 
                 BAB03937.1 
                 51 
                 ○ 
                 V   N   G   GL   I   QV   ADEL 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO:102) 
               
               
                   
               
               
                 12 
                   Rhodococcus  sp. 
                 Q59771.2 
                 31 
                 ○ 
                     N   AG   G     
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 107) 
               
               
                   
               
            
           
         
       
     
     The modified phenylalanine dehydrogenase can be produced by introducing a mutation to a wild-type enzyme having one or more of the motifs (1) to (6). The wild-type enzyme may have two motifs, have three motifs, have four motifs, have five motifs, or six motifs selected from motifs (1) to (6). The modified phenylalanine dehydrogenase can be produced by introducing a mutation to a wild-type enzyme having one or more of the motifs (1a), (2a), (3), (4a), (5), (6a), and (6b). The wild-type enzyme may have two motifs, have three motifs, have four motifs, have five motifs, have six motifs, or have seven motifs selected from motifs (1a), (2a), (3), (4a), (5), (6a), and (6b). 
     Examples of the characteristics of the phenylalanine dehydrogenase related to measurement of phenylalanine include substrate specificity, solubility, and enzyme activity. The modified phenylalanine dehydrogenase may have only one of the characteristics described above or have two or three characteristics of the characteristics described above in combination. 
     The amino acid residues identified by motifs (1) to (6) may be identified by motifs (1a), (2a), (3), (4a), (5), (6a), and (6b) based on the corresponding relations described above. 
     As to the mutation in the six motifs described above, examples of the mutation (a single mutation or a combination with another mutation) improving at least one characteristic selected from substrate specificity, solubility, and enzyme activity include the following: 
     (a) a substitution of leucine in motif (1) with tryptophane, phenylalanine, tyrosine, or methionine; 
     (a+) a substitution of glycine as the fifth amino acid residue in motif (1) with alanine; 
     (a++) a substitution of glycine as the sixth amino acid residue in motif (1) with alanine; 
     (b) a substitution of threonine as the seventh amino acid residue in motif (2) with serine; 
     (c) a substitution of phenylalanine in motif (3) with leucine or isoleucine; 
     (d) a substitution of asparagine as the fourth amino acid residue in motif (4) with glycine, glutamine, threonine, lysine, proline, or serine; 
     (e) a substitution of arginine in motif (5) with aspartic acid or glutamic acid; 
     (f) a substitution of asparagine in motif (6) with valine, aspartic acid, methionine, glutamine, proline, isoleucine, histidine, alanine, threonine, glycine, or cysteine; 
     (g-) a substitution of glycine as the fifth amino acid residue in motif (6) with alanine; 
     (g) a substitution of leucine in motif (6) with phenylalanine, glutamine, histidine, asparagine, isoleucine, aspartic acid, glycine, glutamic acid, threonine, or serine; 
     (h) a substitution of glutamine in motif (6) with aspartic acid, glutamic acid, lysine, asparagine, serine or arginine; and 
     (i) a substitution of valine as the ninth amino acid residue in motif (6) with tyrosine, tryptophane, glutamic acid, asparagine, threonine, isoleucine, lysine, glycine, serine, leucine, methionine, glutamine, phenylalanine, cysteine, or arginine. 
     When the modified phenylalanine dehydrogenase has two or more amino acid residues of the phenylalanine dehydrogenase mutated, at least one or at least two of the mutations of the amino acid residues may be selected from the mutation of the amino acid residue described above. Furthermore, all the mutations of the amino acid residues may be selected from the mutation of the amino acid residue described above. 
     When the modified phenylalanine dehydrogenase includes two or more mutations, one or more of the following mutations can be included: 
     (a) a substitution of leucine in motif (1); 
     (b) a substitution of threonine as the seventh amino acid residue in motif (2); 
     (c) a substitution of phenylalanine in motif (3); 
     (e) a substitution of arginine in motif (5); 
     (f) a substitution of asparagine in motif (6); 
     (g) a substitution of leucine in motif (6); 
     (h) a substitution of glutamine in motif (6); and 
     (i) a substitution of valine as the ninth amino acid residue in motif (6). 
     When the modified phenylalanine dehydrogenase includes two or more mutations, one or more of the following combinations of mutations can be included: 
     (1) a combination of (f) the substitution of asparagine in motif (6) and (b) the substitution of threonine as the seventh amino acid residue in motif (2); 
     (c) the substitution of phenylalanine in motif (3); 
     (e) the substitution of arginine in motif (5); 
     (h) the substitution of glutamine in motif (6); or 
     (i) the substitution of valine as the ninth amino acid residue in motif (6); 
     (2) a combination of (c) the substitution of phenylalanine in motif (3) and (b) the substitution of threonine as the seventh amino acid residue in motif (2); 
     (g) the substitution of leucine in motif (6); 
     (h) the substitution of glutamine in motif (6); or 
     (i) the substitution of valine as the ninth amino acid residue in motif (6); 
     (3) a combination of (b) the substitution of threonine as the seventh amino acid residue in motif (2) and (g) the substitution of leucine in motif (6) or (h) the substitution of glutamine in motif (6); and 
     (4) a combination of (h) the substitution of glutamine in motif (6) and 
     (i) the substitution of valine as the ninth amino acid residue in motif (6). 
     When the modified phenylalanine dehydrogenase includes two or more mutations, one or more of the following mutations can be included: 
     (a) a substitution of leucine in motif (1) with tryptophane; 
     (b) a substitution of threonine as the seventh amino acid residue in motif (2) with serine; 
     (c) a substitution of phenylalanine in motif (3) with leucine or isoleucine; 
     (d) a substitution of arginine in motif (5) with aspartic acid; 
     (e) a substitution of asparagine in motif (6) with aspartic acid, methionine, glutamine, or cysteine; 
     (f) a substitution of leucine in motif (6) with glutamine or asparagine; 
     (g) a substitution of glutamine in motif (6) with aspartic acid; and 
     (h) a substitution of valine as the ninth amino acid residue in motif (6) with glycine, phenylalanine, or arginine. 
     When the modified phenylalanine dehydrogenase includes two or more mutations, one or more of the following combinations of mutations can be included: 
     (1) a combination of (f) the substitution of asparagine in motif (6) with aspartic acid, methionine, glutamine, or cysteine and (b) the substitution of threonine as the seventh amino acid residue in motif (2) with serine; 
     (c) the substitution of phenylalanine in motif (3) with leucine or isoleucine; 
     (e) the substitution of arginine in motif (5) with aspartic acid; 
     (h) the substitution of glutamine in motif (6) with aspartic acid; or 
     (i) the substitution of valine as the ninth amino acid residue in motif (6) with glycine, phenylalanine, or arginine; 
     (2) a combination of (c) the substitution of phenylalanine in motif (3) with leucine or isoleucine and (b) the substitution of threonine as the seventh amino acid residue in motif (2) with serine; 
     (g) the substitution of leucine in motif (6) with glutamine or asparagine; 
     (h) the substitution of glutamine in motif (6) with aspartic acid; or 
     (i) the substitution of valine as the ninth amino acid residue in motif (6) with glycine, phenylalanine, or arginine; 
     (3) a combination of (b) the substitution of threonine as the seventh amino acid residue in motif (2) with serine and (g) the substitution of leucine in motif (6) with glutamine or asparagine or (h) the substitution of glutamine in motif (6) with aspartic acid; and 
     (4) a combination of (h) the substitution of glutamine in motif (6) with aspartic acid and (i) the substitution of valine as the ninth amino acid residue in motif (6) with glycine, phenylalanine, or arginine. 
     When the modified phenylalanine dehydrogenase is one with three or more amino acid residues of the phenylalanine dehydrogenase mutated, at least one, at least two, and at least three of the mutations of the amino acid residues may be selected from the mutation of the amino acid residue described above. All the mutations of the amino acid residues may be selected from the mutation of the amino acid residue described above. 
     When the modified phenylalanine dehydrogenase includes three or more mutations, one or more of the following mutations can be included: 
     (a) a substitution of leucine in motif (1); 
     (b) a substitution of threonine as the seventh amino acid residue in motif (2); 
     (c) a substitution of phenylalanine in motif (3); 
     (e) a substitution of arginine in motif (5); 
     (f) a substitution of asparagine in motif (6); and 
     (i) a substitution of valine as the ninth amino acid residue in motif (6). 
     When the modified phenylalanine dehydrogenase includes three or more mutations, the following combination of mutations can be included: 
     a combination of (f) the substitution of asparagine in motif (6) and (b) the substitution of threonine as the seventh amino acid residue in motif (2), (c) the substitution of phenylalanine in motif (3), or (e) the substitution of arginine in motif (5). 
     When the modified phenylalanine dehydrogenase included three or more mutations, one or more of the following mutations can be included: 
     (a) a substitution of leucine in motif (1) with tryptophane; 
     (b) a substitution of threonine as the seventh amino acid residue in motif (2) with serine; 
     (c) a substitution of phenylalanine in motif (3) with isoleucine; 
     (e) a substitution of arginine in motif (5) with aspartic acid; 
     (f) a substitution of asparagine in motif (6) with aspartic acid or methionine; and 
     (i) a substitution of valine as the ninth amino acid residue in motif (6) with phenylalanine. 
     When the modified phenylalanine dehydrogenase includes three or more mutations, the following combination of mutations can be included: 
     a combination of (f) the substitution of asparagine in motif (6) with aspartic acid or methionine and (b) the substitution of threonine as the seventh amino acid residue in motif (2) with serine, (c) the substitution of phenylalanine in motif (3) with isoleucine, or 
     (e) the substitution of arginine in motif (5) with aspartic acid. 
     When the modified phenylalanine dehydrogenase is one with four or more amino acid residues of the phenylalanine dehydrogenase mutated, at least one, at least two, at least three, and at least four of the mutations of the amino acid residues may be selected from the mutation of the amino acid residue described above. All the mutations of the amino acid residues may be selected from the mutation of the amino acid residue described above. 
     When the modified phenylalanine dehydrogenase includes four or more mutations, one or more of the following mutations can be included: 
     (b) a substitution of threonine as the seventh amino acid residue in motif (2); 
     (c) a substitution of phenylalanine in motif (3); 
     (e) a substitution of arginine in motif (5); and 
     (f) a substitution of asparagine in motif (6). 
     When the modified phenylalanine dehydrogenase includes four or more mutations, the following combination of mutations can be included: 
     a combination of (f) the substitution of asparagine in motif (6) and 
