Evolved -Galactosidases from Geobacillus stearothermophilus with

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2009, p. 6312–6321 0099-2240/09/$08.00⫹0 doi:10.1128/AEM.00714-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 75, No. 19

Evolved ␤-Galactosidases from Geobacillus stearothermophilus with Improved Transgalactosylation Yield for Galacto-Oligosaccharide Production䌤† Gae¨l Placier,1‡ Hildegard Watzlawick,1* Claude Rabiller,2 and Ralf Mattes1 Institut fu ¨r Industrielle Genetik, Universita ¨t Stuttgart, Allmandring 31, 70569 Stuttgart, Germany,1 and Biotechnologie, Biocatalyse, Biore´gulation (UMR CNRS-University U3B 6204), PRES Nantes-Angers-Le Mans University, 2 rue de la Houssinie`re, BP 92208, F-44322-Nantes, France2 Received 26 March 2009/Accepted 18 July 2009

A mutagenesis approach was applied to the ␤-galactosidase BgaB from Geobacillus stearothermophilus KVE39 in order to improve its enzymatic transglycosylation of lactose into oligosaccharides. A simple screening strategy, which was based on the reduction of the hydrolysis of a potential transglycosylation product (lactosucrose), provided mutant enzymes possessing improved synthetic properties for the autocondensation product from nitrophenyl-galactoside and galacto-oligosaccharides (GOS) from lactose. The effects of the mutations on enzyme activity and kinetics were determined. An change of one arginine to lysine (R109K) increased the oligosaccharide yield compared to that for the wild-type BgaB. Subsequently, saturation mutagenesis at this position demonstrated that valine and tryptophan further increased the transglycosylation performance of BgaB. During the transglycosylation reaction with lactose of the evolved ␤-galactosidases, a major trisaccharide was formed. Its structure was characterized as ␤-D-galactopyranosyl-(133)-␤-D-galactopyranosyl-(134)D-glucopyranoside (3ⴕ-galactosyl-lactose). At the lactose concentration of 18% (wt/vol), this trisaccharide was obtained in yields of 11.5% (wt/wt) with GP21 (BgaB R109K), 21% with GP637.2 (BgaB R109V), and only 2% with the wild-type BgaB enzyme. GP643.3 (BgaB R109W) was shown to be the most efficient mutant, with a 3ⴕ-galactosyl-lactose production of 23%. phile amino acid residue, was one attempt to improve the transglycosylation yield. Many glycosynthases which demonstrated improved glycoside synthesis have been reported (8, 10, 12). However, this approach requires knowledge of the catalytically acting nucleophile residues of the glycosidase studied. Furthermore, activated glycosyl donors (e.g., glycosyl fluoride substrates of the opposite anomeric conformation) are needed for their enzymatic reaction. Therefore, their potential for use in industrial processes for large-scale production of GOS seems low, because of their inactivity on natural substrates (e.g., lactose). In a second approach, directed-evolution strategies have been used to enhance transglycosidase activity. Using random mutagenesis and in vitro recombination, Feng et al. (10) were able to diminish the hydrolytic activity of ␤-glycosidase from Thermus thermophilus while significantly increasing the transglycosylation activity. This allowed them to synthesize oligosaccharides through transglycosylation reactions with nitrophenyl-␤-glycosides as donors and various glycosides as acceptors. The enhancement of the transglycosylation activities of ␤-glycosidases toward natural substrates such as lactose was achieved by active-site mutagenesis of ␤-glucosidase from Pyrococcus furiosus (13) or by using a truncated ␤-galactosidase from Bifidobacterium bifidum (19). The application of thermostable ␤-galactosidases is of interest in the conversion of lactose, because at higher temperatures higher lactose concentration can be used, favoring GOS synthesis. Thus, we explored the use of the thermophilic ␤-galactosidase BgaB from Geobacillus stearothermophilus for transglycosylation reaction on lactose to produce specific GOS. BgaB, belonging to glycoside hydrolase family 42 (15), cloned in Escherichia coli is able to hydrolyze lactose, but its GOS

Galacto-oligosaccharides (GOS) are established prebiotic food ingredients and are used to enhance the growth of bifidobacteria and lactobacilli in the large intestine in order to reduce the growth of pathogenic microorganisms (3, 26, 27). The increased interest in these products by consumers heightened the need for good catalysts. Therefore, the development of an efficient and inexpensive GOS production method is highly desirable. One approach is to use lactose as a substrate for the preparation of prebiotic carbohydrates. Lactose is a low-value sugar comprising up to 75% of the total dry material in whey, and it accumulates in quantities of approximately 6 million tons annually worldwide (29). In several studies, GOS syntheses from lactose using different ␤-glycosidases had been reported (21). GOS are usually produced by the transglycosylation reaction during enzymatic hydrolysis of lactose. The proportion of transglycosylation to hydrolysis varies, depending on the different sources of enzymes (20, 22, 30, 31). In most cases yields of oligosaccharides are rather low; presumably the products are substrates for the enzyme and undergo hydrolysis. In order to overcome the hydrolysis problem, attempts to transform a glycosidase into a transglycosidase have been made. The generation of glycosynthases, which are mutants of glycosidases with a nonfunctional catalytically acting nucleo-

* Corresponding author. Mailing address: Institut fu ¨r Industrielle Genetik, Allmandring 31, 70569 Stuttgart, Germany. Phone: 49-711685-66981. Fax: 49-711-685-66973. E-mail: hildegard.watzlawick@iig .uni-stuttgart.de. ‡ Present address: Biotech Tools s.a., Rue de Ransbeek 230, 1120 Brussels, Belgium. † Supplemental material for this article may be found at http://aem .asm.org/. 䌤 Published ahead of print on 7 August 2009. 6312

