Abstract:
Mutated β-glucuronidase enzymes with enhanced enzymatic activity and thermostability as compared to wild type enzyme are provided. The enzymes of the invention advantageously allow for accurate analysis of bodily samples for the presence of drugs in 30 minutes or less, as compared to the several hours needed using prior enzyme preparations. Methods of using the mutated enzymes for hydrolysis of glucuronide substrates, including opiates and benzodiazepines, are also provided.

Description:
RELATED APPLICATIONS 
       [0001]    This application claims priority to and the benefit of U.S. provisional patent application No. 62/056,800, filed on Sep. 29, 2014, the disclosure of which is incorporated herein by reference in its entirety. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    In mammals, glucuronidation is one of the principle means of detoxifying or inactivating compounds using the UDP glucuronyl transferase system. Compounds are conjugated by the glucoronyl transferase system to form glucuronides, which are then secreted in urine or into the lower intestine in bile. Furthermore, microorganisms in the gut, such as  Escherichia coli , have evolved to utilize the excreted β-glucuronides as a carbon source. The β-glucuronidase (BGUS) enzyme catalyzes the hydrolysis of a wide variety of β-glucuronides. Thus, BGUS enzyme activity been reported in those organisms that utilize glucuronidation as a detoxification pathway, as well as in some of their endogenous microbe populations. All vertebrates and many mollusks, as well as certain bacteria, exhibit BGUS enzyme activity, whereas insects and plants that utilize a different detoxification pathway typically do not exhibit BGUS enzyme activity. 
         [0003]    Given the key role of glucuronidation in detoxification of compounds, the BGUS enzyme has been used for detection of drugs in bodily samples, such as to detect the presence of illicit drugs in bodily samples of criminal suspects. For example, a bodily sample can be tested for the presence of a suspected drug by detecting the hydrolysis of the glucuronide form of the drug by BGUS. 
         [0004]    Commercially available preparations of BGUS enzyme, for use for example in drug testing, include crude extract forms of the  E. coli , snail and abalone versions of the enzyme. While these preparations are effective in hydrolyzing glucuronides, they typically include other proteins in addition to the BGUS, which may interfere with enzyme activity. Moreover, importantly, their level of enzyme activity is such that they typically require at least several hours (e.g., three hours or more) to analyze a sample. Including sample preparation time and analysis time, this means that evaluation of a drug sample typically can take at least two days using currently commercially available BGUS preparations. 
         [0005]    Accordingly, there is a need for BGUS enzymes with enhanced activity that are more efficient for use in drug testing. 
       SUMMARY OF THE INVENTION 
       [0006]    The invention provides mutant forms of BGUS enzymes that exhibit enhanced enzymatic activity as compared to wild type enzyme. Moreover, mutant forms of BGUS described herein exhibit higher thermal stability than the wild type enzyme. The enzymes of the invention advantageously allow for accurate analysis of bodily samples for the presence of drugs in 30 minutes or less, as compared to the several hours needed using the current commercially available enzyme preparations, thereby allowing for completion of analyses within a shorter time frame than previously possible. Furthermore, the mutant enzymes of the invention are produced recombinantly and thus can be prepared in a highly purified form without contaminating non-BGUS proteins and with a higher temperature stability. 
         [0007]    In particular, it has been discovered that the single substitution of amino acid residue G559 in the  E. coli  BGUS wild type sequence (shown in SEQ ID NO: 18) with an amino acid having a side chain with a non-aromatic hydroxyl group, such as a serine (G559S) or threonine (G559T), or with histidine (G559H) or asparagine (G559N), leads to significantly increased enzymatic activity as compared to the wild type enzyme. Moreover, addition of a cysteine residue at the C-terminal end of the enzyme has been found to enhance the thermostability of the enzyme. For example, the following sequence can be added at the C-terminal end of the enzyme: Xaa 2-8 -Cys-Xaa 0-2 , wherein Xaa=any amino acid. Still further, a loop region at amino acid residues F385-K390 of the  E. coli  BGUS wild type sequence (shown in SEQ ID NO: 18) has been found to be important for imparting efficient hydrolysis of codeine glucuronide substrates. 
         [0008]    Accordingly, in one aspect, the invention pertains to a packaged formulation comprising a container comprising a preparation of a β-glucuronidase enzyme, wherein: 
         [0009]    (i) the β-glucuronidase enzyme comprises a mutation consisting of a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 with an amino acid comprising a side chain comprising a non-aromatic hydroxyl group or histidine or asparagine; and 
         [0010]    (ii) the preparation of β-glucuronidase enzyme has an enzymatic activity of at least 5,000 Units/ml or 5,000 Units/mg. 
         [0011]    Preferably, the β-glucuronidase enzyme has an enzymatic activity of at least 10,000 Units/ml or 10,000 Units/mg, more preferably at least 25,000 Units/ml or 25,000 Units/mg and even more preferably at least 50,000 Units/ml or 50,000 units/mg. 
         [0012]    In one embodiment, the β-glucuronidase enzyme comprises a mutation consisting of a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 with serine. Preferably, the enzyme having the G559S substitution has an amino acid sequence as shown in SEQ ID NO: 19. In another embodiment, the β-glucuronidase enzyme comprises a mutation consisting of a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 with threonine. Preferably, the enzyme having the G559T substitution has an amino acid sequence as shown in SEQ ID NO: 20. In another embodiment, the β-glucuronidase enzyme comprises a mutation consisting of a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 with histidine. In another embodiment, the β-glucuronidase enzyme comprises a mutation consisting of a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 with asparagine. 
         [0013]    The enzyme preparation in the packaged formulation can be, for example, an aqueous solution or a lyophilized preparation. In one embodiment, the β-glucuronidase enzyme is in an aqueous solution with an enzymatic activity of at least 50,000 Units/ml. In another embodiment, the β-glucuronidase enzyme is in a lyophilized preparation with an enzymatic activity of at least 50,000 Units/mg. In yet another embodiment, the β-glucuronidase enzyme is in a lyophilized preparation that when reconstituted as an aqueous solution has an enzymatic activity of at least 50,000 Units/ml. 
         [0014]    In one embodiment, the preparation is stable at least six months at 2-8° C. In another embodiment, the preparation lacks detectable sulfatase activity. 
         [0015]    Preferably, the β-glucuronidase enzyme is from a bacteria, more preferably from  Escherichia coli , even more preferably from  Escherichia coli  K12 strain. In another embodiment, the β-glucuronidase enzyme is from a mollusk, such as a snail (preferably  Helix pomatia  or  Helix aspersa ) or an abalone (preferably  Haliotis rufescens ). In yet another embodiment, the β-glucuronidase enzyme is from a human. 
         [0016]    In another aspect, the invention provides a method of hydrolyzing a substrate comprising a glucuronide linkage, the method comprising contacting the substrate with a β-glucuronidase enzyme under conditions such that hydrolysis of the glucuronide linkage occurs, wherein the β-glucuronidase enzyme comprises a mutation consisting of a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 with an amino acid comprising a side chain comprising a non-aromatic hydroxyl group or histidine or asparagine. 
         [0017]    In one embodiment, the method uses a β-glucuronidase enzyme comprising a mutation consisting of a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 with serine. Preferably, the enzyme having the G559S mutation comprises an amino acid sequence as shown in SEQ ID NO: 19. In another embodiment, the method uses a β-glucuronidase enzyme comprising a mutation consisting of a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 with threonine. Preferably, the enzyme having the G559T mutation comprises an amino acid sequence as shown in SEQ ID NO: 20. In another embodiment, the method uses a β-glucuronidase enzyme comprising a mutation consisting of a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 with histidine. In another embodiment, the method uses a β-glucuronidase enzyme comprising a mutation consisting of a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 with asparagine. 
         [0018]    In one embodiment, the substrate used in the method is an opiate glucuronide. Non-limiting examples of opiate glucuronide substrates include morphine-3β-D-glucuronide, morphine-6β-D-glucuronide, codeine-6β-D-glucuronide, hydromorphone-3β-D-glucuronide, oxymorphone-3β-D-glucuronide, and combinations thereof. In another embodiment, the substrate used in the method is a benzodiazepine glucuronide. Non-limiting examples of benzodiazepine glucuronide substrates include oxazepam-glucuronide, lorazepam-glucuronide, temazepam-glucuronide, alprazolam, alpha-hydroxy-alprazolam glucuronide, nordiazepam, 7-amino-clonozepam, and combinations thereof. Additional or alternative glucuronide substrates include the glucuronides of buprenorphine, norbuprenorphine, 11-nor-A9-tetrahydrocannabinol-9-carboxylic acid, testosterone, androsterone, tapentadol, cyclobenzaprine, and amitripyline. 
         [0019]    The method of the invention for hydrolyzing a substrate with a glucuronide linkage can be used to test for the presence of drugs in bodily samples, wherein the substrate is present within the sample. Non-limiting examples of bodily samples include samples of blood, urine, tissue or meconium obtained from a subject. 
         [0020]    In another aspect, the invention provides “combination” mutated BGUS enzymes having two or more modifications chosen from: (i) a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 with an amino acid comprising a side chain comprising a non-aromatic hydroxyl group or histidine or asparagine; (ii) an addition of a cysteine residue appended at the carboxy terminus of the enzyme (e.g., addition at the C-terminal end of the enzyme of the sequence: Xaa 2-8 -Cys-Xaa 0-2 , wherein Xaa=any amino acid; and/or (iii) a modification of a region comprising amino acids corresponding to F385 through K390 in SEQ ID NO: 18. In a preferred embodiment, a combination mutant that comprises a substitution at position 559 and that has a cysteine residue appended at or near the C-terminal end has the amino acid sequence shown in SEQ ID NO: 93, wherein position 559 is substituted with Ser (S), Thr (T), His (H) or Asn (N) and wherein the C-terminal end has the following sequence: Xaa 2-8 -Cys-Xaa 0-2 , wherein Xaa=any amino acid. 
         [0021]    In the combination mutants having addition of a cysteine residue appended at the carboxy terminus of the enzyme, in a preferred embodiment the tripeptide Gly-Leu-Cys (GLC) is appended to the C-terminus. In another preferred embodiment, the pentapeptide Gly-Leu-Cys-Gly-Arg (GLCGR) (SEQ ID NO: 95) is added. In the combination mutants having a modification of the region comprising amino acids corresponding to F385 through K390 in SEQ ID NO: 18, in one embodiment this modification is a deletion of the region. In another embodiment, the modification is an insertion of the region, for example into a BGUS sequence from a species that does not naturally contain this region. 
         [0022]    Packed formulations, as described above, that include a combination mutant of the invention are also provided. Methods of using the combination mutants of the invention (having two or more of the above described modifications) for hydrolysis of glucuronide substrates are also provided in which the mutated β-glucuronidase enzyme is contacted with the glucuronide substrate under conditions such that hydrolysis of the glucuronide linkage occurs. 
         [0023]    Other features and aspects of the invention are described in further detail herein. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0024]      FIG. 1  is an alignment of the amino acid sequences of the K1S (SEQ ID NO: 19), K1T (SEQ ID NO: 20), K3 (SEQ ID NO: 21), K3Δ1 (SEQ ID NO: 22), K3Δ2 (SEQ ID NO: 23) and K3Δ2S (SEQ ID NO: 24) mutants as compared to the wild type  E. coli  K12 sequence (SEQ ID NO: 18). The F385 through 5396 modification region, G559S or G559T modifications and C-terminal GLC modification in the mutants are highlighted in bold and underlined. 
           [0025]      FIG. 2  is an alignment of the amino acid sequences of the  E. coli  K3 mutant (K3) (SEQ ID NO: 25), the abalone (Ab) wild type BGUS (SEQ ID NO: 26), the human (Hu) wild type BGUS (SEQ ID NO: 27), the  Lactobacillus brevis  (L. br) wild type BGUS (SEQ ID NO: 28) and the  Staphylococcus  sp. RLH1 (S. rlh) wild type BGUS (SEQ ID NO: 29) across amino acid residues 332-416 ( E. coli  numbering), including across the modification region F385 through S396 ( E. coli  numbering). Identical amino acid residues across the five sequences are highlighted in grey. The conserved glutamic acid residue (E) at position 413 ( E. coli  numbering) within the catalytic site is highlighted in bold, indicating the accuracy of the alignment. The modification region F385 through S396 ( E. coli  numbering) is highlighted in bold and underlined. 