     (b) the substitution of threonine as the seventh amino acid residue in motif (2), 
     (c) the substitution of phenylalanine in motif (3), or 
     (e) the substitution of arginine in motif (5). 
     When the modified phenylalanine dehydrogenase includes four or more mutations, one or more of the following mutations can be included: 
     (b) a substitution of threonine as the seventh amino acid residue in motif (2) with serine; 
     (c) a substitution of phenylalanine in motif (3) with isoleucine; 
     (e) a substitution of arginine in motif (5) with aspartic acid; and 
     (f) a substitution of asparagine in motif (6) with aspartic acid. 
     When the modified phenylalanine dehydrogenase includes four or more mutations, the following combination of mutations can be included: 
     a combination of (f) the substitution of asparagine in motif (6) with aspartic acid and 
     (b) the substitution of threonine as the seventh amino acid residue in motif (2) with serine, 
     (c) the substitution of phenylalanine in motif (3) with isoleucine, or 
     (e) the substitution of arginine in motif (5) with aspartic acid. 
     The phenylalanine dehydrogenase before the mutation may be a phenylalanine dehydrogenase having any of the following amino acid sequences (A) to (C). 
     (A) the amino acid sequence of SEQ ID NO:1, 
     (B) an amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO:1, or 
     (C) an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:1. 
     The amino acid sequence of SEQ ID NO:1 is a wild-type phenylalanine dehydrogenase derived from  Thermoactinomyces intermedius  (TiPheDH (wt)) and is encoded by a codon-optimized nucleotide sequence (SEQ ID NO:2) of TiPheDH (wt), for example. 
     The amino acid residue to be mutated, such as substitution, deletion, insertion, or addition, is normally L-alanine (A), L-asparagine (N), L-cysteine (C), L-glutamine (Q), L-isoleucine (I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophane (W), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), L-lysine (K), or glycine (G) as a natural L-α-amino acid. When the mutation is substitution, addition, or insertion, the amino acid residue to be substituted, added, or inserted is the same as the amino acid residue to be mutated described above. In the present specification, L and a may be omitted in the expression of the amino acid. 
     The amino acid sequence (B) described above may include mutations (for example, substitutions, deletions, insertions, and additions) of one or several amino acid residues. The number of the mutations is, for example, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, or 1 to 25, 1 to 20, 1 to 15, and 1 to 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). 
     The amino acid sequence (C) described above may have at least 90% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO:1. The percentage of the amino acid sequence identity may be 91% or more, 92% or more, 93% or more, or 94% or more, 95% or more or 96% or more, 97% or more, and 98% or more or 99% or more. 
     A protein identified by the amino acid sequences (B) and (C) can have 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more activity of the phenylalanine dehydrogenase activity of a protein having the amino acid sequence (A) described above when measured under the same conditions. 
     The amino acid sequence identity can be determined, for example, using algorithm BLAST by Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) and FASTA by Pearson (Methods Enzymol., 183, 63 (1990)). A program referred to as BLASTP has been developed based on this algorithm BLAST (see www.ncbi.nlm.nih.gov). Thus, the amino acid sequence identity may be calculated using these programs with default settings. Also, for example, a numerical value obtained by calculating Similarity as a percentage using a full-length polypeptide portion encoded in an ORF and using software GENETYX Ver 7.0.9 with a setting of “Gaps are NOT taken into account” or with a setting of Unit Size to Compare=2 from Genetyx Corporation employing Lipman-Pearson method may be used as the amino acid sequence identity. The lowest value among the values derived from these calculations may be employed as the amino acid sequence identity. 
     To prepare (B) the amino acid sequence including substitutions, deletions, insertions, or additions of one or several amino acid residues in the amino acid sequence of SEQ ID NO:1 and (C) the amino acid residue having 90% or more identity to the amino acid sequence of SEQ ID NO:1, when a mutation of an amino acid residue is introduced to the amino acid sequence of SEQ ID NO:1, and when the mutation of the amino acid residue is a substitution, such a substitution of the amino acid residue may be a conservative substitution. The term “conservative substitution” refers to substituting a certain amino acid residue with an amino acid residue having a similar side chain. Families of the amino acid residues having the similar side chain are well-known in the art. Examples of such families include amino acids having a basic side chain (for example, lysine, arginine, and histidine), amino acids having an acidic side chain (for example, aspartic acid and glutamic acid), amino acids having an uncharged polar side chain (for example, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), amino acids having a nonpolar side chain (for example, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophane), amino acids having a β-position branched side chain (for example, threonine, valine, and isoleucine), amino acids having an aromatic side chain (for example, tyrosine, phenylalanine, tryptophane, and histidine), amino acids having a hydroxy group (for example, alcoholic, phenolic)-containing side chain (for example, serine, threonine, and tyrosine), and amino acids having a sulfur-containing side chain (for example, cysteine and methionine). The amino acid having an uncharged polar side chain and the amino acid having a nonpolar side chain may collectively be called a neutral amino acid. The conservative substitution of the amino acid may preferably be the substitution between aspartic acid and glutamic acid, the substitution among arginine, lysine, and histidine, the substitution between tryptophane and phenylalanine, the substitution between phenylalanine and valine, the substitution among leucine, isoleucine, and alanine, and the substitution between glycine and alanine. 
     When the phenylalanine dehydrogenase before the mutation is a phenylalanine dehydrogenase of (A) to (C) described above, the modified phenylalanine dehydrogenase may be a modified phenylalanine dehydrogenase including one or more of the following mutations: 
     R2, R10, Y11, C19, L41, G42, G43, C44, A50, S51, M66, C70, F77, K90, Y112, T115, D116, F124, R129, L137, K139, S140, K144, T147, K173, C200, C210, K216, K220, Q222, N227, R228, C234, C240, R255, C256, L257, N264, R271, Q277, K278, R279, S280, C282, N290, G293, L294, Q296, V297, R326, K328, N329, N331, C335, R340, K347, and/or K348, having enzyme activity, and showing improvement of one or more of the following characteristics: substrate specificity, solubility, and/or enzyme activity. 
     When the phenylalanine dehydrogenase before the mutation is a phenylalanine dehydrogenase of (A) to (C) described above, the modified phenylalanine dehydrogenase include one or more of the following substitutions: 
     R2D, R2E, R10D, R10E, Y11E, Y11D, C19A, C19S, L41W, L41F, L41Y, L41M, G42A, G43A, C44A, C44S, A50D, A50E, S51D, S51E, M66I, M66L, M66V, C70A, C70S, F77L, F77I, F77R, K90E, Y112L, T115S, D116E, F124L, F124I, R129K, L137V, K139E, S140A, K144G, T147A, T147S, T147N, K173E, K173D, C200A, C200S, C210S, C210A, K216D, K216E, K220D, K220E, Q222E, Q222D, N227D, N227E, R228E, R228D, C234A, C234S, C240S, C240A, R255E, R255D, C256A, C256S, L257K, N264G, N264Q, N264T, N264K, N264P, N264S, R271D, R271E, Q277D, K278D, K278E, R279D, R279E, S280D, C282S, C282A, N290V, N290D, N290M, N290Q, N290P, N290I, N290H, N290A, N290T, N290G, N290C, G293A, L294F, L294Q, L294H, L294N, L294I, L294D, L294G, L294E, L294T, L294S, Q296D, Q296E, Q296K, Q296N, Q296S, Q296R, V297Y, V297W, V297E, V297N, V297T, V297I, V297K, V297G, V297S, V297L, V297M, V297Q, V297F, V297C, V297R, R326E, K328E, K328D, N329D, N331E, N331D, C335A, C335S, R340D, R340E, K347D, and/or K348E, having enzyme activity, and showing improvement of one or more of the following characteristics substrate specificity, solubility, and/or enzyme activity. 
     In an embodiment, substrate specificity of the phenylalanine dehydrogenase for phenylalanine is improved. The improvement in the substrate specificity of the phenylalanine dehydrogenase for phenylalanine means that the reactivity of the modified phenylalanine dehydrogenase with phenylalanine further improves as compared with that of a wild-type enzyme. In other words, it means that the reactivity of the modified phenylalanine dehydrogenase with amino acids other than phenylalanine is reduced. Examples of the amino acids other than phenylalanine include L-α-amino acids other than phenylalanine. Specifically, examples of the L-α-amino acids other than phenylalanine include the 19 L-α-amino acids other than phenylalanine that form proteins, such as cystine, taurine, citrulline, ornithine, and α-amino butyric acid. Substrate specificity is measured by comparing the lowest reactivity of the phenylalanine dehydrogenase with an amino acid other than phenylalanine (for example, tyrosine) with the reactivity of phenylalanine dehydrogenase with phenylalanine (relative activity) as an indicator. The reactivity of the phenylalanine dehydrogenase may be measured based on the amount of NADH produced by an enzyme reaction. The extent of the improvement in the substrate specificity of the modified phenylalanine dehydrogenase with respect to the wild-type enzyme is more than 1 when the characteristic of the wild type is 1 and can be more than each of 1.01, 1.03, 1.04, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0. 
     Preferred examples of a mutation suitable for the improvement in substrate specificity include the following: 
     (I) a mutation (for example, a substitution) of at least one amino acid residue in at least one of motifs (1) to (6); 
     (II) one or more of the following substitutions: 
     (a) a substitution of leucine in motif (1); 
     (b) a substitution of threonine as the seventh amino acid residue in motif (2); 
     (c) a substitution of phenylalanine in motif (3); 
     (d) a substitution of asparagine as the fourth amino acid residue in motif (4); 
     (e) a substitution of arginine in motif (5); 
     (f) a substitution of asparagine in motif (6); 
     (g) a substitution of leucine in motif (6); 
     (h) a substitution of glutamine in motif (6); and 
     (i) a substitution of valine as the ninth amino acid residue in motif (6); and 
     (III) one or more of the following mutations (for example, a substitution or substitutions): 
     R10, Y11, C19, L41, G42, G43, C44, M66, C70, F77, Y112, T115, D116, F124, R129, L137, K139, S140, K144, T147, K173, C200, C210, Q222, R228, C234, C240, R255, C256, N264, R271, K278, C282, N290, G293, L294, Q296, V297, and/or C335. 
     Further examples of the mutation suitable for the improvement in substrate specificity include the following: 
     (I) one or more of the following substitutions: 
     (a) a substitution of leucine in motif (1) with tryptophane, phenylalanine, tyrosine, or methionine; 
     (b) a substitution of threonine as the seventh amino acid residue in motif (2) with serine; 
     (c) a substitution of phenylalanine in motif (3) with leucine or isoleucine; 
     (d) a substitution of asparagine as the fourth amino acid residue in motif (4) with glycine, glutamine, threonine, lysine, proline, or serine; 
     (e) a substitution of arginine in motif (5) with aspartic acid; 
