Abstract:
The present invention relates to novel lactose phosphorylase enzymes and the uses thereof. More specifically, the invention relates to lactose phosphorylase enzymes created by mutation of a cellobiose phosphorylase from  Cellulomonas uda . By introducing mutations in this enzyme, the activity can be switched from cellobiose phosphorylase into lactose phosphorylase.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/EP2008/068076, filed Dec. 19, 2008, published in English as International Patent Publication WO 2009/080774 A1 on Jul. 2, 2009, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 07150211.6, filed Dec. 20, 2007, the entire disclosure of each of which is hereby incorporated herein by this reference. 
     TECHNICAL FIELD 
     The present invention relates to novel lactose phosphorylase enzymes and the uses thereof. More specifically, the invention relates to lactose phosphorylase enzymes created by mutation of a cellobiose phosphorylase from  Cellulomonas uda . By introducing mutations in this enzyme, the activity can be switched from cellobiose phosphorylase into lactose phosphorylase. 
     BACKGROUND 
     Monosaccharides that carry an α-linked phosphate group are key intermediates in the Leloir pathway for the synthesis of glycosidic linkages. Indeed, they can be converted into nucleotide sugars that are donor substrates for glycosyltransferases. In vivo, galactose-1-phosphate is converted into UDP-galactose by galactose-1-phosphate uridyl transferase (GALT). Absence of this enzyme results in the accumulation of toxic levels of galactose in the blood, a genetic disorder known as galactosemia (Fridovich-Keil, 2006). UDP-galactose, in turn, is the substrate for galactosyltransferases that are involved in the synthesis of a wide variety of important carbohydrate epitopes in glycoproteins and glycolipids (Varki, 1993). 
     Glycosyl phosphates have traditionally been synthesized by means of conventional chemical catalysis. Starting in 1937, several procedures using different catalysts and different glycosyl or phosphate donors have been described in the literature (Cori et al., 1937; MacDonald, 1961; Inage et al., 1982; Schmidt et al., 1982; Sim et al., 1993). More information can be found in patent EP0553297: “The preparation of glycosyl phosphate triesters.” Chemical phosphorylation of carbohydrates typically consists of multistep reaction schemes, resulting in a low overall yield, and is not very successful in achieving anomeric selectivity. The development of an enzymatic phosphorylation technology is consequently highly desirable, and has the additional benefit of reducing the amount of waste that is generated in the process (green chemistry). 
     Enzymes that phosphorylate monosaccharides belong to the class of kinases (phosphotransferases), which require ATP as phosphate donor. Most of these sugar kinases phosphorylate their substrate at the C6 position and not at the anomeric center. The only known exceptions are galactokinase (EC 2.7.1.6) that produces α-D-galactose-1-phosphate, and fucokinase (EC 2.7.1.52) that produces β-L-fucose-1-phosphate. Interestingly, the specificity of galactokinase has been broadened by means of directed evolution to include D-talose and L-glucose as substrates (Hoffmeister et al., 2003), and a patent describing the use of such galactokinase variants has been published: “Sugar kinases with expanded substrate specificity and their use” (WO2005056786). Although a kinase is available for the production of α-D-galactose-1-phosphate, the need for the unstable and expensive ATP as a phosphate donor is a serious drawback for industrial applications. 