           [0026]      FIG. 3  is an alignment of the amino acid sequences of the  E. coli  K3 mutant (K3)(SEQ ID NO:30), the abalone (Ab) wild type BGUS (SEQ ID NO:31), the human (Hu) wild type BGUS (SEQ ID NO:32), the  Lactobacillus brevis  (L. br.) wild type BGUS (SEQ ID NO: 33) and the  Staphylococcus  sp. RLH1 (S. rlh) wild type BGUS (SEQ ID NO: 34) across amino acid residues 452-606 ( E. coli  numbering), including across the G559S modification ( E. coli  numbering) and the C-terminal GLC modification. Identical amino acid residues across the three sequences are highlighted in grey. The conserved tyrosine residue (Y) at position 468 and the conserved glutamic acid residue (E) at position 504 ( E. coli  numbering) within the catalytic site are highlighted in bold, indicating the accuracy of the alignment. The G559S mutation position ( E. coli  numbering) and C-terminal GLC modifications are highlighted in bold and underlined. 
           [0027]      FIG. 4  is a bar graph showing the specific enzyme activity (in MU/g of protein) of the K1S mutant, as compared to the wild type  E. coli  12 BGUS enzyme, using phenolphthalein-glucuronide as the substrate. Both samples are purified and normalized to protein concentration. 
           [0028]      FIG. 5  is a bar graph showing the specific enzyme activity (as measured by OD at 405 nm) of the K1S mutant, as compared to the wild type  E. coli  12 BGUS enzyme and a thermo-resistant mutant (TR), using 4-nitrophenol glucuronide as the substrate. All three samples are purified and normalized to protein concentration. 
           [0029]      FIG. 6  is a bar graph showing the specific enzyme activity (in MU/g of protein) of the K1T, K3, K3-C606S and K3Δ1 mutants, as compared to the K1S mutant, using phenolphthalein-glucuronide as the substrate. The results were normalized to total protein concentration. 
           [0030]      FIG. 7A  is a graph showing enzyme activity across various pH levels for the BGUS mutant K3. 
           [0031]      FIG. 7B  is a graph showing enzyme activity after overnight storage at different pH levels, following by return of the pH to neutral levels and testing of enzymatic activity for the BGUS mutant K3. 
           [0032]      FIG. 8  is a photograph of an SDS-PAGE gel showing the purity of the recombinant K3 enzyme (lane 2) as compared to commercially available abalone (lane 3), snail (lane 4) and  E. coli  (lane 5) extracts. Molecular weight markers are shown in lane 1. 
           [0033]      FIG. 9A  is an alignment of the wild type abalone BGUS (Wt-Ab) amino acid sequence (SEQ ID NO: 26) with representative loop region insertion mutants Ab1 (SEQ ID NO: 35) and Ab2 (SEQ ID NO: 36) across the loop region F385 through Y395 ( E. coli  numbering). 
           [0034]      FIG. 9B  is an alignment of the wild type human BGUS (Wt-Ab) amino acid sequence (SEQ ID NO: 27) with representative loop region insertion mutants Hu1 (SEQ ID NO: 37) and Hu2 (SEQ ID NO: 38) across the loop region F385 through Y395 ( E. coli  numbering). 
           [0035]      FIG. 10  is a graph showing enzyme activity at four different temperatures for the BGUS mutant K3. 
           [0036]      FIG. 11  is a graph showing enzyme activity for BGUS mutants having the indicated amino acid substitution at position 559, demonstrating enhanced enzyme activity for substitutions with histidine (H), asparagine (N), serine (S) and threonine (T). 
           [0037]      FIG. 12  is a graph showing enzyme activity for BGUS mutants having insertions or substitutions near the carboxy terminus, demonstrating that such mutations do not affect the overall enzyme activity or the thermostability. Purified enzymes were heat treated for one hour prior to measuring their enzymatic activity. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0038]    The invention pertains to mutated β-glucuronidase enzymes having enhanced enzymatic activity as compared to the wild type enzyme, as well as packaged formulations thereof and methods of using the enzymes for hydrolysis of glucuronide linkages. Various aspects of the invention are described in further detail in the following subsections. 
       I. Mutated β-Glucuronidase Enzymes 
       [0039]    A. Position 559 Substitutions 
         [0040]    As used herein, the term β-glucuronidase enzyme“, also referred to as β-glucuronidase” or “BGUS”, refers to an enzyme that hydrolyzes β-glucuronide linkages. A “wild type” BGUS enzyme refers to the naturally occurring form of the enzyme. A “mutated” BGUS enzyme refers to a modified form of the enzyme in which one or more modifications, such as amino acid substitutions, deletions and/or insertions, have been made such that the amino acid sequence of the mutated BGUS enzyme differs from the wild type amino acid sequence. The nucleotide sequence encoding wild type  E. coli  K12 strain BGUS is shown in SEQ ID NO: 1 (NCBI Reference Sequence: NC_000913.2). The amino acid sequence of wild type  E. coli  K12 strain BGUS is shown in SEQ ID NO: 18 and in  FIG. 1 . Cloning of the wild type  E. coli  K12 strain BGUS is described in detail in Example 1. 
         [0041]    It has now been discovered that a single amino acid substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 (wild type  E. coli  BGUS) with an amino acid comprising a side chain comprising a non-aromatic hydroxyl group, or with histidine or asparagine, creates a mutated BGUS enzyme that has significantly enhanced (e.g., at least 3-fold greater) enzymatic activity as compared to the wild type enzyme. As used herein, a “side chain” of an amino acid refers to the “R” group in the standard generic formula for amino acids: H 2 NCHRCOOH. A “non-aromatic hydroxyl group” refers to a side chain structure that contains an —OH group, but that lacks a ring structure. In one embodiment, the amino acid comprising a side chain comprising a non-aromatic hydroxyl group is serine (i.e., the mutation in the enzyme consists of a G559S substitution). In another embodiment, the amino acid comprising a side chain comprising a non-aromatic hydroxyl group is threonine (i.e., the mutation in the enzyme consists of a G559T substitution). In yet other embodiments, the amino acid comprising a side chain comprising a non-aromatic hydroxyl group can be a non-naturally occurring amino acid. Non-limiting examples of non-natural amino acids comprising a side chain comprising a non-aromatic hydroxyl group include L-iso-serine (Sigma Aldrich Product #06054), L-allo-threonine (Sigma Aldrich Product #210269), homoserine (Swiss Side Chain ID# HSER), 3-3-dihydoxyalanine (Swiss Side Chain ID# DDZ) and 2-amino-5-hydroxypentanoic acid (Swiss Side Chain ID# AA4). 
         [0042]    The preparation of mutant BGUS enzymes having either a single G559S substitution (referred to herein as K1 S) or a single G559T substitution (referred to herein as K1T) is described in detail in Example 2. The full-length amino acid sequence of the K1S mutant is shown in SEQ ID: 19. The full-length amino acid sequence of the K1T mutant is shown in SEQ ID NO: 20. The alignments of the K1S and K1T mutant amino acid sequences as compared to wild-type (SEQ ID NO: 18) are shown in  FIG. 1 . 
         [0043]    The enzymatic activity of the K1S enzyme as compared to wild type is described in detail in Example 4 (first experiment) and shown in  FIGS. 4 and 5 . This data demonstrates that the single G559S mutation imparts at least a 3-fold, or greater, enhancement in enzymatic activity of the mutant as compared to the wild type enzyme. The enzymatic activity of the K1T enzyme as compared to the K1S is described in detail in Example 4 (third experiment) and shown in  FIG. 6 . This data demonstrates that the single G559T mutation has a comparable, or even slightly greater enzymatic activity, than the single G559S mutation. 
         [0044]    Xiong, A-S. et al. (2007)  Prot. Eng. Design Select.  20:319-325 has reported the preparation of a mutated BGUS enzyme containing six amino acid substitutions: Q493R, T509A, M532T, N550S, G559S and N566S. This mutant enzyme is reported to have improved thermostability as compared to the wild type enzyme. However, the significantly improved enzymatic activity of the single amino acid substitution, G559S, as reported herein, is not disclosed in or suggested by Xiong et al. A comparison of the enzymatic activity of the K1S mutant (consisting of the G559S substitution) to the thermoresistant six amino acid mutant of Xiong et al. (referred to herein as “TR” and in Xiong et al. as GUS-TR3337) is described in detail in Example 4 (second experiment) and shown in  FIG. 5 . This data demonstrates that the K1S mutant has significantly enhanced (e.g., 3-fold greater, or more) enzymatic activity as compared to the prior art “TR” mutant enzyme. 
         [0045]    Experiments described in Example 7 further demonstrate that substitution of the wild-type G559 position with either histidine (H) or asparagine (N) also leads to enhanced enzymatic activity. The full-length amino acid sequence of a G559H point mutant is shown in SEQ ID: 61. Accordingly, in another aspect, the invention provides a mutated β-glucuronidase enzyme comprising a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 with histidine. The full-length amino acid sequence of a G559N point mutant is shown in SEQ ID NO: 64. Accordingly, in another aspect, the invention provides a mutated β-glucuronidase enzyme comprising a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 with asparagine. 
         [0046]    The term “substitution of an amino acid corresponding to G559 in SEQ ID NO: 18”, as used herein, references the numbering of the wild type  E. coli  strain 12 BGUS enzyme as shown in SEQ ID NO: 18. That is, the wild type sequence shown in SEQ ID NO: 18 has a glycine (G) at position 559 and this amino acid residue, or an amino acid residue corresponding to this residue in another BGUS sequence, is the one that is substituted. 
         [0047]    The sequences of BGUS enzymes from numerous species are known in the art. For example, the amino acid sequence of wild type human ( Homo sapiens ) BGUS (isoform 1 precursor) is shown in SEQ ID NO: 39 (NCBI Reference Sequence NP_000172.2). The amino acid sequence of wild type mouse ( Mus musculus ) BGUS (precursor) is shown in SEQ ID NO: 40 (NCBI Reference Sequence NP_034498.1). The amino acid sequence of wild type  Lactobacillus brevis  BGUS is shown in SEQ ID NO: 41 (Genbank Accession No. ACU21612.1). The amino acid sequence of wild type  Staphylococcus  sp. RLH1 BGUS is shown in SEQ ID NO: 42 (Genbank Accession No. AAK29422.1). Furthermore, the sequences of a number of microbial BGUS enzymes are disclosed in U.S. Pat. No. 6,391,547 and EP Patent EP 1175495B, the entire contents of which, including the sequence listing, are incorporated herein by reference. 
         [0048]    To identify “an amino acid corresponding to G559 in SEQ ID NO: 18” in a BGUS enzyme sequence other than  E. coli  K12 strain, one of skill in the art can align the BGUS enzyme sequence to SEQ ID NO: 18 using standard computer programs for identifying protein homologies to determine the “best fit” alignment to thereby identify an amino acid residue in the non- E. coli  K12 sequence that corresponds to G559 in SEQ ID NO: 18. For example,  FIG. 3  shows a representative alignment of the  E. coli  K12 (K3 mutant), human, abalone,  Lactobacillus brevis  and  Staphylococcus  sp. RLH1 sequences across the region spanning G559 in SEQ ID NO: 18, with the amino acid position corresponding to G559 in the five sequences indicated. Using such an approach, one of skill in the art can easily make a single amino acid substitution at the position corresponding to G559 in BGUS sequences other than  E. coli  K12. 
         [0049]    Accordingly, in one embodiment of the invention, the mutated BGUS enzyme that comprises a mutation consisting of a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 is from a bacteria, preferably  E. coli , more preferably  E. coli  strain K12. In another embodiment, the mutated BGUS enzyme is from a mollusk, such as a snail (preferably  Helix pomatia ) or abalone (preferably  Haliotus rufenscens ). In still another embodiment, the mutated BGUS enzyme is from a mammal, preferably a human. 
         [0050]    B. Carboxy Terminal Cysteine Residue 
         [0051]    It has now been discovered that appending a cysteine residue at or near the carboxy terminal end of the BGUS enzyme enhances the thermostability of the enzyme. Accordingly, in another aspect, the invention provides a mutant BGUS enzyme comprising an addition of a cysteine residue appended at or near the carboxy terminus of the enzyme. As used herein, “the carboxy terminus” (used interchangeably with “C-terminus”, “carboxy terminal end” or “C-terminal end”) of the BGUS enzyme refers to the end of the protein that terminates in a carboxyl group, according to the standard nomenclature for proteins well established in the art. As used herein, “near the C-terminal end” refers to within a few (i.e., 2-4) amino acids of the C-terminal end. 