     (f) a substitution of asparagine in motif (6) with valine, aspartic acid, methionine, glutamine, proline, isoleucine, histidine, alanine, threonine, glycine, or cysteine; 
     (g) a substitution of leucine in motif (6) with phenylalanine, glutamine, histidine, asparagine, isoleucine, aspartic acid, glycine, glutamic acid, threonine, or serine; 
     (h) a substitution of glutamine in motif (6) with aspartic acid, glutamic acid, lysine, asparagine, serine, or arginine; and 
     (i) a substitution of valine as the ninth amino acid residue in motif (6) with tyrosine, tryptophane, glutamic acid, asparagine, threonine, isoleucine, lysine, glycine, serine, leucine, methionine, glutamine, phenylalanine, cysteine, or arginine, and 
     (II) one or more of the following mutations (for example, a substitution or substitutions): 
     R10D, Y11E, C19A, C19S, L41W, L41F, L41Y, L41M, G42A, G43A, C44A, C44S, M66I, M66L, M66V, C70A, C70S, F77L, F77I, F77R, Y112L, T115S, D116E, F124L, F124I, R129K, L137V, K139E, S140A, K144G, T147A, T147S, T147N, K173E, C200A, C210S, C210A, Q222D, R228E, C234A, C234S, C240S, C240A, R255E, C256A, C256S, N264G, N264Q, N264T, N264K, N264P, N264S, R271D, K278D, C282S, C282A, N290V, N290D, N290M, N290Q, N290P, N290I, N290H, N290A, N290T, N290G, N290C, G293A, L294F, L294Q, L294H, L294N, L294I, L294D, L294G, L294E, L294T, L294S, Q296D, Q296E, Q296K, Q296N, Q296S, Q296R, V297Y, V297W, V297E, V297N, V297T, V297I, V297K, V297G, V297S, V297L, V297M, V297Q, V297F, V297C, V297R, C335A, and/or C335S. 
     In another embodiment, solubility of the phenylalanine dehydrogenase is improved. The improvement in the solubility of the phenylalanine dehydrogenase means that the solubility of the modified phenylalanine dehydrogenase further improves as compared with that of a wild-type enzyme. Specifically, the solubility of the phenylalanine dehydrogenase can be measured with the concentration of supernatant fluid when an aqueous phenylalanine dehydrogenase solution is concentrated until it flocculates as an indicator, for example. The extent of the improvement in the solubility of the modified phenylalanine dehydrogenase with respect to the wild-type enzyme is more than 1 when the characteristic of the wild type is 1 and can be more than each of 1.01, 1.03, 1.04, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0. 
     A mutation suitable for the improvement in solubility includes the following: 
     (I) a mutation (for example, a substitution) of at least one amino acid residue in at least one of motifs (1) to (3), (5), and (6); 
     (II) one or more of the following substitutions: 
     (a) a substitution of leucine in motif (1); 
     (b) a substitution of threonine as the seventh amino acid residue in motif (2); 
     (c) a substitution of phenylalanine in motif (3); 
     (e) a substitution of arginine in motif (5); 
     (f) a substitution of asparagine in motif (6); and 
     (i) a substitution of valine as the ninth amino acid residue in motif (6); and 
     (III) one or more of the following mutations (for example, a substitution or substitutions): 
     R2, R10, Y11, C19, L41, C44, A50, S51, C70, K90, T115, F124, K173, C200, C210, K216, K220, Q222, N227, R228, C240, R255, C256, L257, R271, Q277, K278, R279, C282, N290, V297, R326, N329, N331, C335, R340, and/or K348. 
     Further examples of the mutation suitable for the improvement in solubility include the following: 
     (I) one or more of the following substitutions: 
     (a) a substitution of leucine in motif (1) with tryptophane; 
     (b) a substitution of threonine as the seventh amino acid residue in motif (2) with serine; 
     (c) a substitution of phenylalanine in motif (3) with isoleucine; 
     (e) a substitution of arginine in motif (5) with aspartic acid or glutamic acid; 
     (f) a substitution of asparagine in motif (6) with aspartic acid or methionine; and 
     (i) a substitution of valine as the ninth amino acid residue in motif (6) with phenylalanine and 
     (II) one or more of the following mutations (for example, a substitution or substitutions): 
     R2D, R2E, R10D, R10E, Y11E, Y11D, C19A, C19S, L41W, C44A, C44S, A50D, A50E, S51D, S51E, C70A, C70S, K90E, T115S, F124I, K173E, K173D, C200A, C200S, C210S, C210A, K216D, K216E, K220D, K220E, Q222E, Q222D, N227D, N227E, R228E, R228D, C240S, C240A, R255E, R255D, C256A, L257K, R271D, R271E, Q277D, K278D, K278E, R279E, C282S, C282A, N290D, N290M, V297F, R326E, N329D, N331E, N331D, C335A, C335S, R340D, and/or K348E. 
     In still another embodiment, the activity of the phenylalanine dehydrogenase for phenylalanine is improved. The improvement in the activity of the phenylalanine dehydrogenase for phenylalanine means that the activity of the modified phenylalanine dehydrogenase for phenylalanine further improves as compared with that of a wild-type enzyme. Specifically, the improvement in the activity of the phenylalanine dehydrogenase for phenylalanine can be achieved when, with the activity of the wild-type phenylalanine dehydrogenase for phenylalanine at a certain concentration (for example, either concentration of a low concentration or a high concentration) being 100, the activity of the modified phenylalanine dehydrogenase for phenylalanine at the same concentration is larger than 100. Such a modified phenylalanine dehydrogenase enables quick and highly sensitive measurement of phenylalanine and is consequently useful for measurement of phenylalanine. The extent of the improvement in the activity of the modified phenylalanine dehydrogenase with respect to the wild-type enzyme is more than 1 when the characteristic of the wild type is 1 and can be more than each of 1.01, 1.03, 1.04, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0. 
     Preferred examples of a mutation suitable for the improvement in activity include the following: 
     (I) a mutation (for example, a substitution) of at least one amino acid residue in at least one of motifs (3), (5), and (6); 
     (II) one or more of the following substitutions: 
     (c) a substitution of phenylalanine in motif (3); 
     (e) a substitution of arginine in motif (5); 
     (f) a substitution of asparagine in motif (6); 
     (g) a substitution of leucine in motif (6); 
     (h) a substitution of glutamine in motif (6); and 
     (i) a substitution of valine as the ninth amino acid residue in motif (6); and 
     (III) one or more of the following mutations (for example, a substitution or substitutions): 
     R10, Y11, C19, F77, F124, T147, K173, C200, C210, K216, K220, Q222, N227, R228, C234, C240, R255, C256, R271, K278, R279, S280, C282, N290, L294, Q296, V297, K328, N329, N331, C335, R340, and/or K347. 
     Further examples of the mutation suitable for the improvement in activity include the following: 
     (I) one or more of the following substitutions: 
     (c) a substitution of phenylalanine in motif (3) with isoleucine; 
     (e) a substitution of arginine in motif (5) with aspartic acid or glutamic acid; 
     (f) a substitution of asparagine in motif (6) with aspartic acid, threonine, or cysteine; 
     (g) a substitution of leucine in motif (6) with glutamine; 
     (h) a substitution of glutamine in motif (6) with aspartic acid or glutamic acid; and 
     (i) a substitution of valine as the ninth amino acid residue in motif (6) with threonine or glycine and 
     (II) one or more of the following mutations (for example, a substitution or substitutions): 
     R10D, Y11E, C19S, F77L, F124I, T147S, K173E, K173D, C200A, C200S, C210S, K216D, K216E, K220D, K220E, Q222E, Q222D, N227D, N227E, R228E, R228D, C234A, C234S, C240S, C240A, R255E, R255D, C256A, R271D, R271E, K278D, R279D, R279E, S280D, C282S, N290D, N290T, N290C, L294Q, Q296D, Q296E, V297T, V297G, K328E, K328D, N329D, N331D, C335A, R340E, and/or K347D. 
     The modified phenylalanine dehydrogenase may be one having the mutation described above alone or both the mutation described above and a supplemental mutation to include an amino acid sequence having at least 90% or more amino acid sequence identity to the amino acid sequence of the (wild-type) phenylalanine dehydrogenase before the mutation. The percentage of the amino acid sequence identity may be 92% or more, 95% or more, 97% or more, and 98% or more or 99% or more. 
     The modified phenylalanine dehydrogenase may also have another peptide component (for example, a tag moiety) at a C-terminus or an N-terminus. Examples of the other peptide component that can be added to the modified phenylalanine dehydrogenase can include peptide components making purification of an objective protein easy (for example, tag moiety such as histidine tag and strep-tag II; proteins such as glutathione-S-transferase and maltose-binding protein commonly used for the purification of the objective protein), peptide components improving the solubility of the objective protein (for example, Nus-tag), peptide components working as a chaperon (for example, trigger factor), and peptide components as a protein or a domain of the protein having another function or a linker connecting them. 
     The amino acid sequence identity can be determined, for example, using algorithm BLAST by Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) and FASTA by Pearson (Methods Enzymol., 183, 63 (1990)). A program referred to as BLASTP has been developed based on this algorithm BLAST (see www.ncbi.nlm.nih.gov). Thus, the amino acid sequence identity may be calculated using these programs with default settings. Also, for example, a numerical value obtained by calculating Similarity as a percentage using a full length polypeptide portion encoded in an ORF and using software GENETYX Ver 7.0.9 with a setting of Unit Size to Compare=2 from Genetyx Corporation employing Lipman-Pearson method may be used as the amino acid sequence identity. The lowest value among the values derived from these calculations may be employed as the amino acid sequence identity. 
     The position of an amino acid residue at which the supplemental mutation can be introduced in an amino acid sequence is apparent to a person skilled in the art; the supplemental mutation can be introduced with reference to alignment of amino acid sequences, for example. Specifically, a person skilled in the art can 1) compare amino acid sequences of a plurality of homologs (for example, the amino acid sequence of SEQ ID NO:1 and an amino acid sequence of the other homolog) with each other, (2) demonstrate relatively conserved regions and relatively not conserved regions, then (3) predict regions capable of playing a functionally important role and regions incapable of playing a functionally important role from the relatively conserved regions and the relatively not conserved regions, respectively, and thus recognize correlativity between a structure and a function. The analysis result of the three-dimensional structure has been reported for phenylalanine dehydrogenases as described above, and thus a person skilled in the art can introduce the supplemental mutation based on the analysis result of the three-dimensional structure so as to enable the retention of the characteristics described above. The site at which the supplemental mutation is introduced may be an amino acid residue other than the amino acid residue described above. 