     In spite of their name, glycoside phosphorylases do not actually phosphorylate their substrate but, instead, catalyze the phosphorolysis of di- and polysaccharides to produce phosphorylated monosaccharides. These enzymes are highly attractive as biocatalysts because they only require anorganic phosphate as donor, and have long been used for the production of α-D-glucose-1-phosphate from maltodextrin (Griessler et al., 1996) or sucrose (Goedl et al., 2007). More information can be found in U.S. Pat. No. 6,764,841: “Production process of glucose-1-phosphate.” Unfortunately, the specificity of carbohydrate phosphorylases is very limited and only one enzyme is known to produce α-D-galactose-1-phosphate in Nature, i.e., the lacto-N-biose phosphorylase found in Bifidobacteria (Kitaoka et al., 2005). Lacto-N-biose I or β-D-galactosyl-(1,3)-N-acetyl-D-glucosamine is a structural component of oligosaccharides present only in human milk and is not easy to obtain in large quantities (Nishimoto and Kitaoka, 2007). 
     Japanese Patent No. 9224691 discloses the production of sugar phosphate, useful as food material, by reacting chitobiose or lactose with phosphoric acid in the presence of cellobiose phosphorylase from  Cellvibrio gilvus . Starting from lactose, α-D-galactose-1-phosphate is obtained. However, this is only a side activity of the cellobiose phosphorylase, and both long reaction times (48 hours) and the rather low yield make this enzyme, although scientifically interesting, unsuitable for an industrial use. 
     DISCLOSURE 
     Surprisingly, we found that the cellobiose phosphorylase from  Cellulomonas uda  also shows an activity towards lactose, with the production of α-D-galactose-1-phosphate. Even more surprisingly, mutants could be obtained with a strongly increased lactose phosphorylase activity. The mutants do have a specific activity that is at least ten times higher, preferably about 50 times higher, than the specific activity of wild-type  Cellvibrio gilvus.    
     A first aspect of the invention is a lactose phosphorylase enzyme with a specific activity of at least 0.05 units/mg, preferably at least 0.1 units/mg, more preferably 0.2 units/mg, even more preferably at least 0.25 units/mg, as measured on 200 mM lactose in 50 mM MES-buffer pH 6.6 at 37° C. One unit (U) is defined as the amount of enzyme that converts 1 μmole of substrate in 1 minute under these conditions. 
     Preferably, the lactose phosphorylase enzyme is obtained by mutation of a cellobiose phosphorylase. Preferably, the mutation is situated in the region 300-750, even more preferably, in one of the regions 335-375, 395-435, 475-515 and 640-680 of the  Cellulomonas uda  sequence, or in an equivalent region of a homologous enzyme. A “homologous enzyme,” as used herein, is an enzyme with cellobiose phosphorylase activity, and at least 40% identity, preferably 50% identity, more preferably 60% identity, more preferably 70% identity, even more preferably 80% identity, and most preferably 90% identity with the  Cellulomonas uda  sequence, as measured in a BLAST alignment (Tatusova and Madden, 1999). An “equivalent region,” as used herein, means that the region can be identified by an alignment of both sequences, on the base of the conserved residues, by a BLAST alignment (Tatusova and Madden, 1999). Preferably, the lactose phosphorylase is obtained by mutation of the cellobiose phosphorylase of  Cellulomonas uda . More preferably, the lactose phosphorylase enzyme comprises at least the mutation A397 and/or the mutation T508 and/or the mutation N667 of SEQ ID NO:1 ( C. uda  enzyme) or the equivalent mutation in a homologous enzyme, even more preferably, the mutations are selected from the group consisting of A397R, T508A, T5081, N667T and N667A, even more preferably, the enzyme comprises SEQ ID NO:2 (mutant sequence 1), SEQ ID NO:3 (mutant sequence 2) or SEQ ID NO:4 (mutant sequence 3). In one preferred embodiment, the enzyme consists of SEQ ID NO:2. In another preferred embodiment, the enzyme consists of SEQ ID NO:3. In still another preferred embodiment, the enzyme consists of SEQ ID NO:4. 