         [0052]    The cysteine appended to the C-terminus can be contained within a larger peptide. In a preferred embodiment, the cysteine appended to the C-terminus is contained within the following sequence: Xaa 2-8 -Cys-Xaa 0-2 , wherein Xaa=any amino acid (SEQ ID NO: 95). For example, in a preferred embodiment, the cysteine appended to the C-terminal end is part of a tripeptide. A preferred tripeptide for addition onto the C-terminal end has the sequence Gly-Leu-Cys (GLC). Similar tripeptides with conservative substitutions as compared to the GLC tripeptide also can be used. Alternatively, the cysteine appended to the C-terminal end can be part of, for example, a pentapeptide. A preferred pentapeptide for addition to the C-terminal end has the sequence Gly-Leu-Cys-Gly-Arg (GLCGR) (SEQ ID NO: 96). Other exemplary mutant BGUS enzymes having a cysteine appended at or near (within 2 residues of) the C-terminal end have the amino acid sequences shown in SEQ ID NOs: 21, 62, 63, 65, 66-78, 93 and 94. 
         [0053]    The preparation of mutant BGUS enzymes having a cysteine appended at the C-terminus, in the form of the tripeptide GLC is described in detail in Example 2. A representative example is a mutant referred to herein as K3, which contains the G559S mutation that is present in the K1 S mutant and also contains the GLC tripeptide at its C-terminus. The full-length amino acid sequence of the K3 mutant is shown in SEQ ID NO: 21. The alignment of the K3 mutant amino acid sequence as compared to wild-type (SEQ ID NO: 18) is shown in  FIG. 1 . 
         [0054]    The enzymatic activity of the K3 mutant as compared to the K1S mutant (which differ only in the addition of GLC at the C-terminus of K3) is described in detail in Example 4 (third experiment) and shown in  FIG. 6 . This data demonstrates that the K3 enzyme has enzymatic activity that is equal to or even slightly greater than the K1S mutant, which itself has at least 3-fold greater activity than wild type. Furthermore, the thermostability of the K3 mutant as compared to the K1S and K1 T enzymes is described in detail in Examples 5 and 8 and shown in Table 3. This data demonstrates that appending the cysteine at the C-terminal end of the BGUS enzyme enhances its thermostability. 
         [0055]    Further experiments described in Example 8 demonstrate that mutations near the cysteine at or near the carboxy terminus do not affect the overall enzyme activity or thermal stability; rather, that it is only the cysteine residue that is critical for enhanced thermal stability of the enzyme. Various mutant enzymes were made having the cysteine appended to the C-terminus contained within the following sequence: Xaa 2-8 -Cys-Xaa 0-2 , wherein Xaa=any amino acid (SEQ ID NO: 95). A representative full length amino acid sequence is shown in SEQ ID NO: 93. The results of the analysis showed that the various mutations and insertions around the appended cysteine residue did not affect the enzymatic activity or thermal stability. Thus, it is possible to insert amino acids upstream or downstream of the cysteine residue or make amino acid substitutions at positions adjacent to the cysteine residue without affecting overall enzyme activity or thermal stability. 
         [0056]    As discussed above, the sequences of BGUS enzymes from numerous species are known in the art. Accordingly, a cysteine residue (preferably in the form of a GLC tripeptide or a GLCGR pentapeptide) can be appended to the C-terminus of BGUS sequences from  E. coli  as well as from other species. For example,  FIG. 3  shows a representative alignment of the  E. coli  K12 (K3 mutant), human, abalone,  Lactobacillus brevis  and  Staphylococcus  sp. RLH1 sequences with the C-terminal appended cysteine in the K3 mutant indicated. Thus, in one embodiment of the invention, the mutated BGUS enzyme that comprises a cysteine appended to the C-terminus is from a bacteria, preferably  E. coli , more preferably  E. coli  strain K12. In another embodiment, the mutated BGUS enzyme is from a mollusk, such as a snail (preferably  Helix pomatia ) or abalone (preferably  Haliotus rufenscens ). In still another embodiment, the mutated BGUS enzyme is from a mammal, preferably a human. 
         [0057]    C. Modification of Loop Region F385 Through K390 
         [0058]    It has now been discovered that a loop region spanning amino acid residues F385 through K390 in SEQ ID NO: 18 (wild type  E. coli  BGUS) is important for effective hydrolysis of codeine glucuronide substrates. The preparation of mutant BGUS enzymes having this region deleted (referred to herein as K3Δ1, K3A2 and K3A2S) is described in detail in Example 2. The full-length amino acid sequences of the K3Δ1, K3A2 and K3A2S mutants are shown in SEQ ID NOs: 22, 23 and 24, respectively. The alignments of the K3Δ1, K3A2 and K3A2S mutant amino acid sequences as compared to wild-type (SEQ ID NO: 18) are shown in  FIG. 1 . 
         [0059]    The enzymatic activity of the K3Δ1 mutant as compared to the K1S and K3 enzymes, as well as the commercially available snail ( Helix pomatia ) BGUS extract, against two different substrates is described in detail in Example 6 and Table 1. This data demonstrate that deletion of the F385 through K390 region does not significantly affect the enzymatic activity against phenolphthalein-glucuronide as a substrate but it does significantly affect the enzymatic activity against codeine-6-glucuronide as a substrate. In particular, deletion of this loop region reduces the activity of the  E. coli  BGUS for the codeine glucuronide substrate nearly as low as that of the snail BGUS, which lacks this loop region in its wild type form. Thus, this data indicates that this loop region is important for imparting to the BGUS enzyme the ability to efficiently hydrolyze codeine glucuronides. 
         [0060]    Accordingly, in another aspect, the invention provides a BGUS mutant that comprises a modification of a region comprising amino acids corresponding to F385 through K390 in SEQ ID NO: 18. In one embodiment, the modification is a deletion of this region. For example, a BGUS enzyme that contains this region in its wild type form can be mutated to delete this region to thereby reduce the enzymatic activity of the enzyme against codeine glucuronide substrates. Alternatively, in another embodiment, the modification is an insertion of this region. For example, a BGUS enzyme that lacks this region in its wild type form can be mutated to insert this region to thereby impart or enhance enzymatic activity of the enzyme against codeine glucuronide substrates. 
         [0061]    The term “modification of a region comprising amino acids corresponding to F385 through K390 in SEQ ID NO: 18”, as used herein, references the numbering of the wild type  E. coli  K12 strain BGUS enzyme as shown in SEQ ID NO: 18. That is, the wild type sequence shown in SEQ ID NO: 18 has the following residues: F385, E386, A387, G388, N389 and K390, and it is these amino acid residues, or amino acid residues corresponding to these residues in another BGUS sequence, that are to be modified. 
         [0062]    As described above, the sequences of BGUS enzymes from numerous species are known in the art. To identify “a region comprising amino acids corresponding to F385 through K390 in SEQ ID NO: 18” in a BGUS enzyme sequence other than  E. coli  K12 strain, one of skill in the art can align the BGUS enzyme sequence to SEQ ID NO: 18 using standard computer programs for identifying protein homologies to determine the “best fit” alignment to thereby identify an amino acid residue in the non- E. coli  K12 sequence that corresponds to F385 through K390 in SEQ ID NO: 18. For example,  FIG. 2  shows a representative alignment of the  E. coli  K12 (K3 mutant), human, abalone,  Lactobacillus brevis  and  Stapylococcus  sp. RLH1 sequences across the region spanning F385 through K390 in SEQ ID NO: 18, with the amino acid position corresponding to this region in the five sequences indicated. Using such an approach, one of skill in the art can easily make a modification, such as an insertion into the human or abalone sequence that lacks this loop region, at the region corresponding to F385 through K390 in BGUS sequences other than  E. coli  K12. For example,  FIG. 10A  shows two representative insertions mutations (referred to as Ab1 and Ab2) of an amino acid sequence comprising the F385 through K390 sequence of SEQ ID NO: 18 into the wild type abalone sequence at the appropriate insertion region. Similarly,  FIG. 10B  shows two representative insertions mutations (referred to as Hu1 and Hu2) of an amino acid sequence comprising the F385 through K390 sequence of SEQ ID NO: 18 into the wild type human sequence at the appropriate insertion region. 
         [0063]    Accordingly, in one embodiment of the invention, the mutated BGUS enzyme that comprises a modification of a region comprising amino acids corresponding to F385 through K390 in SEQ ID NO: 18 is from a bacteria, preferably  E. coli , more preferably  E. coli  strain K12. In another embodiment, the mutated BGUS enzyme is from a mollusk, such as a snail (preferably  Helix pomatia ) or abalone (preferably  Haliotus rufenscens ). In still another embodiment, the mutated BGUS enzyme is from a mammal, preferably a human. 
         [0064]    D. Combination Mutants 
         [0065]    In another aspect, the invention pertains to mutant BGUS enzymes that contain two or more of the above described modifications, referred to herein as combination mutants. 
         [0066]    Accordingly, in one embodiment, the invention provides a mutated β-glucuronidase enzyme comprising: 
         [0067]    (i) a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 with an amino acid comprising a side chain comprising a non-aromatic hydroxyl group or histidine or asparagine; and 
         [0068]    (ii) an addition of a cysteine residue appended at or near the carboxy terminus of the enzyme (e.g., wherein the carboxy terminus has the sequence: Xaa 2-8 -Cys-Xaa 0-2 , wherein Xaa=any amino acid). 
         [0069]    In another embodiment, the invention provides a mutated β-glucuronidase enzyme comprising: 
         [0070]    (i) a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 with an amino acid comprising a side chain comprising a non-aromatic hydroxyl group or histidine or asparagine; and 
         [0071]    (ii) a modification of a region comprising amino acids corresponding to F385 through K390 in SEQ ID NO: 18. 
         [0072]    In yet another embodiment, the invention provides a mutated β-glucuronidase enzyme comprising: 
         [0073]    (i) a substitution of an amino acid corresponding to G559 in SEQ ID NO: 18 with an amino acid comprising a side chain comprising a non-aromatic hydroxyl group or histidine or asparagine; 
         [0074]    (ii) an addition of a cysteine residue appended at or near the carboxy terminus of the enzyme (e.g., wherein the carboxy terminus has the sequence: Xaa 2-8 -Cys-Xaa 0-2 , wherein Xaa=any amino acid); and 
         [0075]    (iii) a modification of a region comprising amino acids corresponding to F385 through K390 in SEQ ID NO: 18. 
         [0076]    In still another embodiment, the invention provides a mutated β-glucuronidase enzyme comprising: 
         [0077]    (i) an addition of a cysteine residue appended at or near the carboxy terminus of the enzyme (e.g., wherein the carboxy terminus has the sequence: Xaa 2-8 -Cys-Xaa 0-2 , wherein Xaa=any amino acid); and 
         [0078]    (ii) a modification of a region comprising amino acids corresponding to F385 through K390 in SEQ ID NO: 18. 
         [0079]    In these combination mutants having a G559 substitution, in one embodiment, the amino acid corresponding to G559 in SEQ ID NO: 18 is substituted with serine. In another embodiment, the amino acid corresponding to G559 in SEQ ID NO: 18 is substituted with threonine. In yet other embodiments, the amino acid corresponding to G559 in SEQ ID NO: 18 is substituted with a non-natural amino acid as described above in subsection IA. 
         [0080]    In the combination mutants having a cysteine residue appended at the carboxy terminus, preferably a tripeptide Glycine-Leucine-Cysteine (GLC) or a pentapeptide Gly-Leu-Cys-Gly-Arg (GLCGR) is appended at the carboxy terminus. 
         [0081]    A preferred combination mutant has a G559S substitution and a GLC tripeptide appended at the C-terminus. Preferably, this combination mutant has an amino acid sequence as shown in SEQ ID NO: 21. Another combination mutant has a G559H substitution and a GLC tripeptide appended at the C-terminus, such as the mutant having the amino acid sequence shown in SEQ ID NO: 62. Another combination mutant has a G559H substitution and a GLCGR pentapeptide appended at the C-terminus, such as the mutant having the amino acid sequence shown in SEQ ID NO: 63. Another combination mutant has a G559N substitution and a GLC tripeptide appended at the C-terminus, such as the mutant having the amino acid sequence shown in SEQ ID NO: 65. Another combination mutant has a G559N substitution and a GLCGR pentapeptide appended at the C-terminus, such as the mutant having the amino acid sequence shown in SEQ ID NO: 66. 
         [0082]    In a preferred embodiment, a combination mutant that comprises a substitution at position 559 and that has a cysteine residue appended at or near the C-terminal end has the amino acid sequence shown in SEQ ID NO: 93, wherein position 559 is substituted with Ser (S), Thr (T), His (H) or Asn (N) and wherein the C-terminal end has the following sequence: Xaa 2-8 -Cys-Xaa 0-2 , wherein Xaa=any amino acid. 
         [0083]    In the combination mutants having a modification of the region comprising amino acids corresponding to F385 through K390 in SEQ ID NO: 18, in one embodiment this modification is a deletion of the region. Examples of mutant BGUS enzymes having a deletion of this region include those having the amino acid sequence set forth in SEQ ID NOs: 22, 23 and 24. In another embodiment, the modification is an insertion of the region, for example into a BGUS sequence from a species that does not naturally contain this region. 