     When the supplemental mutation of the amino acid residue is a substitution, such a substitution of the amino acid residue may be a conservative substitution. The term “conservative substitution” refers to substituting a certain amino acid residue with an amino acid residue having a similar side chain. Families of the amino acid residues having the similar side chain are well-known in the art. Examples of such families comprise amino acids having a basic side chain (for example, lysine, arginine, and histidine), amino acids having an acidic side chain (for example, aspartic acid and glutamic acid), amino acids having an uncharged polar side chain (for example, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), amino acids having a nonpolar side chain (for example, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophane), amino acids having a β-position branched side chain (for example, threonine, valine, and isoleucine), amino acids having an aromatic side chain (for example, tyrosine, phenylalanine, tryptophane, and histidine), amino acids having a hydroxy group (for example, alcoholic, phenolic)-containing side chain (for example, serine, threonine, and tyrosine), and amino acids having a sulfur-containing side chain (for example, cysteine and methionine). The conservative substitution of the amino acid may preferably be the substitution between aspartic acid and glutamic acid, the substitution among arginine, lysine, and histidine, the substitution between tryptophane and phenylalanine, the substitution between phenylalanine and valine, the substitution among leucine, isoleucine, and alanine, and the substitution between glycine and alanine. 
     The present invention also provides a polynucleotide encoding the modified phenylalanine dehydrogenase. The polynucleotide may be DNA or RNA. 
     The modified phenylalanine dehydrogenase can be prepared using a transformant expressing the modified phenylalanine dehydrogenase or a cell-free system. The transformant can be produced by producing an expression vector and then introducing this expression vector into a host, for example. 
     The expression vector can include the polynucleotide encoding the modified phenylalanine dehydrogenase. 
     The expression vector comprises the polynucleotide (for example, DNA or RNA) encoding the modified phenylalanine dehydrogenase. The expression vector can further include regions encoding a promoter, a terminator, and a drug (for example, tetracycline, ampicillin, kanamycin, hygromycin, or phosphinothricin) resistant gene in addition to the polynucleotide as described herein. The expression vector may be a plasmid or an integrative vector. The expression vector may be a virus vector or a vector for a cell-free system. The expression vector may further include a polynucleotide encoding another peptide component that can be added to the modified phenylalanine dehydrogenase at the 3′ or 5′-end side with respect to the polynucleotide. Examples of the polynucleotide encoding the other peptide component include a polynucleotide encoding the peptide component that renders purification of an objective protein easy as described above, a polynucleotide encoding the peptide component improving the solubility of the objective protein as described above, a polynucleotide encoding the peptide component working as a chaperon, and a polynucleotide encoding the peptide component as a protein or a domain of the protein having another function or a linker connecting them. Various expression vectors including the polynucleotide encoding the other peptide component can be used. Consequently, to produce the expression vector, such expression vectors may be used. Examples thereof include expression vectors including the polynucleotide encoding the peptide component rendering purification of an objective protein easy (for example, pET-15b, pET-51b, pET-41a, and pMAL-p5G), expression vectors including the polynucleotide encoding the peptide component improving the solubility of the objective protein (for example, pET-50b), expression vectors including the polynucleotide encoding the peptide component working as a chaperon (for example, pCold TF), and expression vectors including the polynucleotide encoding the peptide component as a protein or a domain of the protein having another function or a linker connecting them. To enable cleavage between the modified phenylalanine dehydrogenase and the other peptide component added thereto after protein expression, the expression vector may include a region encoding a site to be cleaved by a protease between the polynucleotide encoding the modified phenylalanine dehydrogenase and the polynucleotide encoding the other peptide component. 
     Various prokaryotic cells including bacteria belonging to the genus  Escherichia  such as  Escherichia coli,  bacteria belonging to the genus  Corynebacterium  (for example,  Corynebacterium glutamicum ), and bacteria belonging to the genus  Bacillus  (for example,  Bacillus subtilis ) and various eukaryotic cells including fungi belonging to the genus  Saccharomyces  (for example,  Saccharomyces cerevisiae ), fungi belonging to the genus  Pichia  (for example,  Pichia stipitis ), and fungi belonging to the genus  Aspergillus  (for example,  Aspergillus oryzae ) can be used as the host for expressing the modified phenylalanine dehydrogenase. A strain in which a certain gene has been deleted may be used as the host. Examples of the transformant may include transformants in which the expression vector is retained in its cytoplasm and transformants in which an objective gene is integrated into its genome. 
     The transformant is a host cell that can produce the modified phenylalanine dehydrogenase or can express the polynucleotide to produce the modified phenylalanine dehydrogenase. Specifically, the transformant is a host cell including an expression unit including the polynucleotide. Examples of the host cell including the expression unit including the polynucleotide include a host cell into which the expression vector is introduced as a whole and a host cell in which an expression unit in the expression vector is introduced to its genome. The host cell is not limited to a particular host cell so long as it can express the modified phenylalanine dehydrogenase. The host cell may be homologous or heterologous with respect to the modified phenylalanine dehydrogenase and the polynucleotide and can be heterologous therewith. The host cell may be homologous or heterologous with respect to the promoter and can be heterologous therewith. Examples of the host cell include animal cells, plant cells, insect cells, and microorganisms; microorganisms are a particular example. The host cell for use can be a bacterium or a fungus. The bacterium may be a Gram-positive bacterium or a Gram-negative bacterium. 
     The transformant can be cultured in a medium having a composition described below, for example, using a certain culture apparatus (for example, a test tube, a flask, or a jar fermenter). The culture conditions can be set as appropriate. Specifically, the culture temperature may be 10° C. to 37° C., pH may be 6.5 to 7.5, and the culture time may be 1 hour to 100 hours. Culture may be performed while controlling a dissolved oxygen concentration. In this case, the dissolved oxygen concentration (a DO value) in a culture liquid may be used as an indicator for the control. Ventilation and stirring conditions can be controlled such that a relative dissolved oxygen concentration DO value when the oxygen concentration in the atmosphere is 21% will not be less than 1 to 10%, for example, 3% to 8%. Culture may be batch culture or fed-batch culture. In the case of the fed-batch culture, culture can be continued by adding a solution as a sugar source and a solution containing phosphoric acid to the culture liquid continuously or continually in a successive manner. 
     The host to be transformed is as described above; describing  Escherichia coli  in detail, the host can be selected from  Escherichia coli  K12 subspecies  Escherichia coli  JM109 strain, DH5a strain, HB101 strain, BL21 (DE3) strain, and the like. The method for performing transformation and the method for selecting the transformant are described in Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor press (2001 Jan. 15) and the like. The following describes a method for producing a transformed  Escherichia coli  and producing a certain enzyme using the same more specifically as an example. 
     A promoter generally used for producing a foreign protein in  E. coli  can be used as a promoter for expressing the polynucleotide; examples thereof include potent promoters such as PhoA, PhoC, a T7 promoter, a lac promoter, a trp promoter, a trc promoter, a tac promoter, a PR promoter and a PL promoter of the lambda phage, and a T5 promoter, and PhoA, PhoC, and lac are particular examples. For example, pUC (for example, pUC19 and pUC18), pSTV, pBR (for example, pBR322), pHSG (for example, pHSG299, pHSG298, pHSG399, and pHSG398), RSF (for example, RSF1010), pACYC (for example, pACYC177 and pACYC184), pMW (for example, pMW119, pMW118, pMW219, and pMW218), pQE (for example, pQE30), and derivatives thereof may be used as a vector. A vector from phage DNA may also be utilized as the other vector. Furthermore, an expression vector that includes a promoter and can express an inserted DNA sequence may also be used. The vector may be pUC, pSTV, or pMW. 
     Also, a terminator that is a transcription terminating sequence may be ligated downstream of the polynucleotide. Examples of such a terminator include a T7 terminator, an fd phage terminator, a T4 terminator, a terminator of a tetracycline resistant gene, and a terminator of  Escherichia coli  trpA gene. 
     The vector for introducing the polynucleotide into  Escherichia coli  can be a so-called multicopy type; examples thereof include plasmids having a replication starting point derived from ColE1 such as pUC-based plasmids, pBR322-based plasmids, and derivatives thereof. The “derivative” can one in which modification has been performed on the plasmid by substitution, deletion, insertion, and/or addition of nucleotides. 
     To select the transformant, the vector can have a marker such as an ampicillin resistant gene. Expression vectors having a potent promoter are commercially available as such a plasmid (for example, pUC-based ones (manufactured by Takara Bio Inc.), pPROK-based ones (manufactured by Clontech), and pKK233-2 (manufactured by Clontech)). 
     The modified phenylalanine dehydrogenase can be obtained by transforming  Escherichia coli  using the obtained expression vector and culturing this  Escherichia coli.    
     Media such as M9-casamino acid medium and LB medium generally used for culturing  Escherichia coli  may be used as the medium. The medium may contain a certain carbon source, nitrogen source, and coenzyme (for example, pyridoxine hydrochloride). Specifically, peptone, yeast extract, NaCl, glucose, MgSO 4 , ammonium sulfate, potassium dihydrogen phosphate, ferric sulfate, manganese sulfate, and the like may be used. Culture conditions and production guidance conditions can be selected as appropriate in accordance with the types of a marker and a promoter of the chosen vector, a host bacterium, and the like. 
     The modified phenylalanine dehydrogenase is collected by the following methods or the like. The modified phenylalanine dehydrogenase can be obtained as a pulverized product or a dissolved product by collecting the transformant and then pulverizing (for example, sonication or homogenization) or dissolving (for example, lysozyme treatment) bacterial cells. The modified phenylalanine dehydrogenase can be obtained by subjecting such a pulverized product or dissolved product to a method such as extraction, precipitation, filtration, or column chromatography. 
     A method for analyzing phenylalanine is provided. The method of analysis can include measuring phenylalanine contained in a test sample using the modified phenylalanine dehydrogenase. 
     The test sample is not limited to a particular test sample so long as it is a sample suspected to contain phenylalanine; examples thereof include samples derived from living bodies (for example, blood, urine, saliva, and tear) and food and drink (for example, nutritional drinks and amino acid drinks). Phenylalanine in the test sample may be in a low concentration (for example, a concentration of less than 1 mM such as 1 μM or more and less than 1 mM) or in a high concentration (for example, a concentration of 1 mM or more such as 1 mM or more and less than 1 M). 