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productivity is rather low. Thus, we aimed at increasing the synthetic yield of GOS from lactose exerted by the enzyme. Here we applied a random mutagenic approach together with a screening procedure to obtain mutants possessing higher transgalactosylation activity. MATERIALS AND METHODS Chemicals. Chemicals were purchased from the following companies: ampicillin (Ap) from Biomol Feinbiochemica, Hamburg; protein assay reagent from Bio-Rad Laboratories GmbH, Munich; Bacto agar, yeast extract, and tryptone from Difco Laboratories, Detroit, MI; p-nitrophenyl-␤-D-galactopyranoside (pNP-␤Gal) from Sigma Chemical Co., St. Louis, MO; 5-bromo-4-chloro-3indolyl-␤-D-galactoside (X-Gal) from Carl Roth GmbH; galactose dehydrogenase from Roche; N-methyl-N⬘-nitro-N-nitrosoguanidine (MNNG) from Aldrich; and lactosucrose [4⬘-galactosyl-sucrose, ␤-D-galactopyranosyl-(134)-Dsucrose] and authentic samples of 3⬘-galactosyl-lactose [␤-D-galactopyranosyl(133)-D-lactose] and 4⬘-galactosyl-lactose [␤-D-galactopyranosyl-(134)-D-lactose] from Deutsche Su ¨dzucker AG. All other analytical grade chemicals and reagents were supplied by E. Merck AG, Darmstadt, Germany. Bacterial strains and growth conditions. E. coli JM109 [recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi ⌬(lac-proAB) F⬘ (traD36 proAB⫹ lacIq lacZ⌬M15)] (35) was used as host for expression of wild-type and mutant bgaB genes. The E. coli strain was grown in double yeast tryptone (dYT) medium at 37°C. When necessary, selective antibiotic was added. E. coli transformation was performed according to the transformation and storage solution method (6). Construction of expression plasmid. The bgaB gene was amplified by PCR from the plasmid pCG1 (11), which served as the template. The gene-specific primers S2747 (5⬘-AATACATATGAACGTTTTATC-3⬘) and S2827 (5⬘-GCAA GCTTCTAAACCTTCCCGGC-3⬘) were used in this reaction, which introduced an additional NdeI restriction site at the N-terminal bgaB sequence and a HindIII restriction site just behind the stop codon. After digestion with NdeI and partial digestion with HindIII, the 2,021-bp fragment was inserted into the L-rhamnose (L-Rha)-inducible expression vector pJOE2702 (34), which was cut with the appropriate restriction enzymes to create plasmid pHWG509. Random and directed mutagenesis. (i) MNNG mutagenesis. Random mutations were introduced by adding MNNG to an exponential culture (optical density at 600 nm of 0.5) of E. coli JM109 harboring pHWG509 to final concentrations of 0.1 to 0.3 mg ml⫺1. Following growth overnight at 34°C, plasmid DNA was isolated. (ii) Site-specific saturation mutagenesis at Arg109. For saturation mutagenesis of the codon BgaB109, the mutagenic oligonucleotide S3693 (5⬘-TTTGGA TCCNNNCAACATTATTGTCCTAA-3⬘) was designed to present a codon NNN at position BgaB109 and a silent mutation introducing a recognition site for the restriction enzyme BamHI. The mutated bgaB gene was cloned into the L-Rha-inducible expression vector as follows. Two fragments of the bgaB gene were amplified by PCR with pHWG509 as the template. Fragment A was amplified using the primers S2747 (contains the N-terminal bgaB sequence and an additional NdeI restriction site [see “Construction of expression plasmid” above]) and S3694 (5⬘-AAAAGGATCCAAACGAGAGAATG-3⬘), which introduced a new BamHI restriction site. Fragment B was obtained during amplification with the mutagenic oligonucleotide S3693 and S2827, which contains the C-terminal bgaB sequence (see “Construction of expression plasmid” above). Fragments A and B were then digested with NdeI/BamHI and BamHI/BstEII, respectively, and ligated in pHWG509 previously digested with NdeI/BstEII. E. coli JM109 was transformed with the DNA using the transformation and storage solution method. The resulting transformants were used for plasmid isolation. Plasmids containing the BamHI restriction site were selected and their bgaB sequence verified by sequencing. Screening procedure. E. coli JM109 transformed with random-mutagenized plasmid DNA was plated on MacConkey agar plates containing 1% lactosucrose (19.8 mM), Ap (0.1 mg ml⫺1), and Rha (0.05%). The plates were incubated overnight at 37°C. All colonies displaying a white phenotype were picked and streaked once more on MacConkey/lactosucrose agar plates and on dYT/Ap/ Rha agar plates. An X-Gal overlay assay was used to detect colonies producing active mutants. For this, 4 ml soft agar solution (1% in 10 mM potassium phosphate buffer, pH 7.0) containing 1% sodium dodecyl sulfate (SDS) and 300 ␮l of X-Gal (80 mg ml⫺1 in N,N-dimethylformamide) was spread over the colonies grown on dYT/Ap/Rha plates. The blue phenotype was observed at room temperature after 20 min of incubation. The phenotype of the selected clones was also examined on MacConkey agar plates containing 1% lactose (27.7 mM), 0.1 mg ml⫺1 Ap, and 0.05% Rha. For the detection of activity toward