         [0084]    In one embodiment, a combination mutant as described above is from a bacteria, preferably from  Escherichia coli , more preferably from  Escherichia coli  K12 strain. In another embodiment, the combination mutant is from a mollusk, such as a snail (preferably  Helix pomatia ) or an abalone (preferably  Haliotus rufenscens ). In yet another embodiment, the combination mutant is from a human. 
       II. Preparation of Mutant Enzymes 
       [0085]    The BGUS enzymes of the invention can be prepared using standard recombinant DNA techniques. A preferred method for mutation is to perform overlap extension PCR using primers that incorporate the desired mutation(s), as described in detail in Example 2. Other methods known in the art for protein mutagenesis, however, are also suitable. Once a nucleic acid fragment encoding the desired mutant BGUS enzyme has been obtained, the fragment can be inserted into a suitable expression vector, transformed into a suitable host cell and the mutant protein expressed recombinantly by culturing of the host cell. Representative non-limiting examples of suitable expression vectors and host cells are described in Example 2, although the skilled artisan will appreciate that any of a variety of expression systems known in the art can be used. 
         [0086]    Following recombinant expression of the mutant BGUS enzyme, the protein can be purified using standard protein purification techniques For example, standard affinity chromatography methods, such as immunoaffinity chromatography using an anti-BGUS antibody or metal ion affinity chromatography using nickel, cobalt or copper resin, can be used. As described in detail in Example 6 and  FIG. 9 , recombinant mutant enzyme of the invention exhibits a significantly higher degree of purity than commercially available extracts from abalone, snail or humans. Thus, the recombinant mutant enzymes of the invention advantageously lack contaminating proteins found in commercially available crude extract preparations, which contaminating proteins could interfere with enzyme activity or efficiency. 
       III. Packaged Formulations 
       [0087]    In another aspect, the invention pertains to packaged formulations that comprise a mutant BGUS enzyme of the invention. These packaged formulations comprise a container comprising a preparation of the mutant BGUS enzyme. Non-limiting examples of suitable containers include, bottles, tubes, vials, ampules and the like. Preferably, the container is glass or plastic, although other suitable materials are known in the art. The preparation of the mutant BGUS enzyme can be in liquid or solid form. Thus, in one embodiment, the enzyme preparation is an aqueous solution. In another embodiment, the enzyme preparation is a lyophilized preparation. Lyophilized preparations can be packaged with instructions for reconstituting the enzyme into a liquid solution (e.g., an aqueous solution). 
         [0088]    Preferably, the preparation of β-glucuronidase enzyme in the packaged formulation has an enzymatic activity of at least 5,000 Units/ml or 5,000 Units/mg, more preferably at least 10,000 Units/ml or 10,000 Units/mg, even more preferably at least 25,000 Units/ml or 25,000 Units/mg and even more preferably 50,000 Units/ml or 50,000 Units/mg. In one embodiment, the β-glucuronidase enzyme in the preparation is in an aqueous solution with an enzymatic activity of at least 5,000 Units/ml, or at least 10,000 Units/ml or at least 25,000 Units/ml or at least 50,000 Units/ml. In another embodiment, the β-glucuronidase enzyme in the preparation is in lyophilized form with an enzymatic activity of at least 5,000 Units/mg, or at least 10,000 Units/mg or at least 25,000 Units/mg or at least 50,000 Units/mg. In yet another embodiment, the β-glucuronidase enzyme in the preparation is in lyophilized form that when reconstituted as an aqueous solution has an enzymatic activity of at least 5,000 Units/ml, or at least 10,000 Units/ml or at least 25,000 Units/ml or at least 50,000 Units/ml. 
         [0089]    The specific activity of the enzyme in the preparation, in Units/ml or Units/mg, can be determined using a standardized glucuronide linkage hydrolysis assay using phenolphthalein-glucuronide as the substrate. The standardization of the specific activity of BGUS has been well established in the art. Thus, 1 Unit of BGUS activity is defined as an amount of enzyme that liberates 1 μg of phenolphthalein from phenolphthalein-glucuronide in 1 hour. An exemplary standardized assay that can be used to determine the specific activity (in Units/ml or Units/mg) of an enzyme preparation (e.g., an aqueous solution or lyophilized preparation) is described in further detail in Example 3. The skilled artisan will appreciate that other protocols for the enzyme assay are also suitable (e.g., such as those described by Sigma Aldrich Chemical Co.). The calculation of Units/ml or Units/mg based on the results of the enzymatic assay also is described in detail in Example 3. 
         [0090]    In a preferred embodiment, the preparation containing the mutant BGUS enzyme is substantially free of other non-BGUS proteins. As used herein, “substantially free” refers to less than 5%, preferably less than 3%, even more preferably less than 1% of contamination non-BGUS proteins. In another preferred embodiment, the preparation containing the mutant BGUS enzyme lacks detectable sulfatase activity. In yet another preferred embodiment, the preparation containing the mutant BGUS enzyme is stable at least one month, more preferably at least three months, and even more preferably at least six months at 2-8° C. As used herein, “stable” refers to the mutant BGUS enzyme in the preparation maintaining at least 90%, more preferably at least 95%, even more preferably at least 98% of its enzymatic activity over the indicated time at the indicated temperature. 
       IV. Methods of Use 
       [0091]    The mutant BGUS enzymes of the invention exhibit enhanced ability to hydrolyze glucuronide linkages as compared to the wild type enzyme. Accordingly, the mutant enzymes can be used in methods for hydrolysis of gluruonide substrates. These methods are particularly useful for analyzing bodily samples for the presence of drugs through detection of the glucuronide detoxification products of the drugs. Thus, in another aspect the invention pertains to a method of hydrolyzing a substrate comprising a glucuronide linkage, the method comprising contacting the substrate with a mutant β-glucuronidase enzyme of the invention under conditions such that hydrolysis of the glucuronide linkage occurs. Any of the mutant enzymes of the invention, including those having a single modification and those having more than one modification (i.e., combination mutants) can be used in the method. 
         [0092]    In one embodiment, the substrate is an opiate glucuronide. Non-limiting examples of suitable opiate glucuronide substrates include morphine-3β-D-glucuronide, morphine-6β-D-glucuronide, codeine-6β-D-glucuronide, hydromorphone-3β-D-glucuronide, oxymorphone-3β-D-glucuronide, and combinations thereof. In another embodiment, the substrate is a benzodiazepine glucuronide. Non-limiting examples of suitable benzodiazepine glucuronide substrates include the glucuronides of oxazepam, lorazepam, temazepam, and alpha-hydroxy-alprazolam. Other suitable substrates include the glucuronides of buprenorphine, norbuprenorphine, 11-nor-A9-tetrahydrocannabinol-9-carboxylic acid, testosterone, androsterone, tapentadol, cyclobenzaprine, amitripyline and combinations thereof. 
         [0093]    The methods of the invention can be used on a variety of different bodily samples. Non-limiting examples of suitable bodily samples include blood, urine, tissue or meconium obtained from a subject. Such samples can be obtained, stored and prepared for analysis using standard methods well established in the art. 
         [0094]    Following hydrolysis by the enzyme, the cleavage products in the sample can be analyzed by standard methodologies, such as high performance liquid chromatography (HPLC), gas chromatography (GC) and/or mass spectrometry (MS). Such approaches for analysis of bodily samples for the presence of drugs are well established in the art. For example, a completely automated workflow for the hydrolysis and analysis of urine samples by LC-MS/MS, which can be applied using the mutant enzymes of the invention for hydrolysis, is described in Cabrices, O. G. et al., GERSTEL AppNote AN/2014/4-7. 
         [0095]    In addition to its use in drug testing, a mutated BGUS enzyme of the invention can be used in essentially any other methodology for which the wild type BGUS enzyme can be used. For example, U.S. Pat. No. 5,599,670 describes a gene fusion system in which DNA encoding a BGUS enzyme is fused to DNA encoding a gene of interest to create a reporter gene system that can be used for a wide variety of genetic engineering purposes. Accordingly, the mutated BGUS enzymes of the invention can be used in this gene fusion system to enhance the enzymatic activity of the BGUS portion of the fusion protein. 
         [0096]    The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of Sequence Listing, figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. 
       EXAMPLES 
     Example 1 
     Cloning of  E. coli  β-Glucuronidase (BGUS) Gene 
       [0097]    In this example, the wild type  E. coli  BGUS sequence was cloned by polymerase chain reaction (PCR) based on the published  E. coli  K12 BGUS sequence derived from the  E. coli  genome (NCBI Reference Sequence NC_000913.2), shown in SEQ ID NO: 1. The forward primer used for PCR had the following sequence: GAGAGACATATGTTACGTCCTGTAGAAACCCC (SEQ ID NO: 2). The reverse primer for PCR had the following sequence: GAGAGAAAGCTTTCATTGTTTGCCTCCCTGCT (SEQ ID NO: 3). The forward primer contains an NdeI restriction site and the reverse primer contains a HindIII restriction site. 
         [0098]    To isolate a sample of  E. coli  DH5a strain genomic DNA, 5 ml of LB culture were grown overnight and 1 ml of cells was pelleted in a 1.5 ml tube. The cells were resuspended in 50 μl of water and were heated at 95° C. for 5 minutes to lyse the cells. The debris was pelleted and 10 μl of supernatant was used as a template in the PCR reaction. 
         [0099]    For the PCR reaction, the primers were diluted to 10 μM and 5 μl of each primer was used in a 50 μl PCR reaction (final primer concentration was 1 μM). The 50 μl reaction mixture contained the following: 10× ThermoPol Buffer (5 μl), 10 mM dNTP (1 μl), BGUS forward primer (5 μl), BGUS reverse primer (5 μl),  E. coli  DNA (10 μl), NEB Taq enzyme (1 μl), water (23 μl). The PCR program used for DNA amplification was as follows: 95° C./5 minutes, 40 cycles of 95° C./30 seconds, 50° C./30 seconds, 72° C./2.5 minutes, followed by 72° C./5 minutes, 4° C./00. 
         [0100]    After amplification, the PCR product was digested with NdeI and HindIII for cloning into a bacterial expression vector. The reaction mixture contained the following: PCR product (16 μl), NdeI (20 U/μl) (1 μl), HindIII (20 U/μl) (1 μl), 10×NEB Reaction Buffer 2 (2 μl). The restriction enzyme digestion reaction was carried out at 37° C. for 60 minutes. 
         [0101]    The bacterial vector was prepared by digestion with NdeI and HindIII in a reaction mixture that contained the following: Vector (0.1 μg/μl) (10 μl), NdeI (20 U/μl) (1 μl), HindIII (20 U/μl) (1 μl), 10×NEB Reaction Buffer 2 (2 μl), water (6 μl). The restriction enzyme digestion reaction was carried out at 37° C. for 60 minutes. Then 2 μl of 10× Shrimp Alkaline Phosphatase (SAP) buffer and 1 μl of SAP enzyme was added to the restriction enzyme digestion for the vector and incubated at 37° C. for 30 minutes. All fragments were isolated on a 1% low melting point (LMP) agarose gel in 1× Tris-Acetate-EDTA running buffer. 
         [0102]    The agarose fragments containing the vector and the insert DNA were melted at 68° C. for 10 minutes. A ligation reaction was prepared that contained the following: Vector/NdeI/HindIII/SAP (3 μl), BGUS ORF/NdeI/HindIII (7 μl), 10×NEB T4 DNA Ligase Buffer (5 μl), T4 DNA Ligase (400 U/μl)(1 μl), water (34 μl). The ligation reaction mixture was incubated at room temperature overnight. 
         [0103]    Following the overnight ligation reaction, the T4 DNA ligase was heat inactivated by heating the reaction at 68° C. for 10 minutes. For transformation into competent cells, an aliquot of competent DH5a cells was thawed on ice and 150 μl of the competent cells as added to the ligation reaction and mixed by gentle pipetting. The cells were then incubated on ice for 30 minutes, followed by heat shock at 42° C. for 3 minutes. The cells were then placed back on ice for 2 minutes and then transferred to 1 ml of LB medium in a 14 ml culture tube. The cell culture was incubated at 37° C. for 60 minutes at 250 rpm. Aliquots of the cells were added to LB-Kanamycin plates and grown overnight at 37° C. Colonies were picked for plasmid extraction and analysis by DNA sequencing using standard techniques to identify a plasmid that contained the cloned  E. coli  wild type BGUS gene. 
       Example 2 
     Mutagenesis of  E. coli  β-Glucuronidase (BGUS) Gene 
       [0104]    Overlap extension PCR was used to create mutations in the  E. coli  K12 BGUS ORF using the plasmid encoding the wild type enzyme as the template. Four primers were used to create the K1S mutant (having a G559S single amino acid substitution), as follows: 
         [0000]    
       