     The method of analysis is not limited to a particular method so long as it can measure phenylalanine using the modified phenylalanine dehydrogenase; phenylalanine is measured by mixing the test sample with nicotinamide adenine dinucleotide(NAD + ) in an alkaline or neutral condition, or preferably in an alkaline buffer solution, then subjecting the mixed sample to an enzyme reaction using the modified phenylalanine dehydrogenase, and lastly detecting NADH produced from NAD +  by the action of the modified phenylalanine dehydrogenase, for example. Specifically, in the presence of nicotinamide adenine dinucleotide(NAD + ), in an alkaline buffer solution, the modified phenylalanine dehydrogenase acts on the test sample, whereby an amino group of a substrate contained in a sample derived from a living body is subjected to oxidative deamination, and the reduced form (NADH) is produced from nicotinamide adenine dinucleotide(NAD + ). Thus, quantification of phenylalanine can be performed by detecting NADH through absorbance (340 nm) or the like. The method for measuring amino acids based on such methodology are known (for example, refer to Ueatrongchit T, Asano Y, Anal Biochem., 2011 Mar. 1; 410(1): 44-56). Quantification of phenylalanine can also be performed by reducing a dye with the produced NADH and detecting the color development of the reduced dye as absorbance or the like. Furthermore, NADH can also be detected by an electrochemical method. Phenylalanine can be measured by electrochemically oxidizing NADH produced by causing the modified phenylalanine dehydrogenase to act on the test sample in an alkaline or neutral condition and measuring its oxidation current, or alternatively, by reducing a coexisting electron mediator by the produced NADH and measuring an electrochemical oxidation current of the reduced electron mediator, for example. Electron transfer between NADH and the electron mediator may be mediated by a catalyst. Note that measurement of phenylalanine can be performed by the rate method (the initial rate method). 
     The modified phenylalanine dehydrogenase does not react with amino acids other than phenylalanine or has low reactivity therewith. Consequently, even when not only phenylalanine but also other amino acids are contained in the test sample, the amount of phenylalanine in the test sample can be evaluated using the modified phenylalanine dehydrogenase. 
     Furthermore, a kit for analyzing phenylalanine including the modified phenylalanine dehydrogenase is described. 
     The kit can further include at least one of a buffer solution or a buffer salt for reaction and nicotinamide adenine dinucleotide (NAD + ). 
     The buffer solution or the buffer salt for reaction is used for maintaining pH of a reaction liquid at a value suitable for an objective enzyme reaction. The buffer solution or the buffer salt for reaction is alkaline or neutral, for example, and preferably alkaline. 
     When the kit includes nicotinamide adenine dinucleotide(NAD + ), the kit may further include a dye to be reduced by NADH. In this case, the dye is reduced by NADH produced from NAD +  by the action of the modified phenylalanine dehydrogenase, and the color development of the reduced dye can be detected by absorbance or the like. A substance working as an electron mediator may be involved in the reduction of the dye. 
     An enzyme sensor for analyzing phenylalanine including (a) an electrode for detection and (b) the modified phenylalanine dehydrogenase as described herein immobilized or disposed on the electrode for detection. The modified phenylalanine dehydrogenase is immobilized or disposed on the electrode directly or indirectly. 
     As the electrode for detection, it is possible to use a biosensor directly or indirectly detecting a product or a byproduct (NH 3 +NADH+H + ) produced from phenylalanine by the modified phenylalanine dehydrogenase, for example; more specific examples include an electrode for detection utilizing the modified phenylalanine dehydrogenase and nicotinamide adenine dinucleotide(NAD + ). As such an electrode for detection, those described in WO 2005/075970 and WO 00/57166 can be used, for example. 
     EXAMPLES 
     The following describes the present invention with reference to the following examples in more detail; the present invention is not limited to the following examples. 
     Example 1 Construction of Plasmid for Expressing PheDH (Wild Type) 
     A recombinant expression system of PheDH using  Escherichia coli  was constructed. First, a plasmid for recombinant expression was constructed. As a sequence for insertion to pET-24a (Merck), in a nucleotide sequence (codon-optimized PheDH, SEQ ID NO:2) encoding the amino acid sequence of wild-type PheDH derived from  Thermoactinomyces intermedius  (SEQ ID NO:1), a DNA fragment (SEQ ID NO:112) in which a nucleotide sequence including an NdeI site+an initiation codon+a His-tag code sequence (CATATGCATCACCATCACCACCAC, SEQ ID NO:113) is added to the 5′-end, whereas a nucleotide sequence including a termination codon+a BamHI site (TAATGAGGATCC, SEQ ID NO:114) is added to the 3′-end was produced by chemical synthesis, which was incorporated into restriction enzyme sites of NdeI and BamHI of pET-24a to obtain a plasmid for expressing PheDH (the wild type). Using a standard method for analyzing a DNA sequence with this plasmid as a template, insertion of an objective gene to the plasmid was confirmed. In accordance with a standard method, a transformant of  Escherichia coli  BL21 (DE3) was acquired. 
     In the following, the plasmid including the PheDH sequence with the His-tag added to the N-terminus (SEQ ID NO:111 for the amino acid sequence and SEQ ID NO:112 for the nucleotide sequence) (the plasmid for expressing PheDH) will be called pET24a-PheDH, whereas the transformant of BL21 (DE3) by pET24a-PheDH will be called pET24a-PheDH-BL21 (DE3). 
     Example 2 Construction of Plasmid for Expressing PheDH Mutant 
     A PheDH mutant was prepared as follows. Using KAPA HiFi HS ReadyMix (Kapa Biosystems, Inc.), in accordance with a standard method with pET24a-PheDH as a template, mutation introduction to a PheDH gene was performed. When a plurality of mutations were introduced, supplemental mutations were successively introduced using a plasmid with a mutation introduced as a template. Using a standard method for analyzing a DNA sequence with each expression plasmid as a template, introduction of an objective mutation to the plasmid was confirmed. In accordance with a standard method, a transformant of  Escherichia coli  BL21 (DE3) was acquired. 
     Example 3 Preparation of PheDH 
     Preparation of PheDH for Evaluating Substrate Specificity 
     Preparation of PheDH for evaluating substrate specificity was performed as follows. First, from a glycerol stock of the transformant of  Escherichia coli  BL21 (DE3) acquired in Example 1 and Example 2, the bacteria were inoculated into an LB plate containing 25 μg/mL of kanamycin and were stationarily cultured at 37° C. overnight. An LB liquid medium containing 25 μg/mL of kanamycin in an amount of 2 mL was put into a tube with a volume of 14 mL, and a single colony on the LB plate was inoculated and was cultured by reciprocal shaking at 37° C. overnight. A culture liquid in an amount of 50 μL was added to 4 mL of the LB liquid medium containing 25 μg/mL of kanamycin and was cultured by reciprocal shaking until the value of OD600 at 37° C. reached about 0.9. The culture liquid was left at rest at 30° C. for 30 minutes, isopropyl β-D-thiogalactopyranoside (IPTG) was added thereto so as to have a final concentration of 0.5 mM, the culture liquid was cultured by reciprocal shaking at 30° C. overnight, and then the bacteria were collected into a 2 mL tube. 
     Bacterium cells were suspended in a buffer for pulverization (200 mM Tris-HCl, pH 8.0) and were pulverized using an ultrasonic pulverizer (BIORUPTOR manufactured by Cosmo Bio Co., Ltd.). This pulverized liquid was centrifuged at 6,000×g for 10 minutes to collect PheDH as an objective protein as a supernatant. 
     Preparation of PheDH for Evaluating Solubility and Activity 
     Preparation of PheDH for evaluating solubility and activity was performed as follows. First, from a glycerol stock of the transformant of  Escherichia coli  BL21 (DE3) acquired in Example 1 and Example 2, the bacteria were inoculated into an LB plate containing 25 μg/mL of kanamycin and were stationarily cultured at 37° C. overnight. An LB liquid medium containing 25 μg/mL of kanamycin in an amount of 2 mL was put into a tube with a volume of 14 mL, and a single colony on the LB plate was inoculated and was cultured by reciprocal shaking at 37° C. overnight. A culture liquid in an amount of 300 μL was added to 30 mL of the LB liquid medium containing 25 μg/mL of kanamycin and was cultured by reciprocal shaking until the value of OD600 at 37° C. reached about 0.9. The culture liquid was left at rest at 30° C. for 30 minutes, IPTG was added thereto so as to have a final concentration of 0.5 mM, the culture liquid was cultured by reciprocal shaking at 30° C. overnight, and then the bacteria were collected into a 50 mL tube. 
     Bacterium cells were suspended in a buffer for wash (50 mM HEPES, 500 mM NaCl, 50 mM imidazole, pH 7.5) and were pulverized using an ultrasonic pulverizer (BIORUPTOR manufactured by Cosmo Bio Co., Ltd.). This pulverized liquid was centrifuged at 14,000×g for 10 minutes, and the supernatant was collected, which was then added to Ni Sepharose 6 Fast Flow (manufactured by GE Healthcare Japan Corporation) equilibrated with the buffer for wash and was subjected to mild mixing with inversion at room temperature for 5 minutes, and the solution was removed by free fall using EconoSpin (trademark) Empty Column (manufactured by GeneDesign, Inc.). Subsequently, after being washed with the buffer for wash, PheDH as an objective protein was eluted with an elution buffer (50 mM HEPES, 500 mM NaCl, 500 mM imidazole, pH 7.5). The solvent of the PheDH solution was replaced with a buffer for stock (100 mM Tris-HCl, pH 8.0) by ultrafiltration. 
     Example 4 Evaluation of Substrate Specificity of PheDH 
     Evaluation of the substrate specificity of each of the enzymes prepared in Example 3 was performed in accordance with the following procedure. PheDH was prepared so as to be 0.5 mg/mL, and a change with the lapse of time of the absorbance at a wavelength of 340 nm of a solution obtained by adding 100 μL of 200 mM Glycine-KCl-KOH with PH 10.0, 4 μL of 50 mM NAD +  (manufactured by Fujifilm Wako Pure Chemical Corporation), 20 μL of a 10 mM aqueous L-phenylalanine solution or a 10 mM aqueous L-tyrosine solution, and 56 μL of ultrapure water to 20 μL of PheDH was measured with a microplate reader (SpectraMax M2e manufactured by Molecular Devices) for 5 minutes. Tables 8 and 9 list relative activity at the time of measurement of the aqueous L-tyrosine solution with an absorbance value 5 minutes after the time of measurement of the aqueous L-phenylalanine solution being 100% for each of the wild type and mutants. The results of Tables 8 and 9 were calculated from an average value when an experience was conducted twice for the same sample. For the value of WT, an average value of results of the wild-type PheDH prepared and subjected to substrate specificity evaluation 16 times was used. When a mutated PheDH with multiple mutations introduced is indicated, the introduced mutations are sectioned by/to be continuously described. C19S/N290D, for example, means that it is a mutated PheDH having two mutations of C19S and N290D. WT means that it is of the wild type. 
     It can be seen from the result of Table 8 that introduction of the mutations listed in Table 8 can inhibit the reactivity of PheDH with L-tyrosine. Furthermore, it can be seen from the result of Table 9 that introduction of the multiple mutations listed in Table 9 can inhibit the reactivity of PheDH with L-tyrosine. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Substrate specificity of wild-type and single mutation-introduced PheDH 
               