     Another aspect of the invention is a nucleic acid sequence encoding a lactose phosphorylase enzyme according to the invention. A “nucleic acid,” as used herein, includes, but is not limited to, a DNA sequence, a cDNA sequence or an RNA sequence. Such a nucleic acid sequence can be used, as a non-limiting example, for overproduction of the enzyme in a homologous host organism or for heterologous production of the enzyme in an organism other than  Cellulomonas uda.    
     Another aspect of the invention is a mutant cellobiose phosphorylase enzyme, with increased lactose phosphorylase activity compared to the wild-type enzyme. Lactose phosphorylase activity is measured as described in materials and methods to the examples; preferably, the activity is expressed as specific activity. Preferably, the lactose phosphorylase activity is increased by at least a factor 3, more preferably, by at least a factor 5, even more preferably, by at least a factor 7, and most preferably, by at least a factor 10, compared to the wild-type activity. A “mutant enzyme,” as used herein, is an enzyme whereby at least one amino acid residue is replaced, deleted and/or inserted in the wild-type sequence. Preferably, the mutation is an amino acid replacement, even more preferably, the mutation is a replacement of at least two amino acids, and most preferably, it is a replacement of at least three amino acids. 
     Another aspect of the invention is the use of a lactose phosphorylase enzyme according to the invention for the production of galactose-1-phosphate. Still another aspect of the invention is the use of a lactose phosphorylase enzyme according to the invention for the production of lactose. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     EXAMPLES 
     Materials and Methods to the Examples 
     Bacterial Strains, Plasmids and Growth Conditions 
     The cellobiose phosphorylase gene (accession number AY343322) was cloned from  Cellulomonas uda  DSM20108. The organism was grown in Tryptone Soya Broth medium (TSB: 17 g/L tryptone, 3 g/L papaic digest of soybean meal, 2.5 g/L glucose, 2.5 g/L K 2 HPO 4 , 5 g/L NaCl) at 30° C. The pGEM-T plasmid (Promega) was used for cloning of the PCR fragments and was stored in  E. coli  DH5α. The pTrc99A plasmid (4177 bp, containing the IPTG-inducible trc promoter and an ampicillin resistance gene) was used for construction of the cellobiose phosphorylase expression vector. Ultracompetent  Escherichia coli  XL10-Gold cells (Stratagene) (TetrD(mcrA)183 D(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F′ proAB lacI q ZDM15 Tn10 (Tetr) Amy Cam r ]), were used for transformation with variant libraries. For plasmid isolation,  E. coli  was routinely grown overnight at 37° C. in LB medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, pH 7.0) supplemented with 100 mg/L ampicillin. For expression of recombinant cellobiose phosphorylase, the growth medium was supplemented with isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.1 mM.  E. coli  cells expressing improved enzyme variants were selected in minimal lactose medium (pH 7.4) composed of M9 salts (6 g/L Na 2 HPO 4 , 3 g/L KH 2 PO 4 , 1 g/L NH 4 Cl, 0.5 g/L NaCl), 20 mg/L proline, 0.1 mM CaCl 2 .2H 2 O, 1 mM MgSO 4 , 1 mM thiamine-HCl, 18 μM FeCl 2 .4H 2 O, 6.9 μM ZnCl 2 , 100 mg/L ampicillin, 0.1 mM IPTG and 1% (w/v) lactose. 
     Construction of the Cellobiose Phosphorylase Expression Vector 
       C. uda  was grown in TSB medium for 16 hours, after which genomic DNA was extracted with the GenElute Bacterial Genomic DNA kit (Sigma). The cellobiose phosphorylase gene was amplified from the genomic DNA using the High Fidelity PCR Master kit (Roche) with primers containing restriction sites: 
     
