Patent Application: US-73502308-A

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:
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 . 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 : 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 . 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 : 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 . 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 . 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 , 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 ). 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 , 100 - 500 mm nacl ), gelfiltration ( superdex200 , 300 mm nacl ) and hydrophobic interaction chromatography ( octyl sepharose , 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 . 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 ′- caggaaacagaccatgcaccatcaccatcaccatcgttacgggcacttcg - 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 . 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 upper 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 .). 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 ). 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 ). 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 )). 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 . 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 . 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 . 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 . 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 . cori c . f ., s . p . colowick and g . t . cori ( 1937 ). the isolation and synthesis of glucose - 1 - phosphoric acid . j . biol . chem . 121 , 465 - 477 . fridovich - keil j . l . 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