            
           
           
               
               
               
            
               
                 Relative activity for Tyr  
                 Relative activity for Tyr  
                 Relative activity for Tyr  
               
               
                 with Phe being 100% 
                 with Phe being 100% 
                 with Phe being 100% 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 WT 
                 56.0% 
                 C240S 
                 47.4% 
                 L294G 
                 35.0% 
               
               
                 L41W 
                 7.1% 
                 C240A 
                 48.3% 
                 L294E 
                 41.2% 
               
               
                 L41F 
                 11.0% 
                 N264G 
                 13.3% 
                 L294T 
                 41.7% 
               
               
                 L41Y 
                 12.9% 
                 N264Q 
                 20.0% 
                 L294S 
                 45.3% 
               
               
                 L41M 
                 46.1% 
                 N264T 
                 23.5% 
                 Q296D 
                 22.5% 
               
               
                 G42A 
                 27.3% 
                 N264K 
                 26.8% 
                 Q296E 
                 29.3% 
               
               
                 G43A 
                 24.3% 
                 N264P 
                 27.5% 
                 Q296K 
                 36.1% 
               
               
                 M66I 
                 19.4% 
                 N264S 
                 39.3% 
                 Q296N 
                 36.1% 
               
               
                 M66L 
                 19.7% 
                 N290V 
                 8.5% 
                 Q296S 
                 37.6% 
               
               
                 M66V 
                 24.2% 
                 N290D 
                 10.5% 
                 Q296R 
                 41.4% 
               
               
                 F77L 
                 29.8% 
                 N290M 
                 11.4% 
                 V297Y 
                 11.1% 
               
               
                 F77I 
                 37.7% 
                 N290Q 
                 11.4% 
                 V297W 
                 15.7% 
               
               
                 F77R 
                 41.4% 
                 N290P 
                 11.7% 
                 V297E 
                 15.8% 
               
               
                 Y112L 
                 47.8% 
                 N290I 
                 13.1% 
                 V297N 
                 17.7% 
               
               
                 T115S 
                 24.0% 
                 N290H 
                 14.4% 
                 V297T 
                 18.5% 
               
               
                 D116E 
                 39.9% 
                 N290A 
                 20.8% 
                 V297I 
                 21.4% 
               
               
                 F124L 
                 20.2% 
                 N290T 
                 29.2% 
                 V297K 
                 27.5% 
               
               
                 F124I 
                 26.9% 
                 N290G 
                 43.2% 
                 V297G 
                 27.8% 
               
               
                 R129K 
                 48.3% 
                 N290C 
                 49.0% 
                 V297S 
                 29.9% 
               
               
                 L137V 
                 37.0% 
                 G293A 
                 28.0% 
                 V297L 
                 31.6% 
               
               
                 K139E 
                 44.3% 
                 L294F 
                 10.2% 
                 V297M 
                 34.8% 
               
               
                 S140A 
                 49.8% 
                 L294Q 
                 15.5% 
                 V297Q 
                 37.3% 
               
               
                 K144G 
                 39.1% 
                 L294H 
                 19.3% 
                 V297F 
                 40.6% 
               
               
                 T147A 
                 20.5% 
                 L294N 
                 22.4% 
                 V297C 
                 41.5% 
               
               
                 T147S 
                 28.9% 
                 L294I 
                 23.2% 
                 V297R 
                 47.1% 
               
               
                 T147N 
                 35.2% 
                 L294D 
                 24.7% 
                   
                   
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Substrate specificity of wild-type and multiple mutations-introduced PheDH 
               
            
           
           
               
               
            
               
                 Relative activity for Tyr with Phe 
                 Relative activity for Tyr with Phe 
               
               
                 being 100% 
                 being 100% 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 WT 
                 56.0% 
                 Y11E/R271D/N290D 
                 13.4% 
               
               
                 R10D/N290D 
                 7.1% 
                 Y11E/K278D/N290M 
                 4.9% 
               
               
                 Y11E/N290D 
                 6.5% 
                 Y11E/K278D/N290D 
                 5.7% 
               
               
                 C19S/N290D 
                 7.7% 
                 Y11E/K278D/V297F 
                 9.0% 
               
               
                 C19A/N290D 
                 11.0% 
                 L41W/K173E/K278D 
                 9.0% 
               
               
                 C44S/N290D 
                 11.2% 
                 L41W/R228E/K278D 
                 5.8% 
               
               
                 C44A/N290D 
                 14.5% 
                 K173E/K278D/N290M 
                 4.4% 
               
               
                 C70A/N290D 
                 9.9% 
                 K173E/K278D/N290D 
                 4.7% 
               
               
                 C70S/N290D 
                 13.0% 
                 K173E/K278D/V297F 
                 6.9% 
               
               
                 T115S/F124L 
                 24.0% 
                 R228E/K278D/N290M 
                 6.6% 
               
               
                 T115S/N290C 
                 7.7% 
                 R228E/K278D/N290D 
                 4.9% 
               
               
                 T115S/N290Q 
                 13.5% 
                 R228E/K278D/V297F 
                 5.8% 
               
               
                 T115S/L294Q 
                 11.0% 
                 R10D/Y11E/K173E/N290D 
                 5.9% 
               
               
                 T115S/L294N 
                 12.0% 
                 R10D/Y11E/Q222D/N290D 
                 7.0% 
               
               
                 T115S/Q296D 
                 12.9% 
                 R10D/Y11E/R228E/N290D 
                 5.9% 
               
               
                 F124L/N290C 
                 10.7% 
                 R10D/Y11E/R255E/N290D 
                 6.4% 
               
               
                 F124L/N290Q 
                 12.0% 
                 R10D/Y11E/K278D/N290D 
                 6.3% 
               
               
                 F124I/N290D 
                 27.6% 
                 R10D/T115S/Q222D/N290D 
                 1.9% 
               
               
                 F124L/L294N 
                 20.5% 
                 R10D/T115S/R255E/N290D 
                 1.2% 
               
               
                 F124L/L294Q 
                 25.2% 
                 R10D/F124I/Q222D/N290D 
                 3.2% 
               
               
                 F124L/Q296D 
                 11.2% 
                 R10D/F124I/R255E/N290D 
                 2.5% 
               
               
                 F124L/V297R 
                 33.6% 
                 R10D/K173E/Q222D/N290D 
                 5.3% 
               
               
                 T147S/N290D 
                 18.0% 
                 R10D/K173E/R228E/N290D 
                 4.6% 
               
               
                 K173E/N290D 
                 6.3% 
                 R10D/K173E/R255E/N290D 
                 5.4% 
               
               
                 C200A/N290D 
                 24.6% 
                 R10D/K173E/R271D/N290D 
                 5.8% 
               
               
                 C210A/N290D 
                 15.5% 
                 R10D/K173E/K278D/N290D 
                 5.1% 
               
               
                 C210S/N290D 
                 20.6% 
                 R10D/Q222D/R228E/N290D 
                 5.2% 
               
               
                 Q222D/N290D 
                 6.0% 
                 R10D/Q222D/R255E/N290D 
                 5.5% 
               
               
                 R228E/N290D 
                 4.6% 
                 R10D/Q222D/R271D/N290D 
                 4.4% 
               
               
                 C234A/N290D 
                 24.0% 
                 R10D/Q222D/K278D/N290D 
                 6.1% 
               
               
                 C234S/N290D 
                 35.8% 
                 R10D/R228E/R255E/N290D 
                 5.4% 
               
               
                 C240S/N290D 
                 41.0% 
                 R10D/R228E/K278D/N290D 
                 5.3% 
               
               
                 C240A/N290D 
                 49.8% 
                 R10D/R255E/R271D/N290D 
                 4.9% 
               
               
                 R255E/N290D 
                 5.5% 
                 R10D/R255E/K278D/N290D 
                 7.2% 
               
               
                 C256A/N290D 
                 18.4% 
                 Y11E/T115S/R228E/N290D 
                 2.5% 
               
               
                 C256S/N290D 
                 25.2% 
                 Y11E/T115S/K278D/N290D 
                 4.8% 
               
               
                 R271D/N290D 
                 10.8% 
                 Y11E/F124I/R228E/N290D 
                 3.0% 
               
               
                 K278D/N290D 
                 6.5% 
                 Y11E/F124I/K278D/N290D 
                 3.1% 
               
               
                 C282A/N290D 
                 5.3% 
                 Y11E/K173E/Q222D/N290D 
                 6.2% 
               
               
                 C282S/N290D 
                 12.6% 
                 Y11E/K173E/R228E/N290D 
                 6.1% 
               
               
                 N290C/Q296D 
                 13.7% 
                 Y11E/K173E/R255E/N290D 
                 6.1% 
               
               
                 N290D/V297G 
                 16.6% 
                 Y11E/K173E/R271D/N290D 
                 5.0% 
               
               
                 N290D/C335A 
                 8.6% 
                 Y11E/K173E/K278D/N290D 
                 5.5% 
               
               
                 N290D/C335S 
                 8.7% 
                 Y11E/Q222D/R228E/N290D 
                 6.1% 
               
               
                 Q296D/V297R 
                 36.6% 
                 Y11E/Q222D/K278D/N290D 
                 6.4% 
               
               
                 R10D/L41W/Q222D 
                 5.5% 
                 Y11E/R228E/R255E/N290D 
                 7.2% 
               
               
                 R10D/L41W/R255E 
                 7.2% 
                 Y11E/R228E/R271D/N290D 
                 6.1% 
               
               
                 R10D/K173E/N290D 
                 3.8% 
                 Y11E/R228E/K278D/N290D 
                 6.1% 
               
               
                 R10D/Q222D/N290M 
                 4.6% 
                 Y11E/R255E/K278D/N290D 
                 6.1% 
               
               
                 R10D/Q222D/N290D 
                 4.6% 
                 Y11E/R271D/K278D/N290D 
                 5.5% 
               
               
                 R10D/Q222D/V297F 
                 7.1% 
                 T115S/K173E/K278D/N290D 
                 4.4% 
               
               
                 R10D/R255E/N290M 
                 4.8% 
                 T115S/R228E/K278D/N290D 
                 1.5% 
               
               
                 R10D/R255E/N290D 
                 4.8% 
                 F124I/K173E/K278D/N290D 
                 4.1% 
               
               
                 R10D/R255E/V297F 
                 7.6% 
                 F124I/R228E/K278D/N290D 
                 2.8% 
               
               
                 R10D/R271D/N290D 
                 11.4% 
                 K173E/Q222D/K278D/N290D 
                 4.2% 
               
               
                 Y11E/L41W/R228E 
                 6.5% 
                 K173E/R228E/K278D/N290D 
                 4.2% 
               
               
                 Y11E/L41W/K278D 
                 5.6% 
                 K173E/R255E/K278D/N290D 
                 5.1% 
               
               
                 Y11E/K173E/N290D 
                 5.2% 
                 K173E/R271D/K278D/N290D 
                 4.5% 
               
               
                 Y11E/R228E/N290M 
                 5.7% 
                 Q222D/R228E/K278D/N290D 
                 4.7% 
               
               
                 Y11E/R228E/N290D 
                 5.0% 
                 R228E/R255E/K278D/N290D 
                 5.5% 
               
               
                 Y11E/R228E/V297F 
                 7.2% 
                 R228E/R271D/K278D/N290D 
                 5.5% 
               
               
                   