       
         
               
             
               
               
             
           
               
                 forward primer 
               
             
          
           
               
                 5′- AACGTT ACGGGCACTTCGACGAC-3′; 
                 (SEQ ID NO: 6) 
               
               
                   
               
               
                 reverse primer 
                   
               
               
                 5′-ATT CTGCAG CTAGAGGGTCACGTCGACGC-3′. 
                 (SEQ ID NO: 7) 
               
             
          
         
       
     
     The Psp1406I and PstI restriction sites are underlined in the forward and reverse primers, respectively. DMSO was added to a final concentration of 5% to improve amplification from the GC-rich template. The following PCR cycling conditions were used: 95° C. (10 minutes); 35 cycles of 94° C. (1 minute), 62° C. (1 minute) and 72° C. (3 minutes); 72° C. (7 minutes). A 2477 bp fragment containing the full-length cellobiose phosphorylase gene was thus obtained and ligated into the pGEM-T vector. The resulting plasmid was named pGCP and was used to construct an expression vector for cellobiose phosphorylase. After digestion of 5.3 μg of the pGCP plasmid with 10 units of Psp14061 (Roche) restriction enzyme, the 3531 bp fragment was blunted with 3 units of Klenow polymerase (Roche). The resulting fragment was cut with 10 units of PstI restriction enzyme and the 2467 by used for ligation with the expression vector. 3.1 μg of the pTrc99A expression vector was cut with 10 units of NcoI and the restriction fragment was blunted with 3 units of Klenow polymerase. The resulting fragment was cut with PstI and the 4132 bp fragment used for ligation with the 2467 bp fragment containing the cellobiose phosphorylase gene. The ligation reaction consisted of 63 ng of the 2467 by fragment, 44 ng of the 4132 bp fragment, 5 units T4 DNA polymerase (Fermentas) and 5% PEG4000. After overnight incubation at 22° C.,  E. coli  was transformed with the ligation mixture and plated on LB medium supplemented with ampicillin. The cellobiose phosphorylase expression vector obtained after plasmid extraction was named pXCP. 
     Mutagenesis Methods 
     Random mutagenesis of the cellobiose phosphorylase gene was performed with the GeneMorph II EZClone Domain Mutagenesis Kit (Stratagene) according to the manufacturer&#39;s instructions. Forty nanograms of the pXCP expression vector was used as template and error-prone PCR was performed with the following PCR primers: 
     
       
         
               
             
               
               
             
               
             
               
               
             
           
               
                 (SEQ ID NO: 8) 
               
             
          
           
               
                 5′-CGTTCGTCGGCGCGTACAACTC-3′ 
                 (CPmutF1, forward) 
               
               
                   
               
             
          
           
               
                 (SEQ ID NO: 9) 
               
             
          
           
               
                 5′-ACGACGAGCCCGTCGTACTCC-3′ 
                 (CPmutR1, reverse). 
               
             
          
         
       