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pNP-␤Gal, 4 ml soft agar solution (1%) in 10 mM potassium phosphate buffer (pH 7.0) containing 200 ␮l N,N-dimethylformamide and 800 ␮l pNP-␤Gal (40 mM) was poured on dYT/Ap/Rha agar plates and incubated at 37°C until a yellow color was visible (around 30 min). Production of BgaB wild-type protein and BgaB mutants. E. coli JM109 carrying the respective recombinant plasmid was grown in dYT containing Ap (0.1 mg ml⫺1). For expression of the gene, the culture was grown until the cell density reached an optical density at 600 nm of 0.3 and then induced by adding 0.1% Rha and further grown for 4 h at 30°C. The cells were harvested by centrifugation, washed, and resuspended in 100 mM potassium phosphate buffer, pH 6.5. The proteins of small cultures (5 ml) were extracted by sonication, while cells of larger culture volumes were disrupted by passing them twice through a French pressure cell. The crude cell extract was obtained from the supernatant following centrifugation at 12,000 ⫻ g for 15 min. Enzyme assays. For all ␤-galactosidase assays, 0.1 M potassium phosphate buffer pH 6.5 was employed. (i) Activity toward pNP-␤Gal substrate. Under standard conditions, ␤-galactosidase activity was determined by measuring the rate of pNP-␤Gal hydrolysis. The assay was performed at 37°C in 500 ␮l containing the proper amount of enzyme. After a preincubation period of 5 min for temperature adaptation, the reaction was started with 100 ␮l of pNP-␤Gal solution at 4 mg ml⫺1 (13.27 mM) and stopped after 2 min with 1 ml 0.4 M sodium borate (pH 9.8). The release of p-nitrophenol (pNP) was measured at 405 nm. One unit of enzyme activity was defined as the amount of enzyme catalyzing the liberation of 1 ␮mol of pNP per minute, using an extinction coefficients (ε405, pH 10) of 18.5 ⫻ 103 M⫺1 cm⫺1 for pNP. (ii) Activity toward lactose and lactosucrose substrates. The activity toward lactose was determined by measuring the release of glucose at 37°C. The reaction mixture (200 ␮l) contained 500 mM lactose and 30 ␮l crude extract. After the reaction proceeded, 30-␮l samples were withdrawn at different times, and the reaction was stopped by adding of 4 ␮l of 10% trichloroacetic acid. Precipitated proteins were removed by centrifugation. The amount of released glucose in the supernatant was determined with the glucose oxidase assay as described below. The enzyme activity test for the substrate lactosucrose was performed under similar conditions. The release of galactose was determined by quantitative high-performance liquid chromatography (HPLC) analysis as described below. (iii) Kinetic parameters. The apparent Michaelis constant (Km) and the maximum velocity (Vmax) were determined for each substrate by linear regression analysis of the data in a Hanes-Woolf plot. Lineweaver-Burk plots were used to demonstrate inhibitory effects at high substrate concentrations. The assays were carried out with various concentrations of pNP-␤Gal (0.25 to 20 mM), lactose (2 to 470 mM), or lactosucrose (2 to 100 mM). The amounts of reaction products formed were determined after stopping the reaction using the procedures described above, depending on the substrate used. Each data point was determined in triplicate, and in all cases the initial velocity was used for plotting. (iv) Optimum temperature and stability. The optimum temperature was determined with pNP-␤Gal at between 25°C and 80°C. The proper amounts of crude extract were preincubated for 15 min in 250 ␮l buffer for temperature adaptation, and then a solution of 250 ␮l pNP-␤Gal at 40 mM, also preincubated at the respective temperature, was added. The reaction was stopped with 1 ml 0.4 M sodium borate (pH 9.8). The release of pNP was measured as described for the standard assay with pNP-␤Gal. The stabilities of wild-type BgaB and mutants were determined at 37°C and 60°C as follows: 100 mU of each E. coli crude extract was incubated at either temperature, and aliquots were withdrawn at different times and used for determination of the remaining activity toward pNP-␤Gal substrate under standard conditions. (v) Unit definition. One unit was defined as the amount of enzyme that releases 1 ␮mol of pNP or glucose (from lactose) and galactose (from lactosucrose) per min. Specific activity was expressed as units per mg of protein. The protein quantity was determined by the method of Bradford (4) with bovine serum albumin as a standard. Transglycosylation reactions. All transglycosylation reactions were performed at 37°C. The buffer employed was 0.1 M potassium phosphate buffer, pH 6.5. For a qualitative view of the transglycosylation and hydrolysis products, the reaction mixture was separated by thin-layer chromatography (TLC). For the autocondensation reaction with pNP-␤Gal as the only substrate, the reaction mixtures (300 ␮l) contained 8.85 mM of pNP-␤Gal and 80 mU enzyme (determined under standard conditions). The reaction of the enzyme toward pNP-␤Gal in the presence of a natural acceptor (10 mM cellobiose) was performed in a similar manner. Reactions with lactose were performed in a 500-␮l mixture containing 350 mM lactose and 150 mU enzyme (determined with pNP-␤Gal substrate). Aliquots (4 ␮l) of the enzymatic reaction mixture were withdrawn at different

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times and spotted on TLC plates. Reaction mixtures with lactose were diluted 17.5-fold prior TLC analysis. The standard transglycosylation reaction with lactose was performed in a 400-␮l mixture containing 500 mM lactose (18% [wt/vol]) and 92 mU enzyme (activity determined with 500 mM lactose). Aliquots of 20 ␮l were withdrawn at different times, and the reaction was stopped by heat denaturation (5 min at 95°C). After centrifugation and appropriate dilution (1/100 to 1/5,000 dilution in H2O), the sample was separated by HPLC and individual sugars (residual lactose and trisaccharides) were quantified as described below. The amount of glucose released was determined by the glucose oxidase test. The amount of galactose was determined by an enzymatic test using galactose dehydrogenase. Sugar analysis. (i) TLC. For qualitative analysis of individual sugars, the reaction mixtures were separated by TLC on Silica Gel 60F254 plates (Merck). The migration phase used for pNP-␤Gal substrates was methanol-chloroformacetic acid-H2O (30:60:5:5, vol/vol/vol/vol). The different spots were visualized under UV light at 254 nm. Products of the transglycosylation reaction with lactose were separated with acetone-H2O (87:13, vol/vol). Mono- and oligosaccharides were visualized by spraying with a solution containing 10.5 g ammonium sulfate, 0.5 g cerium sulfate, and 15 ml concentrated sulfuric acid in 245 ml H2O and subsequent blue color development for 15 to 20 min at 80°C. (ii) HPLC. Quantitative analysis of individual sugars was performed by highperformance anion-exchange chromatography with pulsed amperometric detection. The HPLC apparatus consisted of a pump (2200; Bischoff, Leonberg, Germany) and an electrochemical detector (ESA Coulochem II; Bischoff, Leonberg, Germany). The sugars were separated on an RCX-10 column (Hamilton; 250 by 4 mm), using 0.57% sodium hydroxide solution containing 20 mM sodium acetate, pH 5.3, with a flow rate of 0.75 ml min⫺1. The eluted sugars were detected by pulsed amperometry with an analytical cell (ESA 5040; Bischoff, Leonberg, Germany). Individual sugars were identified by comparison of the retention times with those of standards and quantified from the peak area calibrated against sugar standards. (iii) Glucose determination. Glucose was determined by the glucose oxidase method (Granutest; E. Merck AG, Darmstadt, Germany) and quantified according to a glucose standard curve. (iv) Galactose determination. The amount of galactose was determined by coupling the formation of D-galactose to the reduction of NAD⫹ using galactose dehydrogenase. The standard assay solution contained 200 mM Tris-HCl (pH 8.6), 0.35 mM NAD⫹, and 0.05 unit of galactose dehydrogenase in a total volume of 1.0 ml. After 1 h of incubation at 25°C, the assays were read at 340 nm and the quantities of galactose were determined from the appropriate calibration curve determined under the experimental conditions of the assays. (v) Yield calculation. The relative yields of the major trisaccharide produced (3⬘-galactosyl-lactose) were calculated as final carbohydrate concentration (in %, wt/wt) as a percentage of the initial lactose concentration. Isolation of the transglycosylation products for NMR analysis. (i) Autocondensation product. The transglycosylation reaction (500-␮l mixture) was performed with 40 mM pNP-␤Gal and 100 mU of GP21. After a 1.5-h reaction, the reaction mixture was separated on a Biogel P2 (Bio-Rad) column (1 by 20 cm). Products were eluted with H2O. Samples of 250 ␮l were collected and analyzed by TLC as described above. Fractions containing the autocondensation product were pooled, and the substance was isolated by lyophilization. Elucidation of its regioisomer structure was done by means of combined one- and two-dimensional 1 H and 13C nuclear magnetic resonance (NMR) experiments, and it was identified as 4-nitrophenyl-␤-D-Gal-(133)-␤-D-Gal [pNP-␤Gal-(1,3)-Gal]. (ii) Trisaccharide from the reaction with lactose. The transglycosylation reaction (3-ml mixture) was performed with 1 M lactose and 1 U of GP643.3 (activity determined with lactose) for 24 h. The reaction was stopped by heat denaturation. Following centrifugation, the supernatant was applied to a Biogel P2 column (1.5 by 73 cm). Products were eluted with H2O, and their identity was checked by TLC. Fractions containing the trisaccharide were pooled and lyophilized. About 190 mg of powder was obtained from 1.02 g of lactose (⬃ 18.6% [wt/wt]). Its purity was analyzed by HPLC. The elucidation of the structure was done by means of combined one- and two-dimensional 1H- and 13C-NMR experiments, and it was identified as ␤-D-galactopyranosyl-(133)-␤-D-galactopyranosyl-(134)-D-glucopyranoside (3⬘-galactosyl-lactose). NMR spectroscopy and structure elucidation. Analysis of the NMR 1H and 13 C resonances and subsequent structure assignments were carried out using standard two-dimensional sequences (correlation spectroscopy, heteronuclear multiple bond correlation, heteronuclear multiple quantum correlation, and total correlation spectroscopy correlations). Particularly, the structure of ␤-D-galactopyranosyl-(133)-␤-D-galactopyranosyl-(134)-D-glucopyranoside was established on the basis of the identification of the C-3 carbon (shifted at lower fields with respect to the other carbon resonances) according to heteronuclear multiple