               
            
           
         
       
     
     Example 5 Evaluation of Solubility of PheDH 
     Evaluation of the solubility of each of the enzymes prepared in Example 3 was performed as follows. Concentration was performed by ultrafiltration until PheDH the solvent of which had been replaced with a buffer for stock flocculated, and 30 μL of PheDH was put into a tube with a volume of 1.5 mL and was centrifuged at 18,500×g for 60 minutes, and the supernatant was collected. The concentration of the collected supernatant after centrifugation of the wild type and mutants was determined to be solubility. Table 10 and Table 11 list results compared with the solubility of the wild-type PheDH. 
     It can be seen from the result of Table 10 that introduction of the mutations listed in Table 10 increases the solubility of PheDH. Furthermore, it can be seen from the result of Table 11 that introduction of the multiple mutations listed in Table 11 increases the solubility of PheDH. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Solubility of wild-type and single mutation-introduced PheDH 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Solubility 
                   
                 Solubility 
                   
                 Solubility 
               
               
                   
               
               
                 WT 
                 1.8 mg/mL 
                 K173D 
                  2.7 mg/mL 
                 C256A 
                 5.8 mg/mL 
               
               
                 R2D 
                 2.0 mg/mL 
                 C200A 
                 10.9 mg/mL 
                 L257K 
                 2.8 mg/mL 
               
               
                 R2E 
                 3.1 mg/mL 
                 C200S 
                  3.4 mg/mL 
                 R271D 
                 4.8 mg/mL 
               
               
                 R10D 
                 4.0 mg/mL 
                 C210S 
                  4.6 mg/mL 
                 R271E 
                 3.8 mg/mL 
               
               
                 R10E 
                 2.7 mg/mL 
                 C210A 
                  3.3 mg/mL 
                 Q277D 
                 3.5 mg/mL 
               
               
                 Y11E 
                 4.4 mg/mL 
                 K216D 
                  2.9 mg/mL 
                 K278D 
                 6.9 mg/mL 
               
               
                 Y11D 
                 2.4 mg/mL 
                 K216E 
                  2.7 mg/mL 
                 K278E 
                 4.0 mg/mL 
               
               
                 C19A 
                 3.8 mg/mL 
                 K220D 
                  2.7 mg/mL 
                 R279E 
                 2.6 mg/mL 
               
               
                 C19S 
                 3.4 mg/mL 
                 K220E 
                  2.6 mg/mL 
                 C282S 
                 5.1 mg/mL 
               
               
                 C44A 
                 7.4 mg/mL 
                 Q222E 
                  7.4 mg/mL 
                 C282A 
                 4.4 mg/mL 
               
               
                 C44S 
                 4.7 mg/mL 
                 Q222D 
                  7.2 mg/mL 
                 R326E 
                 3.2 mg/mL 
               
               
                 A50D 
                 3.4 mg/mL 
                 N227D 
                  3.6 mg/mL 
                 N329D 
                 2.8 mg/mL 
               
               
                 A50E 
                 2.2 mg/mL 
                 N227E 
                  3.2 mg/mL 
                 N331E 
                 3.3 mg/mL 
               
               
                 S51D 
                 2.8 mg/mL 
                 R228E 
                  6.8 mg/mL 
                 N331D 
                 3.2 mg/mL 
               
               
                 S51E 
                 2.2 mg/mL 
                 R228D 
                  6.6 mg/mL 
                 C335A 
                 6.1 mg/mL 
               
               
                 C70A 
                 3.3 mg/mL 
                 C240S 
                  3.0 mg/mL 
                 C335S 
                 5.2 mg/mL 
               
               
                 C70S 
                 3.3 mg/mL 
                 C240A 
                  2.9 mg/mL 
                 R340D 
                 2.2 mg/mL 
               
               
                 K90E 
                 3.1 mg/mL 
                 R255E 
                  9.8 mg/mL 
                 K348E 
                 2.8 mg/mL 
               
               
                 K173E 
                 5.0 mg/mL 
                 R255D 
                  8.3 mg/mL 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Solubility of wild-type and multiple mutations-introduced PheDH 
               
            
           
           
               
               
               
               
            
               
                   
                 Solubility 
                   
                 Solubility 
               
               
                   
               
               
                 WT 
                  1.8 mg/mL 
                 Y11E/R228E/K278D  
                 10.4 mg/mL 
               
               
                 R10D/Y11E 
                  4.2 mg/mL 
                 Y11E/R228E/N290M 
                  3.7 mg/mL 
               
               
                 R10D/K173E 
                  6.1 mg/mL 
                 Y11E/R228E/N290D 
                  5.8 mg/mL 
               
               
                 R10D/Q222D 
                 11.9 mg/mL 
                 Y11E/R228E/V297F 
                  7.1 mg/mL 
               
               
                 R10D/R228E 
                  5.7 mg/mL 
                 Y11E/R255E/K278D 
                 10.3 mg/mL 
               
               
                 R10D/R255E 
                 15.4 mg/mL 
                 Y11E/R271D/K278D 
                  7.7 mg/mL 
               
               
                 R10D/R271D 
                  7.3 mg/mL 
                 Y11E/K278D/N290M 
                  5.0 mg/mL 
               
               
                 R10D/K278D 
                  5.4 mg/mL 
                 Y11E/K278D/N290D 
                  6.5 mg/mL 
               
               
                 Y11E/K173E 
                  7.2 mg/mL 
                 Y11E/K278D/V297F 
                 12.4 mg/mL 
               
               
                 Y11E/Q222D 
                  3.5 mg/mL 
                 L41W/K173E/K278D 
                  7.1 mg/mL 
               
               
                 Y11E/R228E 
                 34.1 mg/mL 
                 L41W/R228E/K278D 
                  8.1 mg/mL 
               
               
                 Y11E/R255E 
                  3.4 mg/mL 
                 K173E/K278D/N290M 
                  4.6 mg/mL 
               
               
                 Y11E/R271D 
                  6.6 mg/mL 
                 K173E/K278D/N290D 
                 10.5 mg/mL 
               
               
                 Y11E/K278D 
                 12.3 mg/mL 
                 K173E/K278D/V297F 
                 11.6 mg/mL 
               
               
                 K173E/Q222D 
                  3.8 mg/mL 
                 R228E/K278D/N290M 
                  6.2 mg/mL 
               
               
                 K173E/R228E 
                  2.9 mg/mL 
                 R228E/K278D/N290D 
                  9.3 mg/mL 
               
               
                 K173E/R255E 
                  4.6 mg/mL 
                 R228E/K278D/V297F 
                  7.5 mg/mL 
               
               
                 K173E/R271D 
                  2.4 mg/mL 
                 R10D/Y11E/K173E/N290D 
                  7.3 mg/mL 
               
               
                 K173E/K278D 
                  6.8 mg/mL 
                 R10D/Y11E/Q222D/N290D 
                  2.2 mg/mL 
               
               
                 K173E/N290D 
                  7.3 mg/mL 
                 R10D/Y11E/R228E/N290D 
                  3.1 mg/mL 
               
               
                 Q222D/R228E 
                  5.7 mg/mL 
                 R10D/Y11E/R255E/N290D 
                  2.4 mg/mL 
               
               
                 Q222D/R255E 
                  4.3 mg/mL 
                 R10D/Y11E/K278D/N290D 
                  2.3 mg/mL 
               
               
                 Q222D/R271D 
                  2.6 mg/mL 
                 R10D/T115S/Q222D/N290D 
                  4.3 mg/mL 
               
               
                 Q222D/K278D 
                  3.9 mg/mL 
                 R10D/T115S/R255E/N290D 
                  5.2 mg/mL 
               
               
                 Q222D/N290D 
                  3.9 mg/mL 
                 R10D/F124I/Q222D/N290D 
                  4.7 mg/mL 
               
               
                 R228E/R255E 
                  3.4 mg/mL 
                 R10D/F124I/R255E/N290D 
                  4.6 mg/mL 
               
               
                 R228E/R271D 
                  2.3 mg/mL 
                 R10D/K173E/Q222D/N290D 
                  5.7 mg/mL 
               
               
                 R228E/K278D 
                  8.7 mg/mL 
                 R10D/K173E/R228E/N290D 
                 11.7 mg/mL 
               
               
                 R228E/N290D 
                  2.7 mg/mL 
                 R10D/K173E/R255E/N290D 
                  4.7 mg/mL 
               
               
                 R255E/R271D 
                  3.1 mg/mL 
                 R10D/K173E/R271D/N290D 
                  5.0 mg/mL 
               
               
                 R255E/K278D 
                  3.2 mg/mL 
                 R10D/K173E/K278D/N290D 
                  6.9 mg/mL 
               
               
                 R255E/N290D 
                  2.2 mg/mL 
                 R10D/Q222D/R228E/N290D 
                  4.9 mg/mL 
               
               
                 R271D/K278D 
                  4.6 mg/mL 
                 R10D/Q222D/R255E/N290D 
                  4.4 mg/mL 
               
               
                 R10D/Y11E/Q222D 
                  3.2 mg/mL 
                 R10D/Q222D/K278D/N290D 
                  4.2 mg/mL 
               
               
                 R10D/Y11E/R228E 
                  4.9 mg/mL 
                 R10D/R228E/R255E/N290D 
                  5.8 mg/mL 
               
               
                 R10D/Y11E/K278D 
                  3.1 mg/mL 
                 R10D/R228E/K278D/N290D 
                  6.5 mg/mL 
               
               
                 R10D/K173E/Q222D 
                  3.0 mg/mL 
                 R10D/R255E/K278D/N290D 
                  3.8 mg/mL 
               
               
                 R10D/K173E/R255E 
                  7.7 mg/mL 
                 Y11E/T115S/R228E/N290D 
                  2.3 mg/mL 
               
               
                 R10D/K173E/N290D 
                  4.9 mg/mL 
                 Y11E/K173E/Q222D/N290D 
                 11.2 mg/mL 
               
               
                 R10D/Q222D/R228E 
                 12.1 mg/mL 
                 Y11E/K173E/R228E/N290D 
                  3.2 mg/mL 
               
               
                 R10D/Q222D/R255E 
                  8.3 mg/mL 
                 Y11E/K173E/R255E/N290D 
                 10.9 mg/mL 
               
               
                 R10D/Q222D/R271D 
                  5.1 mg/mL 
                 Y11E/K173E/R271D/N290D 
                  8.3 mg/mL 
               
               
                 R10D/Q222D/K278D 
                 10.1 mg/mL 
                 Y11E/K173E/K278D/N290D 
                  7.0 mg/mL 
               