     
     DMSO (1% final concentration) was added to the PCR reaction mixture to improve amplification from the GC-rich template. PCR cycling conditions were as follows: 95° C. (2 minutes); 35 cycles of 95° C. (45 seconds), 65° C. (45 seconds) and 72° C. (2 minutes); 72° C. (10 minutes). The 1624 by PCR fragment was gel-purified and used for the so-called EZClone reaction in which the mutated genes are cloned into the pXCP expression vector by whole-plasmid PCR. The purified PCR fragment was used as megaprimer (500 ng) and wild-type pXCP vector (50 ng) as template. PCR cycling conditions were as follows: 95° C. (1 minute); 30 cycles of 95° C. (50 seconds), 60° C. (50 seconds) and 68° C. (14 minutes). After the reaction, 10 units of DpnI restriction enzyme were added to the reaction mixture and incubated overnight at 37° C. to completely digest parental template DNA. The DpnI-treated PCR mixture was transformed into  E. coli  XL10-Gold and the transformation mixture was plated on LB medium containing ampicillin. Several colonies were picked and sequenced to determine the mutagenesis rate. 
     Site-directed and site-saturation mutagenesis was performed with the QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer&#39;s instructions. The primers contained the appropriate codon for mutagenesis (NNS for saturation), and PCR cycling conditions were as follows: 95° C. (3 minutes); 30 cycles of 95° C. (1 minute), 55° C. (1 minute) and 65° C. (14 minutes). After the reaction, 10 units of DpnI restriction enzyme were added to the reaction mixture and incubated overnight at 37° C. to completely digest parental template DNA. The PCR mixture was transformed into  E. coli  XL10-Gold and the transformation mixture was plated on LB medium containing ampicillin. Several colonies were picked and sequenced to identify the mutated plasmids. 
     Selection for Lactose Phosphorylase Enzyme Variants 
     The mutant DNA library was transformed into  E. coli  XL10-Gold cells and the transformation mixture inoculated in 20 mL LB medium supplemented with 100 mg/L ampicillin. After six hours of growth, IPTG (0.1 mM final concentration) and lactose (1% final concentration) were added and the culture was grown for another 16 hours at 30° C. The culture was then washed with phosphate buffered saline (PBS, 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na 2 HPO 4 , 0.25 g/L KH 2 PO 4 ; pH 7.4) and inoculated (0.25%) in 50 mL lactose minimal medium supplemented with ampicillin and IPTG. The selection culture was grown at 37° C. to an OD 600  of about 1, after which a fresh selection culture was started by inoculation (2%) of the grown culture in 20 mL lactose minimal medium supplemented with ampicillin and IPTG. After four such cycles, an aliquot of the culture was plated on LB medium supplemented with ampicillin. Several colonies were picked and sequenced to identify the mutations. 
     Screening for Lactose Phosphorylase Enzyme Variants 
     The mutant DNA library was transformed into  E. coli  XL10-Gold cells and the transformation mixture was plated on LB medium containing ampicillin. Colonies were picked with an automated colony-picker (QPix2, Genetix) and inoculated into 96-well flat-bottomed microtiter plates containing 175 μL LB medium per well, supplemented with ampicillin. The microtiter plates were incubated for 16 hours at 37° C. and 250 rpm. Recombinant enzyme expression was then induced by inoculation of the grown mini-cultures into new microtiter plates containing 175 μL LB medium per well, supplemented with ampicillin and 0.1 mM IPTG. After incubation for 16 hours at 37° C. and 250 rpm, the microtiter plates were centrifuged at 2500 rpm for 10 minutes, and the pellets frozen at −20° C. The pellets were lysed with 100 μL of lysis buffer composed of 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5% TRITON X-100® (nonionic surfactant), 4 mM MgCl 2 , 50 mM NaCl and 1 mg/mL lysozyme. Lysis was carried out for 30 minutes at 37° C. on a liquid handling robot (Freedom EVO 200, Tecan). After lysis, the plates were centrifuged at 3500 rpm for 10 minutes and the supernatants (crude cell extracts) were used for enzyme screening. Enzyme reactions were carried out at 37° C. in microtiter plates by mixing 30 μL crude cell extract with 170 μL, substrate solution (200 mM lactose and 30 mM KH 2 PO 4  in 50 mM Mes-buffer pH 6.6). After 1 hour incubation, 50 μL samples were taken to determine the amount of released glucose via the glucose oxidase/peroxidase assay (Trinder, 1969). 
     Enzymatic Characterization 