         
               
               
             
               
               
               
             
               
               
             
               
               
               
             
               
               
             
               
               
               
             
               
               
             
               
               
               
             
           
               
                   
                 (SEQ ID NO: 4) 
               
             
          
           
               
                   
                 BGUS Forward: 
                 CTGCTGTCGGCTTTAACCTC 
               
               
                   
                   
               
             
          
           
               
                   
                 (SEQ ID NO: 5) 
               
             
          
           
               
                   
                 G559S Forward: 
                 GACCTCGCAAAGCATATTGCG 
               
               
                   
                   
               
             
          
           
               
                   
                 (SEQ ID NO: 6) 
               
             
          
           
               
                   
                 G559S Reverse: 
                 CGCAATATGCTTTGCGAGGTC 
               
               
                   
                   
               
             
          
           
               
                   
                 (SEQ ID NO: 7) 
               
             
          
           
               
                   
                 Vector Reverse: 
                 CTAGTTATTGCTCAGCGGT 
               
             
          
         
       
     
         [0105]    Two parallel PCR reactions were set up that were designed to produce the 5′ and 3′ parts of the desired mutated DNA fragments. Reaction 1 contained the following: 10× Pfu Buffer (5 μl), 10 mM dNTP (1 μl), 10 μM BGUS forward primer (1 μl), 10 μM G559 reverse primer (1 μl), wild-type plasmid template (0.1 μg/μl) (1 μl), Pfu enzyme (1 μl), water (40 μl). Reaction 2 contained the following: 10× Pfu Buffer (5 μl), 10 mM dNTP (1 μl), 10 μM G559S forward primer (1 μl), 10 μM Vector reverse primer (1 μl), wild type plasmid template (0.1 μg/μl) (1 μl), Pfu enzyme (1 μl), water (40 μl). The PCR program used for DNA amplification was as follows: 95° C./5 minutes, 40 cycles of 95° C./30 seconds, 50° C./30 seconds, 72° C./60 seconds, followed by 72° C./5 minutes, 4° C./∞. 
         [0106]    The DNA fragments were purified using an Omega Cycle Pure Kit and the two purified fragments were mixed together to form the final (mutated) product in a PCR reaction that contained the following: 10× Pfu Buffer (5 μl), 10 mM dNTP (1 μl), 10 μM BGUS forward primer (1 μl), 10 μM Vector reverse primer (1 μl), Fragment 1 (10 μl), Fragment 2 (10 μl), Pfu enzyme (1 μl), water (31 μl). The PCR program used for DNA amplification was as follows: 95° C./5 minutes, 40 cycles of 95° C./30 seconds, 50° C./30 seconds, 72° C./90 seconds, followed by 72° C./5 minutes, 4° C./00. 
         [0107]    The final DNA fragment was purified using an Omega Cycle Pure Kit. The fragment was digested with BstB1 and HindIII restriction enzymes in pUC19 bacterial vector. The recipient bacterial plasmid was digested with BstB1 and HindIII, followed by treatment with SAP. All fragments were isolated on an LMP gel and an in gel ligation reaction was performed using standard techniques. Plasmid DNA was isolated for sequencing to identify the mutant clone. For large-scale expression of the mutant enzyme, the coding sequence of the mutant enzyme was cloned into the bacterial expression vector pSF-TAC (commercially available from Sigma Aldrich) or pD441-NH (commercially available from DNA2.0), both of which vectors contain an IPTG-inducible TAC or T5 promoter and a kanamycin resistance gene. 
         [0108]    The protocol used to produce additional mutants was very similar to that described above for preparing the K1S mutant, except that each mutant had a specific pair of mutagenic primers for use in the overlap extension PCR. To prepare the K1T, K3, K3Δ1, K3Δ2 and K3Δ2S mutants, the following primers were used in overlap extension PCR reactions as described above: 
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 Mutant 
                 Forward Primer 
                 Reverse Primer 
               
               
                   
               
             
             
               
                 K1T 
                 GCGACCTCGCAAACCATATTGCGC 
                 GCGCAATATGGTTTGCGAGGTCGC 
               
               
                   
                 (SEQ ID NO: 8) 
                 SEQ ID NO: 9) 
               
               
                   
               
               
                 K3 
                 CAAACAAGGCCTATGCTAGAAAGGAGAAGCTTG 
                 CAAGCTTCTCCTTTCTAGCATAGGCCTTGTTTG 
               
               
                   
                 (SEQ ID NO: 10) 
                 (SEQ ID NO: 11) 
               
               
                   
               
               
                 K3Δ1 
                 TCTTTAGGCATTGGTCCGAAAGAACTGTAC 
                 GTACAGTTCTTTCGGACCAATGCCTAAAGA 
               
               
                   
                 (SEQ ID NO: 12) 
                 (SEQ ID NO: 13) 
               
               
                   
               
               
                 K3Δ2 
                 TCTTTAGGCATTGGTGAAGAGGCAGTCAAC 
                 GTTGACTGCCTCTTCACCAATGCCTAAAGA 
               
               
                   
                 (SEQ ID NO: 14) 
                 (SEQ ID NO: 15) 
               
               
                   
               
               
                 K3Δ2S 
                 CCGCAGTTCTTCAACGGGGAAACTCAG 
                 GAAGAACTGCGGACCAATGCCTAAAGAG 
               
               
                   
                 (SEQ ID NO: 16) 
                 (SEQ ID NO: 17) 
               
               
                   
               
             
          
         
       
     
         [0109]    For the K1T mutant, the wild type BGUS sequence was used as the PCR template, similar to as described above for the K1S mutant. The resultant K1T mutant contains a G559T single amino acid substitution. 
         [0110]    For the K3 mutant, the K1S mutant sequence was used as the PCR template. The K3 mutant contains a G559S single amino acid substitution and also contains a “GLC” tripeptide added onto the C-terminal end of the protein. 
         [0111]    For the K3Δ1 mutant, the K3 mutant sequence was used as the PCR template. The K3Δ1 mutant contains a G559S single amino acid substitution, a “GLC” tripeptide added onto the C-terminal end of the protein and a deletion of amino acid residues F385 through K390. 
         [0112]    For the K3Δ2 mutant, the K3 mutant sequence was used as the PCR template. The K3Δ2 mutant contains a G559S single amino acid substitution, a “GLC” tripeptide added onto the C-terminal end of the protein and a deletion of amino acid residues F385 through S396. 
         [0113]    For the K3Δ2S mutants, the K3 mutant sequence was used as the PCR template. The K3Δ2S mutant contains a G559S single amino acid substitution, a “GLC” tripeptide added onto the C-terminal end of the protein, a deletion of amino acid residues F385 through S396 and the following additional substitutions: E397P, E398Q, A399F and V400F. 
         [0114]    Following PCR amplification and plasmid ligation as described above, the plasmid DNA was isolated for sequencing to identify the mutant clones. An alignment of the amino acid sequences of the K1S, K1T, K3, K3Δ1, K3Δ2 and K3Δ2S mutants compared to the wild type sequence is shown in  FIG. 1 . 
         [0115]    An alignment of the amino acid sequences of the  E. coli  K3 mutant, the abalone wild type BGUS, the human wild type BGUS, the  Lactobacillus brevis  wild type BGUS and the  Staphylococcus  sp. RLH1 wild type BGUS across amino acid residues 332-416 ( E. coli  numbering), including across the modification region F385 through S396 ( E. coli  numbering), is shown in  FIG. 2 . Identical amino acid residues across the five sequences are highlighted in grey. The conserved glutamic acid residue (E) at position 413 ( E. coli  numbering) within the catalytic site is highlighted in bold, indicating the accuracy of the alignment. This conserved residue within the catalytic domain is described further in  J. Biol. Chem . (1996) vol. 273(51), pp. 34507-34602 and  Proc. Natl. Acad. Sci. USA  (1995) vol. 92(15), pp. 7090-7094. The modification region F385 through S396 ( E. coli  numbering) is highlighted in bold and underlined. 
         [0116]    An alignment of the amino acid sequences of the  E. coli  K3 mutant, the abalone wild type BGUS, the human wild type BGUS, the  Lactobacillus brevis  wild type BGUS and the  Staphylococcus  sp. RLH1 wild type BGUS across amino acid residues 452-606 ( E. coli  numbering), including across the G559S modification ( E. coli  numbering) and the C-terminal GLC modification, is shown in  FIG. 3 . Identical amino acid residues across the five sequences are highlighted in grey. The conserved tyrosine residue (Y) at position 468 and glutamic acid residue (E) at position 504 ( E. coli  numbering) within the catalytic site is highlighted in bold, indicating the accuracy of the alignment. These conserved residues within the catalytic domain are described further in  J. Biol. Chem.  (1996) vol. 273(51), pp. 34507-34602 and  Proc. Natl. Acad. Sci. USA  (1995) vol. 92(15), pp. 7090-7094. The G559S ( E. coli  numbering) and C-terminal GLC modifications are highlighted in bold and underlined. 
       Example 3 
     β-Glucuronidase Enzymatic Activity Assay 
       [0117]    In this example, a standard enzyme activity assay for BGUS is described. The standard reporting format for this assay is in Units/ml for liquid formulations or in Units/mg for lyophilized formulations. 
         [0118]    An activity assay buffer, 20 mM potassium phosphate buffer, pH 6.8, was prepared. The substrate solution used was 1 mM phenolphthaleine-glucuronide (PT-gluc) in water, prepared fresh. 400 μl of activity buffer was pipetted into a clean 1.5 ml microfuge tube. 4 μl of enzyme solution was added to the buffer to achieve a 1:100 dilution of the enzyme. Then, 30 μl of the diluted enzyme solution was pipetted in each well of a 96-well plate, with each enzyme solution performed in triplicate. 30 μl of diluted control enzyme solution was pipetted into control wells in triplicate. 30 μl of the PT-gluc substrate solution was pipetted into the wells with the enzyme solution. The plates were incubated for 30 minutes at 25° C. 180 μl of glycine was added to stop the reaction and develop color in each well. The absorbance at 540 nm was measured by standard methods. 
         [0119]    1 Unit of BGUS activity is defined as an amount of enzyme that liberates 1 μg of phenolphthalein from phenolphthalein-glucuronide in 1 hour. Thus, to determine Units/ml of enzyme, first a standard curve was prepared by plotting background-subtracted absorbance at 540 nm for the phenolphthalein (PT) standards. Assuming a linear plot for the standard curve, the formula for determining the concentration of PT liberated by the enzyme is as follows: 
         [0000]      [conc. PT in μg]=[(corrected absorbance at 540 nm)−( y  intercept value)]/slope
 
         [0120]    The specific activity of the enzyme was determined by correcting for time and dilution factors, divided by the volume of enzyme used. Thus, to calculate the specific activity in Units/ml using the assay protocol above, the following formula was used: 
         [0000]      Units/mL=(μg of PT released)×2×100/0.03
 