APPL. ENVIRON. MICROBIOL. quantum correlation and heteronuclear multiple bond correlation 1H/13C resonance correlations. The attribution of the 13C resonance absorptions required a complete analysis of the 1H-NMR spectra, condition solved by means of total correlation spectroscopy sequences, which allowed the identification of each monosaccharide unit by selective irradiation of the corresponding anomeric proton. The resonance of the H-3⬘ proton, easily attributed at ␦ ⫽ 3.82 ppm (see data below), correlates with the 13C resonance located at 84.6 ppm (lower fields than all other carbon resonances except the anomeric ones), thus showing the existence of the ␤-(133) glycosidic bond. The spectra were recorded with a Bruker DRX500 spectrometer operating at 500 MHz for 1H and 126 MHz for 13 C. Due to their solubility in water, the spectra of the saccharides were recorded in D2O, and the chemical shifts (in ppm) were quoted from the resonance of methyl protons of sodium 3-(trimethylsilyl)-propansulfonate used as an internal reference. The NMR parameters of the autocondensation product allowed its identification as 4-nitrophenyl-␤-D-galactopyranosyl-(133)-␤-D-galactopyranoside by comparison with previous published data (5). NMR data for ␤-D-galactopyranosyl-(133)-␤-D-galactopyranosyl-(134)-Dglucopyranoside. 1H-NMR [500 MHz, D2O, reference: (CH3)3Si-CH2-CH2CH2-SO3Na]: ␦ 5.22 (1H, d, J ⫽ 3.4 Hz, H-1␣), 4.65 (1H, d, J ⫽ 7.9 Hz, H-1␤), 4.60 (1H, d, J ⫽ 7.8 Hz, H-1”), 4.50 (1H, d, J ⫽ 7.9 Hz, H-1⬘), 4.18 (1H, d, J ⫽ 3.4 Hz, H-4⬘), 3.95 (1H, dd, J ⫽ 2.3, ⫺12.3 Hz, H-6␤), 3.91 (1H, d, J ⫽ 3.3, H-4⬙), 3.82 (1H, dd, J ⫽ 9.9, 3.4 Hz, H-3⬘), 3.79 (1H, dd, J ⫽ 5.1, ⫺12.3 Hz, H-6␤), 3.71 (1H, H-5⬘, or H-5⬙), 3.69 (1H, dd, J ⫽ 9.9, 7.9 Hz, H-2⬘), 3.68 (1H, H-4␣), 3.67 (1H, dd, J ⫽ 8.7, 9.6, Hz, H-4␤), 3.65 (1H, dd, J ⫽ 9.9, 7.3 Hz, H-3⬙), 3.63 (1H, dd, J ⫽ 9.0, 8.7 Hz, H-3␤), 3.61 (1H, H-5⬘ or H-5⬙), 3.59 (1H, dd, J ⫽ 7.8, 9.9, Hz, H-2⬙), 3.28 (1H, dd, J ⫽ 9.0, 7.9 Hz, H-2␤). 13C NMR (125 MHz, D2O, reference: (CH3)3Si-CH2-CH2-CH2-SO3Na]: ␦ 107.1 (C-1⬙), 105.3 (C-1⬘), 98.5 (C-1␤), 94.6 (C-1␣), 84.6 (C-3⬘), 81.1 (C-4␣), 81.0 (C-4␤), 77.8 (C-2⬘), 77.7 (C-2⬙), 77.5 (C-5␤), 77.1 (C-3␤), 76.6 (C-2␤), 75.3 (C-3⬙), 74.1 (C-3␣ or C-5␣), 73.9 (C-3␣ or C-5␣), 73.8 (C-5⬘ or C-5⬙), 72.9 (C-5⬘ or C-5⬙), 72.8 (C-2␣), 71.3 (C-4⬙), 71.2 (C-4⬘), 63.7 (C-6⬘ and C-6⬙), 62.9 (C-6␤), 62.7 (C-6␣). DNA sequence analysis. DNA sequencing reactions were carried out according to the dideoxy-chain termination method. The DNA was analyzed with an automated laser fluorescence ALF sequencer (Pharmacia) using labeled primers or labeled dATP. The resulting nucleotide sequences were analyzed with sequence analysis software (Seqman II from DNASTAR Inc.).