     For a thorough characterization of the wild-type enzyme or improved variant, the corresponding expression vector was used to transform  E. coli  XL10-Gold and the resulting transformant was picked and grown at 37° C. in LB medium supplemented with ampicillin. Expression of the recombinant enzyme was induced by adding IPTG to a final concentration of 0.1 mM when the optical density at 600 nm of the culture reached 0.6. After six hours of induction, the culture was centrifuged for 10 minutes at 15000 g and the pellet was frozen at −20° C. Crude cell extracts were prepared using the EasyLyse Bacterial Protein Extraction Solution (Epicentre). These cell extracts contain the phosphorylase enzyme that was used in an assay, either directly or in a purified form. 
     The purification was performed in 50 mM Tris-HCl buffer pH 7.5 with equipment from GE Healthcare, and consisted of a combination of anion-exchange (Q-SEPHAROSE FASTFLOW® (crosslinked polysaccharide polymer), 100-500 mM NaCl), gelfiltration (Superdex200, 300 mM NaCl) and hydrophobic interaction chromatography (OCTYL SEPHAROSE® (crosslinked polysaccharide polymer), 10 mM NaCl and 2.5% ammoniumsulphate). 
     Enzyme reactions were performed using 30 mM KH 2 PO 4  and either 30 mM cellobiose or 200 mM lactose in 50 mM MES-buffer pH 6.6 at 37° C. At regular intervals, samples were inactivated by boiling for five minutes and the released glucose was measured via the glucose oxidase/peroxidase assay (Trinder, 1969). One unit (U) of enzyme activity was defined as the amount of enzyme that converts 1 μmole of substrate in one minute under these conditions. 
     Cloning and Purification of his-Tagged Enzyme 
     For simplifying the purification, a His6-tag was inserted in the pXCP-vector between the first and second codon by means of PCR (using the primer sequence 5′-caggaaacagaccat gcaccatcaccatcaccat cgttacgggcacttcg-3′ (SEQ ID NO:10)) with the QuikChange XL site-directed mutagenesis kit from Stratagene (La Jolla, Calif., USA). The variant enzymes were cloned into this vector by conventional cloning procedures. The enzymes were purified with the IMAC QuickPick kit from Bio-Nobile Oy (Turku, Finland) according to the manufacturer&#39;s instructions. 
     Example 1 
     Expression and Characterization of the Wild-Type Cellobiose Phosphorylase 
     A cellobiose phosphorylase expression vector (pXCP) was successfully constructed by ligation of the cellobiose phosphorylase gene from  Cellulomonas uda  into a pTrc99A expression plasmid, as described in the materials and methods section. After transformation of  E. coli  XL10-Gold and induction, crude cell extracts were prepared by cell lysis and centrifugation. An activity of about 5 U and 0.02 U per mL of cell extract was obtained on 30 mM cellobiose and 200 mM lactose, respectively (pH 6.6 and 37° C.). 
     The wild-type enzyme was purified to electrophoretic homogeneity by a combination of chromatographic techniques. The first step was anion-exchange chromatography, in which the enzyme eluted with a salt concentration of 500 mM. Subsequent gelfiltration yielded an enzyme sample that was almost completely pure. Hydrophobic interaction chromatography was used as a final step, in which the enzyme did not bind to the column but eluted with 2.5% ammoniumsulphate. A specific activity of 12.66 U/mg and 0.0406 U/mg was obtained on 30 mM cellobiose and 200 mM lactose, respectively (pH 6.6 and 37° C.). 
     Example 2 
     Development of a Lactose Phosphorylase by Random Mutagenesis 
     The pXCP plasmid was used as template for random mutagenesis of the cellobiose phosphorylase gene via error-prone PCR. Using structural information from the homologous  Cellvibrio gilvus  cellobiose phosphorylase (Hidaka et al., 2006; PDB 2CQT), we restricted random mutagenesis to the residues between T216 and V757. This resulted in the amplification of a 1624 by DNA fragment that was cloned into the pXCP expression plasmid. The generated DNA library was used to transform  E. coli  XL10-Gold, which was plated on LB medium containing ampicillin to determine the mutation frequency of the library. An average mutation frequency of 6.5 DNA mutations per 1000 by was obtained. 
     In order to find enzyme variants with improved lactose phosphorylase activity, an in vivo selection system was developed. Since  E. coli  XL10-Gold does not express a β-galactosidase, only the cells that are transformed with an active lactose phosphorylase should be able to grow in minimal medium with lactose as the sole carbon source. Moreover, the cells expressing the highest lactose phosphorylase activity will grow fastest, meaning that the best enzyme present in the library can be selected using an enrichment culture. We performed four cycles of growth in lactose minimal medium and plated an aliquot of the culture on LB medium. Three colonies were picked and grown for plasmid extraction. Sequencing of the plasmids revealed that they all contained the same mutations, meaning that one variant was indeed enriched in the selection culture and this variant was called LP1. Nine DNA mutations were identified and these resulted in six amino acid substitutions (Table 1). 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Overview of mutations found in the LP1 variant obtained after 
               