       Example 4 
     Enzymatic Activity of β-Glucuronidase Mutants 
       [0121]    In this example, the enzymatic assay of the BGUS mutants was examined in a series of experiments comparing their activity to wild type  E. coli  BGUS, as well as to another mutant previously described in the art or to a commercially available  Helix pomatia  (snail) BGUS enzyme extract. Different glucuronide substrates were examined, as described further below, using the enzymatic activity assay described in Example 3 
         [0122]    In a first experiment, the specific activity of the K1S mutant, as compared to the wild type  E. coli  enzyme, was tested using the enzymatic activity assay described in Example 3 that uses phenolphthalein-glucuronide as the substrate. The results are shown in  FIG. 4 . The results demonstrate that the K1S mutant exhibits significantly increased specific enzyme activity (over 3-fold greater activity) compared to the wild type enzyme. 
         [0123]    In a second experiment, the specific activity of the K1S mutant was compared to the wild type  E. coli  BGUS enzyme and to a thermo-resistant mutant that was reported in Xiong, A-S. et al. (2007)  Prot. Eng. Design Select.  20:319-325. The thermoresistant mutant described in Xiong et al. (referred to in  FIG. 5  as “TR” and referred to in Xiong et al. as GUS-TR3337) contains the G559S substitution but also contains five other single residue substitutions: Q439R, T509A, M532T, N550S and N566S. The enzyme reaction used approximately 500 ng of purified enzyme incubated with 750 μM concentration of 4-nitrophenol glucuronide as the substrate, followed by measurement of absorbance at 405 nm. The results are shown in  FIG. 5 . The results demonstrate that the K1S mutant exhibits significantly increased specific enzyme activity (over 3-fold greater activity) compared to both the wild type enzyme and the previously-reported TR (GUS-TR3337) mutant. 
         [0124]    In a third experiment, the specific activity of the K1T, K3, K3-C606S and K3Δ1 mutants, as compared to the K1S mutant, was tested. The results are shown in  FIG. 6 . The results demonstrate that the K1T and K3 mutants exhibit specific enzyme activity that is equal to or greater than that of the K1S mutant, whereas the K3-C606S and K3Δ1 mutants exhibit specific activity that is lower than that of the K1S mutant, although still higher than the wild type enzyme. 
         [0125]    The K3-C606S mutant differs from the K3 mutant in that the C-terminal cysteine in K3 has been substituted with a serine. Thus, the results with K3-C606S as compared to K3 demonstrate the contribution of the C-terminal cysteine to the enzymatic activity. The K3Δ1 mutant differs from the K3 mutant in that residues F385 through K390 have been deleted. Thus, the results with K3Δ1 as compared to K3 demonstrate the contribution of the F385-K390 loop region to the enzymatic activity. 
         [0126]    In a fourth experiment, the role of the F385-K390 loop region was further examined by comparing hydrolysis of two different substrates, phenolphthalein-glucuronide and codeine-6-glucuronide, by the K1S, K3 and K3Δ1 mutants as compared to commercially available wild type  Helix pomatia  BGUS enzyme. The specific activities of the enzymes (in kU/ml) was determined using the phenolphthalein-glucuronide hydrolysis assay described above. For the codeine-6-glucuronide hydrolysis, the reaction mixtures contained 200 μl of drug-free urine sample, 40 μl enzyme, 50 μl rapid hydrolysis buffer and 10 μl codeine-D 6  (10 ppm) in acetonitrile. The reactions were incubated at 55° C. for 60 minutes. The results are shown below in Table 1: 
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Specific enzyme 
                   
               
               
                   
                 activity 
               
               
                   
                 (kU/ml) 
                 codeine (ppb) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 No enzyme 
                 NA 
                 NF 
               
               
                   
                 K1S 
                 73 
                 935.0 
               
               
                   
                 K3 
                 83 
                 881.8 
               
               
                   
                 K3Δ1 
                 78 
                 345.1 
               
               
                   
                   Helix pomatia  without 
                 114 
                 NF 
               
               
                   
                 incubation 
               
               
                   
                   Helix pomatia  with 30 min. 
                 114 
                 129.1 
               
               
                   
                 incubation 
               
               
                   
                   
               
               
                   
                 NF = not found, 
               
               
                   
                 NA = not applicable, 
               
               
                   
                 ppb = parts per billion 
               
             
          
         
       
     
         [0127]    The results showed that for the phenolphthalein-glucuronide substrate, the BGUS mutants and the commercially available snail extract had similar specific activities, with the snail extract exhibiting slightly higher activity than the  E. coli  BGUS mutants. 
         [0128]    The hydrolysis of codeine-6-glucuronide, however, differed among the enzymes tested. Both the K1S and K3 mutants exhibited efficient hydrolysis of codeine-6-glucuronide, recovering 935.0 and 881.8 ppb codeine, respectively, from 1000 ppb of codeine-6-glucuronide substrate. In contrast, the K3Δ1 mutant, which differs from K3 in that K3Δ1 has a deletion of the loop region F385-K390, only recovered 345.1 ppb codeine from 1000 ppb codeine-6-glucuronide substrate. This result demonstrates that deletion of this loop region depresses the hydrolysis of the codeine glucuronides. In comparison, the  Helix pomatia  (snail) enzyme had a very low hydrolysis of codeine-6-glucuronide (129.1 ppb recovered from 1000 ppb substrate), despite having a higher specific activity than the  E. coli  mutants for the phenolphthalein-glucuronide substrate. This result is consistent with the F385-K390 loop region being important for hydrolysis of codeine glucuronides, since the  Helix pomatia  amino acid sequence lacks a region corresponding to the F385-K390 loop region of  E. coli . Thus, based on these studies, one can mimic the level of snail BGUS activity for codeine glucuronides by replacing of this loop region in BGUS enzymes that contain the loop (e.g.,  E. coli ). Moreover, one can improve the level of BGUS activity for codeine glucuronides by inserting this loop region into BGUS enzymes that lack the loop (e.g., snail, abalone or human versions of BGUS). 
       Example 5 
     Thermostability of β-Glucuronidase Mutants 
       [0129]    To examine the heat stability of the mutants, the enzymes were either unheated, or heated for 30 or 60 minutes at 65° C., and the percent of original enzyme activity left after heating was measured. In a first series of experiments, the thermostability of the K1 S, K3, K3-C606S and K1T enzymes were compared. The results demonstrate that the K3 mutant, which has a C-terminal cysteine residue modification, exhibits a higher thermostability that the K1S and K1T mutants, which lacks this C-terminal cysteine. The mutation of the cysteine residue at the carboxy terminus of the K3 mutant to serine, to form the K3-C606S variant, eliminates this heat stability, thereby demonstrating the contribution of the C-terminal cysteine to the thermostability. Wild type enzyme had similar heat stability as K1 S. 
         [0130]    The thermostability of the K3 mutant was further examined at four different temperatures using a 2 hour incubation period. Enzyme mixtures, containing buffer and analytes, were incubated at either 4° C., 20° C., 37° C. or 55° C. Enzyme activity was measured both pre- and post-incubation. The results are shown in  FIG. 10 , expressed as kU/mL, comparing the pre- and post-incubation enzymatic activity at the four different temperatures. The results demonstrate the stability of the purified, recombinant K3 mutant enzyme at the four different temperatures, showing no loss of enzyme activity over time at any of the temperatures tested. 
         [0131]    A further analysis of the effect of C-terminal mutations on the thermostability of the BGUS enzyme is described in Example 8 below. 
       Example 6 
     Further Characterization of β-Glucuronidase K3 Mutant 
       [0132]    In this example, the effects of pH on the activity of the BGUS K3 mutant, as well as the purity of the protein, were further characterized. 
         [0133]    Effect of pH 
         [0134]    The relative enzyme activity was measured at various pH levels to examine the effect of pH on the enzyme activity. The results are shown in  FIGS. 7A and 7B  for the K3 mutant. For the results shown in  FIG. 7A , the enzyme activity dropped significantly when the buffer solution pH was below 6. The optimal pH range for enzyme activity was within pH 7-8. For the results shown in  FIG. 7B , the enzyme was stored overnight at different pH levels and then enzyme activity was tested by bringing the pH back to neutral levels. Thus comparing the results in  FIGS. 7A and 7B , the results showed that although the pH level affects the enzymatic activity, it does not affect the stability of the enzyme in the short term during storage when the pH is returned to neutral levels for testing enzyme activity. 
         [0135]    Purity 
         [0136]    The recombinant K3 enzyme was purified by standard affinity purification and the resultant purified protein was examined by SDS-PAGE as compared to commercially available preparations of BGUS protein from different species. The results are shown in  FIG. 8 , in which lane 1 shows the molecular weight markers, lane 2 shows the K3 mutant, lane 3 shows a commercially available abalone BGUS preparation, lane 4 shows a commercially available snail BGUS preparation and lane 5 shows a commercially available  E. coli  BGUS preparation. The results show that each of the commercially available BGUS preparations contained extraneous proteins that may interfere with downstream analytical processing of test samples, whereas the purified recombinant K3 preparation did not contain these extraneous proteins. 
       Example 7 
     Further Analysis of β-Glucuronidase Position 559 Mutants 
       [0137]    In this example, the wild-type glycine at position 559 of BGUS was substituted with each of the 19 other natural amino acids to determine the effect of the substitutions on enzyme activity. Each 559 position mutant also included the “GLC” addition at the C-terminal end (as in the K3 enzyme) to enhance thermostability. 
         [0138]    In addition to the G559S and G559T mutations described above, the 17 other point mutations at position 559 were prepared using the following forward primers in overlap extension PCR reactions as described above. All reactions used the following as the reverse primer: GCAAAATCGGCGAAATTC (SEQ ID NO: 43). 
         [0000]                                            Mutant   Forward Primer                           G559A   GACCTCGCAA GCA ATATTGCGCGTTG               (SEQ ID NO: 44)                       G559C   GACCTCGCAA TGT ATATTGCGCGTTG               (SEQ ID NO: 45)                       G559D   GACCTCGCAA GAT ATATTGCGCGTTG               (SEQ ID NO: 46)                       G559E   GACCTCGCAA GAA ATATTGCGCGTTG               (SEQ ID NO: 47)                       G559F   GACCTCGCAA TTC ATATTGCGCGTTG               (SEQ ID NO: 48)                       G559H   GACCTCGCAA CAT ATATTGCGCGTTG               (SEQ ID NO: 49)                       G559I   GACCTCGCAA ATC ATATTGCGCGTTG               (SEQ ID NO: 50)                       G559K   GACCTCGCAA AAA ATATTGCGCGTTG               (SEQ ID NO: 51)                       G559L   GACCTCGCAA CTG ATATTGCGCGTTG               (SEQ ID NO: 52)                       G559M   GACCTCGCAA ATG ATATTGCGCGTTG               (SEQ ID NO: 53)                       G559N   GACCTCGCAA AAC ATATTGCGCGTTG               (SEQ ID NO: 54)                       G559P   GACCTCGCAA CCG ATATTGCGCGTTG               (SEQ ID NO: 55)                       G559Q   GACCTCGCAA CAG ATATTGCGCGTTG               (SEQ ID NO: 56)                       G559R   GACCTCGCAA CGT ATATTGCGCGTTG               (SEQ ID NO: 57)                       G559V   GACCTCGCAA GTG ATATTGCGCGTTG               (SEQ ID NO: 58)                       G559W   GACCTCGCAA TGG ATATTGCGCGTTG               (SEQ ID NO: 59)                       G559Y   GACCTCGCAA TAC ATATTGCGCGTTG               (SEQ ID NO: 60)                        
Enzyme activity was tested as described in the previous examples. The results for the 19 different position 559 point mutations, as compared to the wild type glycine (G), are shown in  FIG. 11 . The results demonstrate that substitution at position 559 with either serine (S), threonine (T), histidine (H) or asparagine (N) (as compared to the wild-type glycine (G)) resulted in enhanced enzyme activity.
 
       Example 8 
     Further Analysis of β-Glucuronidase C-Terminal Mutants 
       [0139]    In this example, the C-terminal end of the BGUS enzyme was mutated with various substitutions and/or insertions to determine the effect on overall enzyme activity and thermostability. The mutations made at the C-terminal end are shown below in Table 2. Each mutant also included the G559S mutation. The entire amino acid sequence of each mutant is shown in the Sequence Listing at the indicated SEQ ID NOs. Additionally, the forward and reverse primers used to prepare the mutants in overlap extension PCR reactions are shown in the Sequence Listing at the indicated SEQ ID NOs. 
         [0000]                                    TABLE 2                   Mutant Portion   Mutant   Forward   Reverse       Mutation Type   of BGUS   SEQ ID NO:   Primer SEQ   Primer SEQ                   Serine (S)     SS CXX   67   79   85               Glutamic Acid (E)     EE CXX   68   80   85               Lysine (K)     KK CXX   69   81   85               Alanine (A)     AA CXX   70   82   85               Tryptophan (W)     WW CXX   71   83   85               Proline (P)     PP CXX   72   84   85               1   GL G CXX   73   86   92               2   GL GG CXX   74   87   92               3   GL GGG CXX   75   88   92               4   GL GGGG CXX   76   89   92               5   GL GGGGG CXX   77   90   92               6   GL GGGGGG CXX   78   91   92                    
Enzyme activity was tested as described in the previous examples. Purified enzymes were heat treated for one hour prior to measuring their enzymatic activity. The results are shown in  FIG. 12 . All substitution and insertion mutations tested showed similar enzymatic activity as that of the positive control, thereby demonstrating that the mutations near the carboxy terminus do not affect the overall enzyme activity. Furthermore, the heat pre-treatment did not affect the enzymatic activity, thereby demonstrating that the substitutions and insertions around the cysteine residue did not affect the thermostability. Thus, these results indicate that the length and type of amino acid variation prior to the cysteine residue near the carboxy terminus is not critical for maintaining enzymatic activity and thermostability.
 