RESULTS Cloning of the ␤-galactosidase gene from Geobacillus stearothermophilus KVE39. Geobacillus stearothermophilus strain KVE39 produced two isoenzymes of both ␤-galactosidase and ␣-galactosidase (11). Previously, a chromosomal DNA fragment of this strain was cloned in plasmid pCG1, and it was demonstrated that the respective recombinant plasmid coded for the ␤- and ␣-galactosidase activities (11). In the course of the present study, a region of plasmid pCG1 which encoded the ␤-galactosidase activity (BgaB) was sequenced. The bgaB gene showed a high degree of homology (99.5% identity) to the ␤-galactosidase I gene bac_bgab from Geobacillus stearothermophilus (GenBank accession no. M13466) originally described by Hirata et al. (17). The two gene sequences showed only 10 base pair differences, which resulted in two alterations at the amino acid level: V54M and T576A. In order to achieve efficient expression, bgaB was inserted in the L-Rha-inducible expression vector pJOE2702 (34), giving plasmid pHWG509. The expression of bgaB was confirmed by SDS-polyacrylamide gel electrophoresis analysis, which demonstrated that in crude cell extracts of the induced E. coli JM109/pHWG509 cells a prominent protein band with an expected size of 78 kDa was present, which was absent in the extracts prepared from uninduced cells (data not shown). Screening of evolved ␤-galactosidases. The bgaB gene was mutated using MNNG in order to improve the transglycosylation/hydrolysis ratio of BgaB. Variants were screened for diminished hydrolytic activity toward lactosucrose, a potential

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FIG. 1. Transglycosylation reaction of ␤-galactosidases with pNP-␤Gal (autocondensation). (A) Wild-type BgaB and the mutants obtained by the random mutagenesis approach, represented by GP21 (BgaB R109K). (B) The site-specific mutants GP637.2 (BgaB R109V) and GP643.3 (BgaB R109W). Aliquots of the enzymatic reaction mixture were withdrawn at different times. The reaction components were separated by TLC and visualized under UV light at 254 nm.

transglycosylation product. This was performed in two steps. In the first step, reduced hydrolysis of lactosucrose was screened on an indicator medium (MacConkey agar) containing lactosucrose. In this system, the formation of galactose, a hydrolysis product of lactosucrose, resulted in the formation of red colonies. In contrast, colonies without hydrolytic activity toward this oligosaccharide displayed a colorless-white phenotype. In the second step of the screening process, the white colonies from the first screening step were tested for their catalytic activities toward the chromogenic substrate X-Gal and toward the natural donor substrate lactose in order to test that they were still catalytically active. The screening of the mutant library of pHWG509 yielded about 10% of white colonies on MacConkey/lactosucrose agar plates. In contrast, all colonies obtained from a control culture which had not been treated with MNNG were homogenously red. From the mutant library, about 500 colonies with a white phenotype were subsequently tested for their hydrolytic activity on X-Gal agar plates. In parallel, their activity on lactose was investigated on MacConkey/lactose agar plates. Among the 500 colonies obtained from the first screening, only nine colonies formed on X-Gal agar plates a similar degree of blue color as the recombinant E. coli strain synthesizing the wildtype form of BgaB. Surprisingly, all nine selected mutants displayed only a pink phenotype on MacConkey/lactose agar plates, whereas the wild type formed intensely red colonies. Characterization of the transglycosylation activities of the evolved ␤-galactosidases. Cell extracts from the nine mutants were initially tested with pNP-␤Gal for transglycosylation activities. The reaction mixtures were analyzed by TLC, and it was found that the nine clones were able to transfer the galactosyl residue to the substrate pNP-␤Gal (autocondensation reaction). As shown in Fig. 1A, the mutants, represented by GP21, formed more autocondensation products than the wildtype enzyme. All the other mutants formed similar amounts of the autocondensation disaccharide (data not shown), which was identified as pNP-␤Gal-(1,3)-Gal and which disappeared again after a longer incubation time. Next, transglycosylation reactions were performed with pNP-␤Gal in the presence of cellobiose, in order to test if the

mutants exhibited transfer activity toward a natural disaccharide acceptor rather than performing the autocondensation reaction. Analysis of the products by TLC revealed that the selected mutants, represented by GP21 (Fig. 2A), were able to produce transglycosylation products, which consist of galactosyl-cellobiose since these spots were not UV fluorescent. Remarkably, after total conversion of the donor pNP-␤Gal, the transglycosylation products were quite stable, in contrast to the autocondensation product shown in Fig. 1A. No transglycosylation product was detectable with the wild-type enzyme. These results indicated the enhancement of the transglycosylation/ hydrolysis ratio for the selected mutants. Finally, transglycosylation reactions were performed with lactose acting simultaneously as substrate and as donor. The product formation was followed qualitatively by TLC. Analysis of the products revealed that five mutants (GP13, GP17, GP21, GP23, and GP30) formed more transglycosylation products than the wild-type enzyme. All mutants converted lactose and accumulated glucose, as represented by GP21 shown in Fig. 2B. In comparison to the wild-type enzyme, the mutants formed only barely detectable amounts of galactose. This indicated that the galactosyl residue was preferentially transferred to lactose and yielded large amounts of GOS, which were detected as a new spot in TLC. Sequence analysis of the mutants and saturation mutagenesis. Sequence analysis of the five evolved BgaB variants revealed the presence of one to three mutations (Table 1). Arginine 109 was replaced by a lysine in all the mutants, and mutant GP21 carried only this amino acid exchange. Therefore, a saturating mutagenesis was performed at position 109. The recombinant clones were sequenced, and 16 BgaB variants were obtained. These mutants comprised all the possible amino acid residues at this position except the amino acids asparagine, methionine, and proline. The phenotypes of these recombinant clones were tested on the agar plates with the same composition as used for the initial screening experiments. Most of the clones exhibited a white phenotype on MacConkey/ lactosucrose agar and a pink phenotype on MacConkey/lactose agar and appeared blue on X-Gal agar plates. In contrast, E. coli JM109/pGP637.2, which encoded BgaB R109V, turned red

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APPL. ENVIRON. MICROBIOL.

FIG. 2. Qualitative view of the transglycosylation reaction of the ␤-galactosidases with pNP-␤Gal as substrate and cellobiose as acceptor (A) and with lactose (B). Reactions were performed with wild-type BgaB and BgaB mutant GP21 as described in Materials and Methods. The reaction components from different times of the enzymatic reaction were separated by TLC, and sugars were visualized by spraying as described in Materials and Methods.

on MacConkey/lactose plates, and E. coli JM109/pGP643.3, which encoded BgaB R109W, exhibited a white phenotype on all three types of plates, suggesting the presence of an inactive enzyme. Surprisingly, with the substrate pNP-␤Gal E. coli JM109/pGP643.3 displayed the same yellow phenotype as the other clones, which suggested that BgaB R109W was still active on pNP-␤Gal. In order to examine the transglycosylation capacities of BgaB R109V and BgaB R109W, crude extracts were tested for the autocondensation reaction with pNP-␤Gal by following their reaction products by TLC analysis (see Fig. 1B). Thus, it was found that BgaB R109V (GP637.2) and BgaB R109W (GP643.3) produced the same autocondensation product, pNP-␤Gal-(1,3)-Gal, as the mutants which had been obtained by random mutagenesis (see above). Additionally, BgaB R109W formed a new product which was not hydrolyzed after total conversion of the substrate pNP-␤Gal (see Fig. 1B). The condensation performances of BgaB R109V and BgaB R109W on lactose were tested and analyzed by TLC, and transglycosylation rates similar to those with the mutant GP21 were observed (Fig. 2B). Characteristics of the evolved ␤-galactosidases. The mutants GP13, GP17, GP21, GP23, GP30, BgaB R109V, and