               
                 R10D/Q222D/N290M 
                  5.4 mg/mL 
                 Y11E/Q222D/R228E/N290D 
                 15.3 mg/mL 
               
               
                 R10D/Q222D/N290D 
                  8.6 mg/mL 
                 Y11E/Q222D/K278D/N290D 
                  6.2 mg/mL 
               
               
                 R10D/Q222D/V297F 
                 10.5 mg/mL 
                 Y11E/R228E/R255E/N290D 
                  8.7 mg/mL 
               
               
                 R10D/R228E/R255E 
                  8.7 mg/mL 
                 Y11E/R228E/R271D/N290D 
                  6.5 mg/mL 
               
               
                 R10D/R255E/R271D 
                  6.2 mg/mL 
                 Y11E/R228E/K278D/N290D 
                  6.1 mg/mL 
               
               
                 R10D/R255E/K278D 
                 11.3 mg/mL 
                 Y11E/R255E/K278D/N290D 
                 16.0 mg/mL 
               
               
                 R10D/R255E/N290M 
                 17.2 mg/mL 
                 Y11E/R271D/K278D/N290D 
                  3.1 mg/mL 
               
               
                 R10D/R255E/N290D 
                  7.7 mg/mL 
                 T115S/K173E/K278D/N290D 
                  4.4 mg/mL 
               
               
                 R10D/R255E/V297F 
                  9.8 mg/mL 
                 T115S/R228E/K278D/N290D 
                  5.0 mg/mL 
               
               
                 Y11E/L41W/R228E 
                  4.6 mg/mL 
                 F124I/K173E/K278D/N290D 
                  2.0 mg/mL 
               
               
                 Y11E/L41W/K278D 
                 11.5 mg/mL 
                 F124I/R228E/K278D/N290D 
                  2.5 mg/mL 
               
               
                 Y11E/K173E/R228E 
                  5.5 mg/mL 
                 K173E/Q222D/K278D/N290D 
                  5.8 mg/mL 
               
               
                 Y11E/K173E/K278D 
                 10.6 mg/mL 
                 K173E/R228E/K278D/N290D 
                  5.1 mg/mL 
               
               
                 Y11E/K173E/N290D 
                  5.4 mg/mL 
                 K173E/R255E/K278D/N290D 
                  3.6 mg/mL 
               
               
                 Y11E/Q222D/R228E 
                  9.4 mg/mL 
                 Q222D/R228E/K278D/N290D 
                  6.8 mg/mL 
               
               
                 Y11E/Q222D/K278D 
                  8.5 mg/mL 
                 R228E/R255E/K278D/N290D 
                  6.1 mg/mL 
               
               
                 Y11E/R228E/R255E 
                  6.6 mg/mL 
                 R228E/R271D/K278D/N290D 
                  2.3 mg/mL 
               
               
                 Y11E/R228E/R271D 
                 11.9 mg/mL 
               
               
                   
               
            
           
         
       
     
     Example 6 Evaluation of Enzyme Activity of PheDH 
     Evaluation of the activity of each of the enzymes prepared in Example 3 was performed in accordance with the following procedure. PheDH the solvent of which had been replaced with a buffer for stock was prepared so as to be 0.1 mg/mL, and a change with the lapse of time of the absorbance at a wavelength of 340 nm of a solution obtained by adding 100 μL of 200 mM Glycine-KCl-KOH with PH 10.0, 4 μL of 50 mM NAD +  (manufactured by Fujifilm Wako Pure Chemical Corporation), 20 μL of a 10 mM aqueous L-phenylalanine solution, and 56 μL of ultrapure water to 20 μL of PheDH was measured with a microplate reader (SpectraMax M2e manufactured by Molecular Devices) for 5 minutes. Tables 12 and 13 list relative activity compared with the value of the wild-type PheDH as a control. The results of Tables 12 and 13 were calculated from an average value when an experience was conducted twice for the same sample. 
     It can be seen from the result of Table 12 that introduction of the mutations listed in Table 12 can improve the reactivity of PheDH with L-phenylalanine. Furthermore, it can be seen from the result of Table 13 that introduction of the multiple mutations listed in Table 13 can improve the reactivity of PheDH with L-phenylalanine. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Activity of wild-type and single mutation-introduced PheDH 
               
            
           
           
               
               
               
            
               
                 Relative activity 
                 Relative activity 
                 Relative activity 
               
               
                 compared with  
                 compared with  
                 compared with  
               
               
                 control (WT)  
                 control (WT)  
                 control (WT) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 WT 
                 100% 
                 N227D 
                 121% 
                 S280D 
                 124% 
               
               
                 C19S 
                 131% 
                 N227E 
                 119% 
                 C282S 
                 106% 
               
               
                 F77L 
                 144% 
                 R228E 
                 160% 
                 N290C 
                 140% 
               
               
                 F124I 
                 106% 
                 R228D 
                 118% 
                 N290T 
                 111% 
               
               
                 T147S 
                 160% 
                 C234A 
                 131% 
                 L294Q 
                 111% 
               
               
                 K173E 
                 122% 
                 C234S 
                 123% 
                 Q296D 
                 134% 
               
               
                 K173D 
                 106% 
                 C240S 
                 164% 
                 Q296E 
                 127% 
               
               
                 C200A 
                 115% 
                 C240A 
                 140% 
                 V297T 
                 128% 
               
               
                 C200S 
                 113% 
                 R255E 
                 135% 
                 V297G 
                 111% 
               
               
                 K216D 
                 160% 
                 R255D 
                 126% 
                 K328E 
                 114% 
               
               
                 K216E 
                 122% 
                 C256A 
                 109% 
                 K328D 
                 105% 
               
               
                 K220E 
                 152% 
                 R271E 
                 107% 
                 N329D 
                 117% 
               
               
                 K220D 
                 136% 
                 K278D 
                 139% 
                 N331D 
                 122% 
               
               
                 Q222D 
                 152% 
                 R279D 
                 111% 
                 R340E 
                 108% 
               
               
                 Q222E 
                 127% 
                 R279E 
                 108% 
                 K347D 
                 109% 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Activity of wild-type and multiple mutations-introduced PheDH 
               
            
           
           
               
               
            
               
                 Relative activity compared with 
                 Relative activity  
               
               
                 control (WT) 
                 compared with control (WT) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 WT 
                 100% 
                 R10D/Q222D/R255E 
                 106% 
               
               
                 K173E/Q222D 
                 110% 
                 R10D/Q222D/R271D 
                 113% 
               
               
                 C210S/N290D 
                 120% 
                 R10D/R228E/R255E 
                 127% 
               
               
                 Q222D/R228E 
                 112% 
                 R10D/R255E/R271D 
                 124% 
               
               
                 R228E/R255E 
                 111% 
                 Y11E/K173E/R228E 
                 111% 
               
               
                 N290D/C335A 
                 122% 
                 Y11E/K173E/N290D 
                 116% 
               
               
                 R10D/Y11E/Q222D 
                 121% 
                 Y11E/R228E/N290D 
                 112% 
               
               
                 R10D/Y11E/R255E 
                 115% 
                 Y11E/K278D/N290D 
                 122% 
               
               
                 R10D/Y11E/K278D 
                 108% 
                 R10D/Y11E/R255E/N290D 
                 111% 
               
               
                 R10D/K173E/Q222D 
                 129% 
                 R10D/R228E/R255E/N290D 
                 144% 
               
               
                 R10D/K173E/R255E 
                 118% 
                 Y11E/K173E/R255E/N290D 
                 166% 
               
               
                 R10D/Q222D/R228E 
                 124% 
                 Y11E/R255E/K278D/N290D 
                 119% 
               
               
                   
               
            
           
         
       
     
     INDUSTRIAL APPLICABILITY 
     The modified phenylalanine dehydrogenase is useful for quick, high-precision, and highly sensitive measurement of phenylalanine and/or production of phenylpyruvate. The modified phenylalanine dehydrogenase is also useful as a liquid reagent. The modified phenylalanine dehydrogenase is useful as a liquid reagent in particular. The method of analysis is useful for diagnosis of diseases such as phenylketonuria and measurement of a phenylalanine content in food, for example. 
     SEQUENCE LISTING FREE TEXT 
     SEQ ID NO:1 indicates the amino acid sequence of the  Thermoactinomyces intermedius  phenylalanine dehydrogenase (PheDH). 
     SEQ ID NO:2 indicates the codon-optimized nucleotide sequence encoding the amino acid sequence of the  Thermoactinomyces intermedius  PheDH (SEQ ID NO:1). 
     SEQ ID NOS:3 to 6 indicate the amino acid sequences of the respective motifs in PheDH. 
     SEQ ID NOS:7 to 9 indicate the amino acid sequences of the respective motifs indicated by the respective shorter amino acid sequences in PheDH. 
     SEQ ID NOS:10 to 12 indicate amino acid sequences near the respective motifs in the  Thermoactinomyces intermedius  PheDH. 
     SEQ ID NOS:13 to 15 indicate consensus amino acid sequences (amino acid sequences having a high degree of commonness) near the respective motifs in PheDH. 
     SEQ ID NOS:16, 26, 35, 44, 54, 62, 70, 79, 88, 97, and 106 indicate the amino acid sequences of PheDH derived from the respective species. 
     SEQ ID NOS:17 to 22, 27 to 31, 36 to 40, 45 to 50, 55 to 58, 63 to 66, 71 to 75, 80 to 84, 89 to 93, 98 to 102, and 107 indicate the amino acid sequences of conservations regions corresponding to the respective motifs in PheDH derived from the respective species. 
     SEQ ID NOS:23 to 25, 32 to 34, 41 to 43, 51 to 53, 59 to 61, 67 to 69, 76 to 78, 85 to 87, 94 to 96, 103 to 105, and 108 to 110 indicate amino acid sequences near the respective motifs in PheDH derived from the respective species. 
     SEQ ID NO:111 indicates the amino acid sequence of  Thermoactinomyces intermedius  PheDH in which the His-tag is added to the N-terminus. 
     SEQ ID NO:112 indicates a codon-optimized nucleotide sequence encoding the amino acid sequence of  Thermoactinomyces intermedius  PheDH in which the His-tag is added to the N-terminus (SEQ ID NO:111). 
     SEQ ID NO:113 indicates a linker nucleotide sequence (an NdeI site+an initiation codon+a His-tag code sequence) added to the 5′-end of the nucleotide sequence of SEQ ID NO:2 in order to form a DNA fragment including the nucleotide sequence of SEQ ID NO:112. 
     SEQ ID NO:114 indicates a linker nucleotide sequence (a termination codon+a BamHI site) added to the 3′-end of the nucleotide sequence of SEQ ID NO:2 in order to form the DNA fragment including the nucleotide sequence of SEQ ID NO:112.