         [0140]    The thermostability of C-terminal mutations was further examined by incubating various mutants at 65° C. for 30 or 60 minutes. The results are shown below in Table 3: 
         [0000]                                                                    TABLE 3                               Percent Activity after               Incubation at 65° C.                Mutant   30 Minutes   60 Minutes                            K1T   0   0                       K1S   0.53   ND                       K1S + G   0   ND                       K1S + GL   0.55   ND                       K1S + GLC (K3)   19.0   ND                       K1S + GLCG   22.0   ND                       K1S + GLCGR   27.6   6.0                       K1S + GLSGR   0   0                       ND = not determined            
First, the results in Table 3 confirm the previously reported results that adding the “GLC” tripeptide to the C-terminus (shown in the table as K1S+GLC (K3)) significantly increases the thermostability as compared to enzymes that lack this tripeptide (K1S or K1 T) or that only include a “G” (K1 S+G) or “GL” (K1 S+GL) addition to the C-terminus. Furthermore, adding one residue (K1 S+GLCG) or two residues (K1S+GLCGR) on the C-terminal side of the added cysteine residue maintains this enhanced thermostability. The mutant with two residues added beyond the cysteine residue (K1S+GLCGR) exhibited the greatest thermostability of all mutants tested. Finally, substitution of the cysteine with a serine (K1S+GLSGR) abolished the enhanced thermal stability, thereby demonstrating that the cysteine residue is critical for the enhanced thermal stability.
 
       EQUIVALENTS 
       [0141]    Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. 
         [0000]    
       
         
               
             
               
               
             
           
               
                   
               
               
                 SUMMARY OF SEQUENCE LISTING 
               
             
          
           
               
                 SEQ ID NO: 
                 DESCRIPTION 
               
               
                   
               
               
                  1 
                   E. coli  K12 BGUS wild type nucleic acid sequence 
               
               
                   
                 ATGTTACGTCCTGTAGAAACCCCAACCCGTGAAATCAAAAAACTCGACGGCCTGTGGGCATTCAGTCTGGAT 
               
               
                   
                 CGCGAAAACTGTGGAATTGATCAGCGTTGGTGGGAAAGCGCGTTACAAGAAAGCCGGGCAATTGCTGTGCCA 
               
               
                   
                 GGCAGTTTTAACGATCAGTTCGCCGATGCAGATATTCGTAATTATGCGGGCAACGTCTGGTATCAGCGCGAA 
               
               
                   
                 GTCTTTATACCGAAAGGTTGGGCAGGCCAGCGTATCGTGCTGCGTTTCGATGCGGTCACTCATTACGGCAAA 
               
               
                   
                 GTGTGGGTCAATAATCAGGAAGTGATGGAGCATCAGGGCGGCTATACGCCATTTGAAGCCGATGTCACGCCG 
               
               
                   
                 TATGTTATTGCCGGGAAAAGTGTACGTATCACCGTTTGTGTGAACAACGAACTGAACTGGCAGACTATCCCG 
               
               
                   
                 CCGGGAATGGTGATTACCGACGAAAACGGCAAGAAAAAGCAGTCTTACTTCCATGATTTCTTTAACTATGCC 
               
               
                   
                 GGGATCCATCGCAGCGTAATGCTCTACACCACGCCGAACACCTGGGTGGACGATATCACCGTGGTGACGCAT 
               
               
                   
                 GTCGCGCAAGACTGTAACCACGCGTCTGTTGACTGGCAGGTGGTGGCCAATGGTGATGTCAGCGTTGAACTG 
               
               
                   
                 CGTGATGCGGATCAACAGGTGGTTGCAACTGGACAAGGCACTAGCGGGACTTTGCAAGTGGTGAATCCGCAC 
               
               
                   
                 CTCTGGCAACCGGGTGAAGGTTATCTCTATGAACTGTGCGTCACAGCCAAAAGCCAGACAGAGTGTGATATC 
               
               
                   
                 TACCCGCTTCGCGTCGGCATCCGGTCAGTGGCAGTGAAGGGCGAACAGTTCCTGATTAACCACAAACCGTTC 
               
               
                   
                 TACTTTACTGGCTTTGGTCGTCATGAAGATGCGGACTTGCGTGGCAAAGGATTCGATAACGTGCTGATGGTG 
               
               
                   
                 CACGACCACGCATTAATGGACTGGATTGGGGCCAACTCCTACCGTACCTCGCATTACCCTTACGCTGAAGAG 
               
               
                   
                 ATGCTCGACTGGGCAGATGAACATGGCATCGTGGTGATTGATGAAACTGCTGCTGTCGGCTTTAACCTCTCT 
               
               
                   
                 TTAGGCATTGGTTTCGAAGCGGGCAACAAGCCGAAAGAACTGTACAGCGAAGAGGCAGTCAACGGGGAAACT 
               
               
                   
                 CAGCAAGCGCACTTACAGGCGATTAAAGAGCTGATAGCGCGTGACAAAAACCACCCAAGCGTGGTGATGTGG 
               
               
                   
                 AGTATTGCCAACGAACCGGATACCCGTCCGCAAGGTGCACGGGAATATTTCGCGCCACTGGCGGAAGCAACG 
               
               
                   
                 CGTAAACTCGACCCGACGCGTCCGATCACCTGCGTCAATGTAATGTTCTGCGACGCTCACACCGATACCATC 
               
               
                   
                 AGCGATCTCTTTGATGTGCTGTGCCTGAACCGTTATTACGGATGGTATGTCCAAAGCGGCGATTTGGAAACG 
               
               
                   
                 GCAGAGAAGGTACTGGAAAAAGAACTTCTGGCCTGGCAGGAGAAACTGCATCAGCCGATTATCATCACCGAA 
               
               
                   
                 TACGGCGTGGATACGTTAGCCGGGCTGCACTCAATGTACACCGACATGTGGAGTGAAGAGTATCAGTGTGCA 
               
               
                   
                 TGGCTGGATATGTATCACCGCGTCTTTGATCGCGTCAGCGCCGTCGTCGGTGAACAGGTATGGAATTTCGCC 
               
               
                   
                 GATTTTGCGACCTCGCAAGGCATATTGCGCGTTGGCGGTAACAAGAAAGGGATCTTCACTCGCGACCGCAAA 
               
               
                   
                 CCGAAGTCGGCGGCTTTTCTGCTGCAAAAACGCTGGACTGGCATGAACTTCGGTGAAAAACCGCAGCAGGGA 
               
               
                   
                 GGCAAACAATGA 
               
               
                   
               
               
                  2 
                 GAGAGACATATGTTACGTCCTGTAGAAACCCC 
               
               
                   
               
               
                  3 
                 GAGAGAAAGCTTTCATTGTTTGCCTCCCTGCT 
               
               
                   
               
               
                  4 
                 CTGCTGTCGGCTTTAACCTC 
               
               
                   
               
               
                  5 
                 GACCTCGCAAAGCATATTGCG 
               
               
                   
               
               
                  6 
                 CGCAATATGCTTTGCGAGGTC 
               
               
                   
               
               
                  7 
                 CTAGTTATTGCTCAGCGGT 
               
               
                   
               
               
                  8 
                 GCGACCTCGCAAACCATATTGCGC 
               
               
                   
               
               
                  9 
                 GCGCAATATGGTTTGCGAGGTCGC 
               
               
                   
               
               
                 10 
                 CAAACAAGGCCTATGCTAGAAAGGAGAAGCTTG 
               
               
                   
               
               
                 11 
                 CAAGCTTCTCCTTTCTAGCATAGGCCTTGTTTG 
               
               
                   
               
               
                 12 
                 TCTTTAGGCATTGGTCCGAAAGAACTGTAC 
               
               
                   
               
               
                 13 
                 GTACAGTTCTTTCGGACCAATGCCTAAAGA 
               
               
                   
               
               
                 14 
                 TCTTTAGGCATTGGTGAAGAGGCAGTCAAC 
               
               
                   
               
               
                 15 
                 GTTGACTGCCTCTTCACCAATGCCTAAAGA 
               
               
                   
               
               
                 16 
                 CCGCAGTTCTTCAACGGGGAAACTCAG 
               
               
                   
               
               
                 17 
                 GAAGAACTGCGGACCAATGCCTAAAGAG 
               
               
                   
               
               
                 18 
                 Wild type  E. coli  K12 BGUS full length amino acid sequence (FIG. 1) 
               
               
                   
               
               
                 19 
                 K1S mutant full length amino acid sequence (FIG. 1) 
               
               
                   
               
               
                 20 
                 K1T mutant full length amino acid sequence (FIG. 1) 
               
               
                   
               
               
                 21 
                 K3 mutant full length amino acid sequence (FIG. 1) 
               
               
                   
               
               
                 22 
                 K3Δ1 mutant full length amino acid sequence (FIG. 1) 
               
               
                   
               
               
                 23 
                 K3Δ2 mutant full length amino acid sequence (FIG. 1) 
               
               
                   
               
               
                 24 
                 K3Δ2S mutant full length amino acid sequence (FIG. 1) 
               
               
                   
               
               
                 25 
                   E. coli  K3 mutant partial amino acid sequence, residues 332-416 (FIG. 2) 
               
               
                   
               
               
                 26 
                 Abalone BGUS partial wild-type amino acid sequence (FIG. 2) 
               
               
                   
               
               
                 27 
                 Human BGUS partial wild-type amino acid seqeuence (FIG. 2) 
               
               
                   
               
               
                 28 
                   Lactobacillus brevis  BGUS partial wild-type amino acid sequence (FIG. 2) 
               
               
                   
               
               
                 29 
                   Staphylococcus sp.  RLH1 partial wild-type amino acid sequence (FIG. 2) 
               
               
                   
               
               
                 30 
                   E. coli  K3 mutant partial amino acid sequence, residues 452-606 (FIG. 3) 
               
               
                   
               
               
                 31 
                 Abalone BGUS partial wild-type amino acid sequence (FIG. 3) 
               
               
                   
               
               
                 32 
                 Human BGUS partial wild-type amino acid seqeuence (FIG. 3) 
               
               
                   
               
               
                 33 
                   Lactobacillus brevis  BGUS partial wild-type amino acid sequence (FIG. 3) 
               
               
                   
               
               
                 34 
                   Staphylococcus sp.  RLH1 partial wild-type amino acid sequence (FIG. 3) 
               
               
                   
               
               
                 35 
                 Abalone BGUS partial mutant amino acid sequence Ab1 (FIG. 9) 
               
               
                   
               
               
                 36 
                 Abalone BGUS partial mutant amino acid sequence Ab 2 (FIG. 9) 
               
               
                   
               
               
                 37 
                 Human BGUS partial mutant amino acid sequence Hu1 (FIG. 9) 
               
               
                   
               
               
                 38 
                 Human BGUS partial mutant amino acid sequence Hu 2 (FIG. 9) 
               
               
                   
               
               
                 39 
                 Human BGUS wild type amino acid sequence 
               
               
                   
                 margsavawaalgpllwgcalglqggmlypqespsreckeldglwsfradfsdnrrrgfeeqwyrrplwesg 
               
               
                   
                 ptvdmpvpssfndisqdwrlrhfvgwvwyerevilperwtqdlrtrvvlrigsahsyaivwvngvdtleheg 
               
               
                   
                 gylpfeadisnlvqvgplpsrlritiainntltpttlppgtiqyltdtskypkgyfvqntyfdffnyaglqr 
               
               
                   
                 svllyttpttyidditvttsveqdsglvnyqisvkgsnlfklevrlldaenkvvangtgtqgqlkvpgvslw 
               
               
                   
                 wpylmherpaylyslevqltaqtslgpvsdfytlpvgirtvavtksqflingkpfyfhgvnkhedadirgkg 
               
               
                   
                 fdwpllvkdfnllrwlganafrtshypyaeevmqmcdrygivvidecpgvglalpqffnnvslhhhmqvmee 
               
               
                   
                 vvrrdknhpavvmwsvanepashlesagyylkmviahtksldpsrpvtfvsnsnyaadkgapyvdviclnsy 
               
               
                   
                 yswyhdyghleliqlqlatqfenwykkyqkpiiqseygaetiagfhqdpplmfteeyqkslleqyhlgldqk 
               
               
                   
                 rrkyvvgeliwnfadfmteqsptrvlgnkkgiftrqrqpksaafllrerywkianetryphsvaksqclens 
               
               
                   
                 lft 
               
               
                   