BgaB R109W were further characterized because cell extracts from these strains exhibited specific activities toward the substrate pNP-␤Gal that were similar to those of the wild-type enzyme. The enzymes were expressed in E. coli JM109. SDSpolyacrylamide gel electrophoresis analysis of crude extracts revealed amounts of the recombinant enzyme that were similar to those in crude extracts from E. coli JM109/pHWG509 cells (data not shown). This indicated that the mutations did not affect the expression level of the BgaB variants in comparison to the wild type. The wild-type enzyme and the mutants were tested for their optimum temperature for the hydrolysis of pNP-␤Gal (Fig. 3). The wild-type enzyme exhibited an optimum temperature at 65°C. In contrast, some mutants demonstrated a lower optimum temperature. The storage stabilities of the enzyme variants were tested at 37°C. No detectable loss of activity was

TABLE 1. Amino acid substitutions in the evolved ␤-galactosidases GP13, GP17, GP21, GP23, and GP30, which display improved synthesis capability Amino acida at residue: BgaB

Wild type GP13 GP17 GP21 GP23 GP30

80

109

140

181

424

446

534

557

T T I T T T

R K K K K K

L L L L L F

E G E E E E

A A V A A A

T T T T T I

T T T T M T

G G G G E G

a The arginine-to-lysine mutation at residue 109 is indicated in bold; other mutations are in italic.

FIG. 3. Effect of temperature on activity of wild-type BgaB (HWG509) and the mutants GP13, GP17, GP21, GP23, and GP30. ␤-Galactosidase activity toward the substrate pNP-␤Gal (2.65 mM) was determined under standard conditions.


VOL. 75, 2009 TABLE 2. Kinetic parameters of wild-type BgaB and different mutants for the substrates lactose and lactosucrose and their specific activities toward pNP-␤Gal at different concentrationsa

BgaB

Wild-type GP13 GP17 GP21 (BgaB R109K) GP23 GP30 GP637.2 (BgaB R109V) GP643.3 (BgaB R109W) a b c

Sp act (U mg⫺1) for pNP␤Gal at:

Lactose

Lactosucrose

2.65 mM

20 mM

Km (mM)

Vmax (U mg⫺1)b

Km (mM)

Vmax (U mg⫺1)c

0.50 0.19 0.41 0.22

0.19 0.45 0.79 0.47

1.78 65 73 84

0.34 0.45 0.29 0.42

5 165 150 136

0.43 0.45 0.50 0.42

0.23 0.18 1.52

0.57 0.43 4.90

63 55 100

0.31 0.40 0.72

200 155 142

0.50 0.40 0.39

0.60

1.79

114

0.05

160

0.03

The reactions were performed at 37°C. Amount of glucose released per minute per mg crude extract protein. Amount of galactose released per minute per mg crude extract protein.

observed after incubation for 6 days. Therefore, all further experiments were carried out at 37°C. The enzyme kinetics were compared with pNP-␤Gal as a substrate. Initial velocities at different substrate concentrations obeyed the Michaelis-Menten equation. For the BgaB wildtype enzyme, inhibition by the substrate appeared to occur at substrate concentrations of above 3 mM pNP-␤Gal, since the velocity decreased with increased substrate concentrations (see Fig. S1A in the supplemental material). In contrast, no inhibition was observed with the mutants. The mutants exhibited increased hydrolytic activities in the presence of 20 mM pNP␤Gal compared to the concentration of pNP-␤Gal normally used in the activity test (2.65 mM). Most of the mutants demonstrated in the presence of 20 mM pNP-␤Gal similar specific activities as the wild type after addition of 2.65 mM substrate (Table 2). The enzyme kinetics were also determined with the substrates lactose and lactosucrose. In these reactions the release of glucose was measured in order to exclude the transglycosylation reaction of galactose. Thus, it was found that the wild-type enzyme was inhibited at substrate concentrations of higher than 15 mM for lactose (see Fig. S1B in the supplemental material) and 30 mM for lactosucrose (data not shown). In contrast, with the mutants, no inhibitory effect was detected. A summary of the kinetic parameters is given in Table 2. The apparent Km values of the evolved enzymes toward the natural substrates (lactose and lactosucrose) were strongly increased (30- to 42-fold) compared to those of the wild-type enzyme. However, for most of the mutants the relative activities found in the cell extracts were quite similar to those of the wild-type enzyme. The main exception was BgaB R109V, which exhibited an about 10-fold-lower enzyme activity. As a result, for all the mutant enzymes, decreased catalytic efficiencies (Vmax/Km) toward the substrates lactose and lactosucrose were observed.

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Quantification of the transgalactosylation product. The transfer capabilities of the mutants GP21 (BgaB R109K), GP637.2 (BgaB R109V), and GP643.3 (BgaB R109W) were examined by using the standard transglycosylation reaction with lactose and following the most abundant GOS-trisaccharide (identified as 3⬘-galactosyl-lactose, as described below) produced. Glucose and galactose were formed from lactose by hydrolysis, and GOS were formed by transgalactosylation. The latter products can also serve as substrate. The components of the reaction mixture were separated by HPLC and quantified by pulsed amperometric detection. Because this method did not separate glucose and galactose, both of these monosaccharides were quantified by enzymatic tests. The time courses of typical reactions are shown in Fig. 4. The BgaB wild-type enzyme produced only very small amounts (5 mM; yield by weight, 2%) of a trisaccharide. After the conversion of about 70% of the initially added lactose, the amounts of galactose and glucose were almost the same and almost stoichiometric to the amount of the lactose consumed, since GOS were not detectable. The enzyme variants GP21 (BgaB R109K), GP637.2 (BgaB R109V), and GP643.3 (BgaB R109W) produced significantly more of this trisaccharide (Fig. 4). Furthermore, during all experiments with the evolved enzymes, the glucose concentration was higher than the galactose concentration. This indicated that the “missing” galactose was used in the transgalactosylation reaction to form GOS and also other by-products such as disaccharides (data not shown). The production of the major trisaccharide occurred within approximately 6 h from the start of the reaction. As the reaction proceeded, considerable amounts of monosaccharides (galactose and glucose) were produced with the mutants GP21 and BgaB R109V, because the transglycosylation product was hydrolyzed. The extracts from BgaB R109V demonstrated significant hydrolysis of the trisaccharide almost immediately after the maximal amount of this GOS had been formed. The maximal yields of this trisaccharide were 41 mM (yield by weight, 11.5%) with GP21 and 75 mM (yield by weight, 21%) with BgaB R109V. The most efficient mutant was BgaB R109W, which produced 82 mM of the trisaccharide (yield by weight, 23%). Furthermore, with this mutant, no hydrolysis of the trisaccharide was observed after the maximal yield of trisaccharide had been reached. This demonstrated that BgaB R109W exhibited a very low hydrolytic activity toward this trisaccharide. Identification of the Gal-GOS produced. The major transglycosylation product produced by the evolved enzymes had the same retention time as an authentic sample of 3⬘-galactosyllactose when subjected to high-performance anion-exchange chromatography with pulsed amperometric detection. In order to confirm its identity, a transglycosylation reaction with lactose was performed with the mutant GP643.3 (BgaB R109W). The reaction mixture was analyzed by HPLC (Fig. 5A), and the product mixture was separated by size exclusion chromatography on Biogel P2. The isolated carbohydrate fractions were analyzed by HPLC and revealed mixtures of mono-, di-, and trisaccharides and, in addition, small amounts of tetrasaccharides and other oligosaccharides (data not shown). The trisaccharide mixture comprised 3⬘-galactosyl-lactose and small amounts of 4⬘-galactosyl-lactose (Fig. 5B). The former product