               
                 selection 
               
             
          
           
               
                   
                   
                   
                 Distance to donor 
               
               
                   
                 DNA mutation 
                 Amino acid substitution 
                 (Å)* 
               
               
                   
               
               
                   
                 g735a 
                 — 
                 — 
               
               
                   
                 g930a 
                 — 
                 — 
               
               
                   
                 c1190t 
                 A397V 
                 23.7 
               
               
                   
                 c1410t 
                 — 
                 — 
               
               
                   
                 a1522g 
                 T508A 
                 17.1 
               
               
                   
                 g1534a 
                 A512T 
                 22.9 
               
               
                   
                 g1669a 
                 D557N 
                 26.5 
               
               
                   
                 a2000c 
                 N667T 
                 9.6 
               
               
                   
                 g2041a 
                 G681S 
                 26.8 
               
               
                   
               
               
                 *Distance from the Cα atom of the amino acid to the C1 atom of glycerol in the 3D structure of the homologous  C .  gilvus  cellobiose phosphorylase (PDB 2CQT) 
               
             
          
         
       
     
     After enzyme production and extraction, a five-fold increase in lactose phosphorylase activity could be detected for the LP1 variant while the cellobiose phosphorylase activity had decreased four-fold compared to the wild-type enzyme (Table 2). We investigated the importance of each amino acid substitution in the LP1 variant by reverting it to the wild-type amino acid. These experiments revealed that only T508A and N667T contribute to the improved activity on lactose. Interestingly, two of the other mutations were found to have a negative effect on lactose phosphorylase activity: A397V and G681S. Based on these results, we constructed the double mutant T508A/N667T, which was called LP2 and has about two times more lactose phosphorylase activity than the parent LP1 variant. The results are summarized in Table 2(a). 
     
       
         
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Activity of the recombinant enzymes (wild-type and mutants) in crude 
               
               
                 cell extracts (a), and as his-tagged purified enzyme (b) 
               
               
                   
               
             
             
               
                 (a) 
               
             
          
           
               
                   
                 Activity (U/mL cell extract) 
                 cellobiose 
                 lactose 
               
               
                   
               
               
                   
                 WT 
                 5.42 ± 0.31 
                 0.020 ± 0.002 
               
               
                   
                 LP1 
                 1.34 ± 0.21 
                 0.098 ± 0.006 
               
               
                   
                 LP2 
                 1.63 ± 0.27 
                 0.217 ± 0.007 
               
               
                   
                 LP3 
                 2.16 ± 0.23 
                 0.298 ± 0.006 
               
               
                   
               
             
          
           
               
                 (b) 
               
             
          
           
               
                   
                 Activity (U/mg pure enzyme) 
                 cellobiose 
                 lactose 
               
               
                   
               
               
                   
                 WT 
                 7.78 ± 0.34 
                 0.033 ± 0.003 
               
               
                   
                 LP1 
                 1.35 ± 0.11 
                 0.100 ± 0.008 
               
               
                   
                 LP2 
                 1.71 ± 0.13 
                 0.171 ± 0.014 
               
               
                   
                 LP3 
                 2.61 ± 0.21 
                 0.249 ± 0.017 
               
               
                   
               
             
          
         
       
     
     A His6-tag was inserted before the start codon of the genes, and the enzymes were purified with the IMAC QuickPick kit from Bio-Nobile Oy (Turku, Finland) according to the manufacturer&#39;s instructions. The specific activity of the pure his tagged enzymes were measured on 200 mM lactose, in 30 mM KH 2 PO 4 -50 mM MES buffer, pH 6.6, at 37° C. The specific activity of the LP2 double mutant was 0.171 units/mg, as compared with 0.033 units/mg for the purified  C. uda  wild-type enzyme (Table 2(b)). Activities on cellobiose are given as comparison. 
     The two mutant positions were at random mutated into any other amino acid, and the resulting combinations were screened on activity. One variant, T5081/N667A (indicated as LP3) showed about 50% higher activity than the LP2 variant, as measured both on crude extract (Table 2(a)) as on purified enzyme (Table 2(b)). 
     Example 3 
     Comparison of the Activity of the Mutant  Cellulomonas uda  Enzyme with the Activity of  Cellvibrio gilvus    
     The gene coding for the cellobiose phosphorylase from  C. gilvus  (SEQ ID NO:5) was synthesized by Genscript (Piscataway, N.J., USA). The cloning, expression, mutagenesis, extraction and measurement of the enzymatic activity was performed as described for enzyme from  Cellulomonas uda . The results are summarized in Table 3. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 analysis of the activity of cellobiose phosphorylase of 
               
               
                   Cellvibrio gilvus  (wt and T508I mutant) on cellobiose and lactose 
               
             
          
           
               
                   
                 activity 
                   
                   
               
               
                   
                 (U/mg crude extract) 
                 cellobiose 
                 lactose 
               
               
                   
               
               
                   
                 Wild-type 
                 1.046 ± 0.052 
                 0.0036 ± 0.0001 
               
               
                   