               
               
                 40 
                 Mouse BGUS wild type amino acid sequence 
               
               
                   
                 mslkwsacwvalgqllcscalalkggmlfpkespsrelkaldglwhfradlsnnrlqgfeqqwyrqplresg 
               
               
                   
                 pvldmpvpssfnditqeaalrdfigwvwyereailprrwtqdtdmrvvlrinsahyyavvwvngihvveheg 
               
               
                   
                 ghlpfeadisklvqsgplttcritiainntltphtlppgtivyktdtsmypkgyfvqdtsfdffnyaglhrs 
               
               
                   
                 vvlyttpttyidditvitnveqdiglvtywisvqgsehfqlevqlldeggkvvahgtgnqgqlqvpsanlww 
               
               
                   
                 pylmhehpaymyslevkvtttesvtdyytlpigirtvavtkskflingkpfyfqgvnkhedsdirgkgfdwp 
               
               
                   
                 llvkdfnllrwlgansfrtshypyseevlqlcdrygivvidecpgvgivlpqsfgneslrhhlevmeelvrr 
               
               
                   
                 dknhpavvmwsvanepssalkpaayyfktlithtkaldltrpvtfvsnakydadlgapyvdvicvnsyfswy 
               
               
                   
                 hdyghleviqpqlnsqfenwykthqkpiiqseygadaipgihedpprmfseeyqkavlenyhsvldqkrkey 
               
               
                   
                 vvgeliwnfadfmtnqsplrvignkkgiftrqrqpktsafilrerywrianetgghgsgprtqcfgsrpftf 
               
               
                   
               
               
                 41 
                   Lactobacillus brevis  BGUS wild type amino acid sequence 
               
               
                   
                 mlypmetasrvvldlsgvwrfmidkeqipvdvtrplpatlsmavpasfndqtaskeirehvgyvwyercfel 
               
               
                   
                 pqllrgerlvlrfgsatheawvylnghlithhkggftpfeveinddlvtgenrltvklsnmldyttlpvghy 
               
               
                   
                 ketqnetgqrvrqldenfdffnyaglqrpvkiystphsyirditltpkvnltnhsavvngeietvgdveqvv 
               
               
                   
                 vtildednqvvgttsgktlaielnsvhlwqpgkaylyrakvelyqagqvidtyietfgirqiavkagkflin 
               
               
                   
                 gqpfyfkgfgkhedayihgrglsepqnvldlslmkqmgansfrtshypyseemmrlcdregivvidevpavg 
               
               
                   
                 lmlsftfdvsalekddfeddtweklrtaeahrqaitemidrdknhasvvmwsisneaanfskgayeyfkplf 
               
               
                   
                 dlarkldpqqrpctstsimmttlktdrclaladvialnryygwymgngdlkaaetatreellayqakfpdkp 
               
               
                   
                 imyteygadtiaglhsnydepfseefqedyyrmcsrvfdevtnfvgeqlwnfadfqtkfgiqrgqgnkkgif 
               
               
                   
                 trarepkmvvryltqrwrnipdfnykk 
               
               
                   
               
               
                 42 
                   Staphylococcus   sp . RLH1 BGUS wild type amino acid sequence 
               
               
                   
                 mlypintetrgvfdlngvwnfkldygkgleekwyeskltdtismavpssyndigvtkeirnhigyvwyeref 
               
               
                   
                 tvpaylkdqrivlrfgsathkaivyvngelvvehkggflpfeaeinnslrdgmnrvtvavdnilddstlpvg 
               
               
                   
                 lyserheeglgkvirnkpnfdffnyaglhrpvkiyttpftyvedisvvtdfngptgtvtytvdfqgkaetvk 
               
               
                   
                 vsvvdeegkvvasteglsgnveipnvilweplntylyqikvelvndgltidvyeepfgvrtvevndgkflin 
               
               
                   
                 nkpfyfkgfgkhedtpingrgfneasnvmdfnilkwigansfrtahypyseelmrladreglvvidetpavg 
               
               
                   
                 vhlnfmattglgegservstwekirtfehhqdvlrelvsrdknhpsvvmwsianeaateeegayeyfkplve 
               
               
                   
                 ltkeldpqkrpvtivlfvmatpetdkvaelidvialnryngwyfdggdleaakvhlrqefhawnkrcpgkpi 
               
               
                   
                 miteygadtvagfhdidpvmfteeyqveyyqanhvvfdefenfvgeqawnfadfatsqgvmrvqgnkkgvft 
               
               
                   
                 rdrkpklaahvfrerwtnipdfgykn 
               
               
                   
               
               
                 43 
                 GCAAAATCGGCGAAATTC 
               
               
                   
               
               
                 44 
                 GACCTCGCAA GCA ATATTGCGCGTTG 
               
               
                   
               
               
                 45 
                 GACCTCGCAA TGT ATATTGCGCGTTG 
               
               
                   
               
               
                 46 
                 GACCTCGCAA GAT ATATTGCGCGTTG 
               
               
                   
               
               
                 47 
                 GACCTCGCAA GAA ATATTGCGCGTTG 
               
               
                   
               
               
                 48 
                 GACCTCGCAA TTC ATATTGCGCGTTG 
               
               
                   
               
               
                 49 
                 GACCTCGCAA CAT ATATTGCGCGTTG 
               
               
                   
               
               
                 50 
                 GACCTCGCAA ATC ATATTGCGCGTTG 
               
               
                   
               
               
                 51 
                 GACCTCGCAA AAA ATATTGCGCGTTG 
               
               
                   
               
               
                 52 
                 GACCTCGCAA CTG ATATTGCGCGTTG 
               
               
                   
               
               
                 53 
                 GACCTCGCAA ATG ATATTGCGCGTTG 
               
               
                   
               
               
                 54 
                 GACCTCGCAA AAC ATATTGCGCGTTG 
               
               
                   
               
               
                 55 
                 GACCTCGCAA CCG ATATTGCGCGTTG 
               
               
                   
               
               
                 56 
                 GACCTCGCAA CAG ATATTGCGCGTTG 
               
               
                   
               
               
                 57 
                 GACCTCGCAA CGT ATATTGCGCGTTG 
               
               
                   
               
               
                 58 
                 GACCTCGCAA GTG ATATTGCGCGTTG 
               
               
                   
               
               
                 59 
                 GACCTCGCAA TGG ATATTGCGCGTTG 
               
               
                   
               
               
                 60 
                 GACCTCGCAA TAC ATATTGCGCGTTG 
               
               
                   
               
               
                 61 
                   E. coli  K12 BGUS G559H mutant amino acid sequence 
               
               
                   
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwygre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgeqflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   h   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq 
               
               
                   
               
               
                 62 
                   E. coli  K12 BGUS G559H + GLC C-terminal mutant amino acid sequence 
               
               
                   
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwygre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgegflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   h   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   glc     
               
               
                   
               
               
                 63 
                   E. coli  K12 BGUS G559H + GLCGR C-terminal mutant amino acid sequence 
               
               
                   
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwyqre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgegflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   h   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   glcgr     
               
               
                   
               
               
                 64 
                   E. coli  K12 BGUS G559N mutant amino acid sequence 
               
               
                   
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwyqre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgegflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   n   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq 
               
               
                   
               
               
                 65 
                   E. coli  K12 BGUS G559N + GLC C-terminal mutant amino acid sequence 
               
               
                   
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwyqre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgeqflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   n   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   glc     
               
               
                   
               
               
                 66 
                   E. coli  K12 BGUS G559N + GLCGR C-terminal mutant amino acid sequence 
               
               
                   
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwyqre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritycvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgeqflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrysavvgeqvwnfadfatsq   n   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   glcgr     
               
               
                   
               
               
                 67 
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwyqre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritycvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgeqflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyahtdtisd 
               
               
                   
                 lfaplaeatrkldptrpitcvnvmfcdfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrysavvgeqvwnfadfatsq   s   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   sscxx     
               
               
                   
               
               
                 68 
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwyqre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgeqflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   s   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   eecxx     
               
               
                   
               
               
                 69 
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwygre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgeqflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   s   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqg 
               
               
                   
                 gkq   kkcxx     
               
               
                   
               
               
                 70 
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwyqre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgeqflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   s   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   aacxx     
               
               
                   
               
               
                 71 
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwygre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgeqflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   s   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   wwcxx     
               
               
                   
               
               
                 72 
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwygre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgeqflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   s   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   ppcxx     
               
               
                   
               
               
                 73 
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwygre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgeqflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   s   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   glgcxx     
               
               
                   
               
               
                 74 
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwyqre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgeqflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   s   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   glggcxx     
               
               
                   
               
               
                 75 
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwyqre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgegflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   s   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   glgggcxx     
               
               
                   
               
               
                 76 
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwyqre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgeqflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   s   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   glggggcxx     
               
               
                   
               
               
                 77 
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwyqre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgeqflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   s   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   glgggggcxx     
               
               
                   
               
               
                 78 
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwygre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgeqflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   s   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   glggggggcxx     
               
               
                   
               
               
                 79 
                 AGCAGCTGCGGCCGGTAGAAAGGAG 
               
               
                   
               
               
                 80 
                 GAAGAATGCGGCCGGTAGAAAGGAG 
               
               
                   
               
               
                 81 
                 AAAAAATGCGGCCGGTAGAAAGGAG 
               
               
                   
               
               
                 82 
                 GCGGCGTGCGGCCGGTAGAAAGGAG 
               
               
                   
               
               
                 83 
                 TGGTGGTGCGGCCGGTAGAAAGGAG 
               
               
                   
               
               
                 84 
                 CCGCCGTGCGGCCGGTAGAAAGGAG 
               
               
                   
               
               
                 85 
                 TTGTTTGCCTCCCTGCTG 
               
               
                   
               
               
                 86 
                 GGCTGCGGCCGGTAGAAAGGAG 
               
               
                   
               
               
                 87 
                 GGCGGCTGCGGCCGGTAGAAAGGAG 
               
               
                   
               
               
                 88 
                 GGCGGCGGCTGCGGCCGGTAGAAAGGAG 
               
               
                   
               
               
                 89 
                 GGCGGCGGCGGCTGCGGCCGGTAGAAAGGAG 
               
               
                   
               
               
                 90 
                 GGCGGCGGCGGCGGCTGCGGCCGGTAGAAAGGAG 
               
               
                   
               
               
                 91 
                 GGCGGCGGCGGCGGCGGCTGCGGCCGGTAGAAAGGAG 
               
               
                   
               
               
                 92 
                 TAGGCCTTGTTTGCCTCCC 
               
               
                   
               
               
                 93 
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwygre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgeqflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   z   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   x     2-8     cx     0-2 , 
               
               
                   
                 wherein z = s, t, h or n, and x = any a.a. 
               
               
                   
               
               
                 94 
                 mlrpvetptreikkldglwafsldrencgidqrwwesalqesraiavpgsfndqfadadirnyagnvwygre 
               
               
                   
                 vfipkgwagqrivlrfdavthygkvwvnnqevmehqggytpfeadvtpyviagksvritvcvnnelnwqtip 
               
               
                   
                 pgmvitdengkkkqsyfhdffnyagihrsvmlyttpntwvdditvvthvaqdcnhasvdwqvvangdvsvel 
               
               
                   
                 rdadqqvvatgqgtsgtlqvvnphlwqpgegylyelcvtaksqtecdiyplrvgirsvavkgegflinhkpf 
               
               
                   
                 yftgfgrhedadlrgkgfdnvlmvhdhalmdwigansyrtshypyaeemldwadehgivvidetaavgfnls 
               
               
                   
                 lgigfeagnkpkelyseeavngetqqahlqaikeliardknhpsvvmwsianepdtrpqgareyfaplaeat 
               
               
                   
                 rkldptrpitcvnvmfcdahtdtisdlfdvlclnryygwyvqsgdletaekvlekellawqeklhqpiiite 
               
               
                   
                 ygvdtlaglhsmytdmwseeyqcawldmyhrvfdrvsavvgeqvwnfadfatsq   s   ilrvggnkkgiftrdrk 
               
               
                   
                 pksaafllqkrwtgmnfgekpqqggkq   glcgr     
               
               
                   
               
               
                 95 
                 Xaa 2-8 -Cys-Xaa 0-2 , wherein Xaa = any amino acid 
               
               
                   
               
               
                 96 
                 glcgr