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FIG. 4. Time course of GOS production during lactose hydrolysis by wild-type BgaB and the mutants GP21 (BgaB R109K), GP637.2 (BgaB R109V), and GP643.3 (BgaB R109W). The standard transglycosylation reaction was applied, the components of the reaction mixture were separated by HPLC, and individual sugars were quantified as described in Materials and Methods. Abbreviations: Gal, galactose; Glc, glucose; Lac, lactose; trisaccharide, 3⬘-galactosyl-lactose.

was isolated by preparative HPLC. Its structure was established by means of NMR to be 3⬘-galactosyl-lactose. DISCUSSION Glycosidases have emerged as a useful tool in the enzymatic synthesis of oligosaccharides through transglycosylation reactions. Due to their availability and stability, glycosidases are more advantageous than glycosyltransferases (EC 2.4) and chemical synthesis methods. The retaining glycosidases use a double-displacement mechanism in which an active-site nucleophile attacks the anomeric center to generate a covalent glycosyl-enzyme intermediate, which is subsequently hydrolyzed in a general acid/base-catalyzed manner (7). In the presence of a suitable nucleophile other than water, e.g., the substrate lactose, a new glycosidic

linkage is formed through transglycosylation activity. However, the concurrent hydrolysis of substrate and product decreases the final oligosaccharide yield. To overcome the drawbacks of glycosidases for oligosaccharide synthesis, we attempted to reduce the hydrolysis of the oligosaccharide product formed during the transglycosylation reaction. Screening of evolved BgaB. One prerequisite for the screening procedure was to provide a stable recombinant strain for expression of the ␤-galactosidase gene from Geobacillus stearothermophilus. This was achieved by the application of the LRha-inducible expression vector in E. coli. The expression of the bgaB gene was strictly dependent on the induction with Rha. Induced cultures of E. coli JM109/pHWG509 produced high levels of BgaB and displayed a homogenous red phenotype on MacConkey/lactose or MacConkey/lactosucrose agar plates supplemented with 0.05% Rha. This disclosed the pos-

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FIG. 5. HPLC chromatograms. (A) The reaction mixture obtained after the transgylcosylation reaction performed with GP643.3 (BgaB R109W) and lactose. The experimental conditions are described in Materials and Methods. (B) The trisaccharide fraction obtained after separation the transglycosylation reaction on Biogel P2. Peaks were identified by comparison of the retention time (RT) with those of authentic samples, as follows: peak 1, glucose and galactose; peak 2, lactose; peak 3, unknown; peak 4, 4⬘-galactosyl-lactose; peak 5, 3⬘-galactosyl-lactose.

sibility of screening BgaB mutants with reduced hydrolytic activity toward lactosucrose, which was thought to mimic a transglycosylation product. Screening of a mutant library of E. coli JM109/pHWG509 provided mutants with a white phenotype on MacConkey/lactosucrose agar plates while keeping catalytic activity for the chromogenic substrate X-Gal. These candidates displayed a pink phenotype on MacConkey/lactose agar plates, which suggested a reduced activity for the natural donor lactose compared to the wild-type enzyme. However, the use of the mutants in a transglycosylation reaction with lactose proved them to be active on lactose, comparable to the wildtype enzyme. The elucidation of the enzymes kinetics toward the substrate lactose may explain their observed phenotype on the MacConkey/lactose agar plates. Due to a drastically increased Km value, the catalytic efficiency of the mutants was reduced, leading to a lower catalytic activity on lactose, especially in the presence of small amounts of substrate. A similar explanation could be made for their white phenotype on MacConkey lactosucrose agar plates. Synthetic performance of the evolved enzymes. The evolved BgaB mutants obtained were used to analyze the transglycosylation reaction with lactose in more detail. Quantitative determination of the reaction products revealed that the amounts of galactose were always lower than those of glucose. This supported their increased synthesis capability, in which galactose was transferred to donors such as lactose and yielded GOS. Moreover, the galactose and glucose quantities did not reach the amount of lactose used, which suggested that galactose and glucose may also act as acceptors to form disaccharides, which are considered by-products. Such products may represent the unidentified substances within the HPLC chromatogram presented in Fig. 5A (peak 3). Thus, the apparent maximal velocities of the mutants do not reflect the real catalytic activities toward lactose and lactosucrose, since the galactosyl residue is preferentially transferred on acceptors such as lactose but also on glucose and galactose. The decreased amounts of monosaccharides in comparison to the lactose bioconversion were even more remarkable in case of GP643.3 (BgaB R109W). During the application of this enzyme for transglycosylation reaction with a 500 mM lactose