                 T508I 
                 0.699 ± 0.029 
                 0.0044 ± 0.0002 
               
               
                   
                 N667A 
                 0.891 ± 0.027 
                 0.0041 ± 0.0002 
               
               
                   
                 T508I + N667A 
                 0.529 ± 0.020 
                 0.0050 ± 0.0003 
               
               
                   
               
             
          
         
       
     
     In order to evaluate the effect of mutations at positions T508 and N667 (equivalent of the position T508, respectively, the position N667 in  Cellulomonas uda ) of the  Cellvibrio gilvus  enzyme, the T at position 508 was replaced by I, and the N at position 667 was replaced by A in a similar way as described for  Cellulomonas uda . Similar to what was noticed in  Cellulomonas uda , introducing a mutation at these positions increased the activity, but the resulting activity for both the mutations and for the double mutation was still significantly lower than was obtained for  Cellulomonas uda.    
     Example 4 
     Optimization of the Lactose Phosphorylase Activity 
     The wild-type cellobiose phosphorylase from  Cellulomonas uda  displays little activity on 200 mM lactose at pH 6.6 and 37° C. (although far higher than the activity measured for  Cellvibrio gilvus ). The lactose phosphorylase activity has already been increased ten-fold by introducing the mutations N667T and T508A, but additional enzyme engineering will further improve the efficiency of the production of α-D-galactose-1-phosphate. 
     Libraries of variant enzymes are produced by both random mutagenesis and site-saturation mutagenesis, as described in the materials and methods section. For the random mutagenesis, epPCR followed by selection in lactose minimal medium is used. For the site-saturation mutagenesis, active-site residues are targeted and the effect is evaluated by high-throughput screening. Variants with increased lactose phosphorylase activity are sequenced and the individual effect of all identified mutations is determined by means of site-directed mutagenesis. The beneficial mutations are pooled into one enzyme that is used as a starting point for a new round of directed evolution. 
     Example 5 
     Application of the Improved Enzyme Variants in Production Process 
     The best enzyme variant at the end of each cycle of directed evolution is produced at a larger scale and characterized more thoroughly. Its specific activity on 200 mM lactose and 30 mM phosphate in 50 mM MES buffer pH 6.6 at 37° C. is determined. Furthermore, its kinetic parameters and the optimal substrate concentration for maximal efficiency of α-D-galactose-1-phosphate production are determined. Because the cellobiose phosphorylase from  Cellvibrio gilvus  has been reported to be moderately active on lactose, a comparative analysis is performed with the improved enzyme variants. In those experiments, the test conditions described in Japanese Patent No. 9224691 are used: mixing 2 U/ml of enzyme with 10 mM lactose and 10 mM phosphate in Tris/HCL-buffer pH 7. After 48 hours of reaction at 37° C., 1.5 mM of α-galactose-1-phosphate was produced with the enzyme of  Cellvibrio gilvus.    
     Because the phosphorolysis of disaccharides constitutes a reversible reaction, a lactose phosphorylase is also useful for the enzymatic synthesis of lactose from α-galactose-1-phosphate and glucose. Such lactose is very interesting for the manufacture of pharmaceutical formulations since it is not derived from animal sources, and hence guaranteed BSE-free. The synthetic capacities of our improved variants are tested by mixing enzyme with substrate, inactivating samples at regular intervals by boiling for five minutes, and measuring the lactose concentration by means of HPLC. The specific activity on 200 mM α-D-galactose-1-phosphate and 30 mM glucose in 50 mM MES-buffer pH 6.6 at 37° C. is determined, as well as the kinetic parameters and the optimal substrate concentration for maximal efficiency of lactose production. 
     Example 6 
     Because residue 397 clearly influences the lactose phosphorylase activity of the LP1-variant (albeit negatively in the case of the mutation A397V), we decided to saturate this position in the LP3-variant. After screening one microtiter plate (96 colonies), we could indeed identify a variant with increased activity on lactose. This enzyme contains the mutation A397R and has a specific activity of 0.177±0.007 U/mg crude cell extract, compared to 0.161±0.006 U/mg crude cell extract for the LP3-variant. 
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