solution (18% [wt/vol]), the maximum yield by weight of the most abundant GOS-trisaccharide, 3⬘-galactosyl-lactose, reached 23% (⬃41.6 mg ml⫺1), near 80% of lactose conversion. Further GOS such as 4⬘-galactosyl-lactose and GOStetrasaccharides were detected in the reaction mixture in minor quantities (Fig. 5A) and increased the total yield of GOS to ⬃30%. Even though the comparison of the results reported for various enzymes would be difficult due to different reaction conditions used in other studies, the total amount of GOS produced by GP643.3 seemed to be comparable to those for the relevant enzymes derived from Bifidobacterium bifidum (29%) (9), Sterigmatomyces elviae CBS8119 (34%) (24), Rhodotorula minuta IFO879 (35%) (25), and Enterobacter agglomerans 38% (20). The increased synthesis of GOS by the evolved enzymes observed in this study can be due either to an increase in the transglycosylation/hydrolysis ratio of the mutant enzyme or to a lower rate of hydrolysis of the oligosaccharide products compared to the wild-type enzyme. For the mutants BgaB R109K and BgaB R109V, the reaction with substrate pNP-␤Gal indicates that a breakdown of the autocondensation product occurred after a maximum yield was reached (Fig. 1B). Similar results were observed during the reaction with lactose, where degradation of the trisaccharide was noticed after it reached a maximum. This suggests that the increased trisaccharide yield by both these mutants was due to an increase in the transglycosylation/hydrolysis ratio. These enzymes favor the reaction of the galactosyl-enzyme with acceptors (for example, lactose) more than the hydrolysis. These enzymes can be applied for transgalactosylation reactions with other natural carbohydrates as acceptors (for example, cellobiose) (Fig. 2A). In contrast, with BgaB R109W the newly synthesized autocondensation product can be detected during the reaction, and it was not degraded even after total conversion of the pNP-␤Gal substrate (Fig. 1B). The application of this mutant for the transgalactosylation reaction with lactose yielded a trisaccharide, which was only slightly degraded after the reaction proceeded. This suggested that the higher yield on transglycosylation products was due to a lower rate of hydrolysis of the oligosaccharide products by the mutant enzymes than by the wild-type enzyme.

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The main GOS that was produced by the evolved enzymes was identified to be 3⬘-galactosyl-lactose. The synthesis of a Gal-␤(1,3) linkage by BgaB is interesting, since other ␤-galactosidases from different sources predominantly formed Gal␤(1,4) (23, 24, 28, 33, 36) or Gal-␤(1,6) (14, 18) linkages. Gal-␤((1,3) linkages play an important physiological role, e.g., in the carbohydrate structure of Lewis antigen (Lea) (32) or in glycoproteins such as cell adhesion molecules (1) and immunoglobulin A (2). Thus, the design of better catalytic tools for synthesis of sugars containing the Gal-␤(1,3) linkage is of first interest. Structural basis of the evolved BgaB. After the random mutagenesis approach, the best-evolved enzymes obtained bore a common amino acid alteration (R109K). The mutant GP21 carries solely this amino acid exchange, and R109K may be responsible for the increased synthetic capacity. This residue is highly conserved among ␤-galactosidases belonging to the same family of glycoside hydrolases (GH 42 [15]; see alignment in Fig. S2 in the supplemental material). Moreover, the crystal structure of one member of GH 42, ␤-galactosidase from Thermus thermophilus A4, has been resolved (16). Its three-dimensional structure in complex with galactose offers a basis for interpretation of the results. In Thermus thermophilus ␤-galactosidase, the conserved arginine (Arg 102) is located in the active-site pocket and is involved in the binding of the oxygens O3 and O4 of the galactose molecule by direct hydrogen bond interactions. The mutated residue at this site may disturb the interaction network and may change the hydrolytic capability of the ␤-galactosidase. Moreover, saturation mutagenesis at this site revealed that it can be replaced by several different amino acids, and this provided mutants with transglycosylation potentialities similar to those of GP21 (BgaB R109K). As no mutant enzyme with properties similar to those of the wild-type BgaB was found, it could be speculated that the Arg residue at this position is necessary to keep the hydrolytic activity of family GH 42 glycoside hydrolases. The results obtained after saturation mutagenesis at position BgaB109 suggested that this residue is one of the best targets to improve the transgalactosylation yield of family 42 glycoside hydrolases. This speculation was proved on another ␤-galactosidase target. The exchange of the equivalent amino acid residue R102 of Thermus thermophilus BgaT ␤-galactosidase led to similar results, which we will present in the future. Feng et al. (10) also described the involvement of altered active-site residues in the improvement of transglycosylation activities of evolved ␤-glycosidases from Thermus thermophilus. Here, the most efficient mutation was located in front of the subsite (⫺1) within the three-dimensional structure of Thermus thermophilus ␤-glycosidase. Slight improvement of the transglycosylation activity was observed with Pyrococcus furiosus ␤-glucosidase after mutation of the homologous position (13), which targets this position to be most effective to improve transglycosidase activity in the glycoside hydrolase family 1. However, in order to optimize the transglycosylation activity of glycosidases in a rational way, the influence of the active-site structure on the catalytic properties of the enzyme has to be understood. In conclusion, the screening procedure proved to be efficient and could provide mutant enzymes possessing an increased synthesis capability. Experiments to examine further members


of GH 42 family and their behavior after the alteration of the BgaB R109 homologous position are in progress. REFERENCES 1. Asada, M., K. Furukawa, C. Kantor, C. G. Gahmberg, and A. Kobata. 1991. Structural study of the sugar chains of human-leukocyte cell-adhesion molecule-CD11/CD18. Biochemistry 30:1561–1571. 2. Baenziger, J., and S. Kornfeld. 1974. Structure of carbohydrate units of IgA1 immunoglobulin. II. Structure of O-glycosidically linked oligosaccharide units. J. Biol. Chem. 249:7270–7281. 3. Bouhnik, Y., L. Raskine, G. Simoneau, E. Vicaut, C. Neut, B. Flourie, F. Brouns, and F. R. Bornet. 2004. The capacity of nondigestible carbohydrates to stimulate fecal bifidobacteria in healthy humans: a double-blind, randomized, placebo-controlled, parallel-group, dose-response relation study. Am. J. Clin. Nutr. 80:1658–1664. 4. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. 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Purification and properties of a beta-D-galactosidase from Bifidobacterium bifidum exhibiting a transgalactosylation reaction. Biotechnol. Appl. Biochem. 19:341–354. 10. Feng, H. Y., J. Drone, L. Hoffmann, V. Tran, C. Tellier, C. Rabiller, and M. Dion. 2005. Converting a beta-glycosidase into a beta-transglycosidase by directed evolution. J. Biol. Chem. 280:37088–37097. 11. Ganter, C., A. Bock, P. Buckel, and R. Mattes. 1988. Production of thermostable, recombinant alpha-galactosidase suitable for raffinose elimination from sugar beet syrup. J. Biotechnol. 8:301–310. 12. Hancock, S. M., M. D. Vaughan, and S. G. Withers. 2006. Engineering of glycosidases and glycosyltransferases. Curr. Opin. Chem. Biol. 10:509–519. 13. Hansson, T., T. Kaper, O. J. van Der, W. M. de Vos, and P. Adlercreutz. 2001. Improved oligosaccharide synthesis by protein engineering of betaglucosidase CelB from hyperthermophilic Pyrococcus furiosus. Biotechnol. Bioeng. 73:203–210. 14. Hedbys, L., P. O. Larsson, K. 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26.

27. 28.

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