Modulation of the Regioselectivity of a Thermotoga Neapolitana Β-Glucosidase by Site-Directed Mutagenesis

J. Microbiol. Biotechnol. (2008), 18(5), 901–907 Modulation of the Regioselectivity of a Thermotoga neapolitana β-Glucosidase by Site-Directed Mutagenesis Choi, Ki-Won1, Kyung-Min Park1, So-Young Jun1, Cheon-Seok Park2, Kwan-Hwa Park3, and Jaeho Cha1* 1Department of Microbiology, College of Natural Sciences, Pusan National University, Busan 609-735, Korea 2Department of Food Science and Technology, Graduate School of Biotechnology and Institute of Life Sciences and Resources, KyungHee University, Yongin 449-701, Korea 3Department of Food Science and Biotechnology, School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea Received: August 16, 2007 / Accepted: December 18, 2007 Thermotoga neapolitana β-glucosidase (BglA) was subjected influence their physicochemical and pharmacological to site-directed mutagenesis in an effort to increase its ability properties, such as transport through membrane barriers to synthesize arbutin derivatives by transglycosylation. The and transport by body fluids [17]. Glycosylation also often transglycosylation reaction of the wild-type enzyme displays reduces the irritation and toxicity caused by the aglycone major β(1,6) and minor β(1,3) or β(1,4) regioselectivity. The [17]. three mutants, N291T, F412S, and N291T/F412S, increased Enzymatic synthesis of glycosides has an advantage the ratio of transglycosylation/hydrolysis compared with over chemical synthesis because it can control the regio- the wild-type enzyme when pNPG and arbutin were used and stereospecificities of the reaction [19]. Glycosidases are as a substrate and an acceptor, respectively. N291T and used in reactions of reverse hydrolysis or transglycosylation N219T/F412S had transglycosylation/hydrolysis ratios about [3, 5, 16]. Family 1 glycosyl hydrolase (GH1), which can 3- and 8-fold higher, respectively, than that of the wild-type catalyze the transglycosylation reaction as well as hydrolysis, enzyme. This is due to the decreased hydrolytic activity of is ideal for use in the synthesis of glycoside derivatives the mutant rather than increased transglycosylation activity. [22]. Specifically, β-glucosidases have received great interest Interestingly, N291T showed altered regioselectivity, as because of their biosynthetic abilities and their various well as increased transglycosylation products. TLC analysis biological functions [9, 10, 20, 23]. In our early reports, of the transglycosylation products indicated that N291T we described the Thermotoga neapolitana β-glucosidase- retained its β(1,3) regioselectivity, but lost its β(1,4) and catalyzed synthesis of arbutin derivatives [21]. However, β(1,6) regioselectivity. The altered regioselectivity of N291T in the previous studies, the yield of the arbutin derivatives using two other acceptors, esculin and salicin, was also was very low. The low yield of the reaction is a demerit of confirmed by TLC. The major transglycosylation products the reaction of GH1 enzymes [14]. Moreover, the presence of the wild type and N291T mutant were clearly different. of undesired regioisomers, which make the purification This result suggests that Asn-291 is highly involved in the step difficult, has encouraged us to improve the properties catalytic mechanism by controlling the transglycosylation of the enzyme using different strategies. Many review reaction. articles have been published about the use of glycoside Keywords: Arbutin derivatives, β-glucosidase, regioselectivity, synthesis for the purpose of increasing the yield and highly Thermotoga neapolitana, transglycosylation regioselective synthesis of glycosides [1, 6, 7, 19, 22]. The β-glycosidase of Thermus thermophilus increased its transglycosylation ability to synthesize oligosaccharides through random mutagenesis [11]. The most efficient mutants, Many of the glycosides that naturally occur in plants have F401S and N282T, increased the yield of trisaccharides biological activities [17]. These activities are primarily due by 6-fold compared with the wild-type enzyme. Mutations to the aglycones of the glycosides. The water solubilities of M424K and F426Y also significantly improved (18-40%) hydrophobic molecules containing one or more hydroxyl the synthesis of galactooligosaccharides from the Pyrococcus groups can be increased by glycosylation, which can furiosus β-glucosidase [13]. Changes in the yield of transglycosylation products *Corresponding author Phone: 82-51-510-2196; Fax: 82-51-514-1778; usually affect the regioselectivity in the enzymatic synthesis E-mail: [email protected] of the glycosides. In general, glycosidases exhibit low 902 Choi et al. regioselectivity for the hydroxyl linkage of acceptor substrates GTGACCTGAATAGTAGGTCAGCCCAACGAAGTC-3'), N291T-R in the transglycosylation reaction. It is also known that the (5'-GACTTCGTTGGGCTGACCTACTATTCAGGTCAC-3'), F412S- regioselectivity in the enzymatic synthesis of oligosaccharides F (5'-CACGTACACTATACCGGACCTCTTCGAATATCC-3'), and using glycosidases is dependent on the origin of the microbe F412S-R (5'-GGATATTCGAA GAGGTCCGGTATAGTGTACGTG- and the selection of acceptors [2, 18]. Therefore, it is important 3'), were designed based on the result of directed evolution of Thermus thermophilus β-glycosidase [11]. The mutagenic primers to determine what controls the regioselectivity of glycoside contained the desired mutation and annealed to the same sequence synthesis in the enzyme reaction. Although regioselectivity on opposite strands of the plasmid. Each primer (10 pmol) was in the synthesis of glycosides using glycosidases is known mixed with the p6xhis119-bglAp6 plasmid (10 ng) in a 50-µl PCR. to be very low, transglycosylation products were shown to The reaction conditions were as follows: 10× Pfu reaction buffer, be highly regioselective when glycosidases that showed 2.5 mM dNTP, 1.5 mM MgCl2, and 2.5 units of PfuUltra HF DNA high specificity were used in the hydrolysis reaction [1]. polymerase. PCR amplication was carried out on a thermal cycler In this paper, we report our success in isolating BglA (GeneAmp PCR System 2400, Perkin Elmer) with a program mutants that show increased transglycosylation activity consisting of predenaturation for 30 s at 95oC, followed by 30 cycles compared with the wild-type enzyme. Moreover, one of the of denaturation at 95oC for 1 min, annealing at 55oC for 1 min, and o BglA mutants was identified as being strongly involved in extension at 68 C for 6 min. Five µl of each PCR product was the regioselectivity of glycoside synthesis. Thus, directed analyzed on 0.8% (w/v) agarose gel containing ethidium bromide (0.25 mg/ml). Mutagenized PCR products were digested by a DpnI evolution is an efficient approach to the synthesis of various restriction enzyme at 37oC for 3 h to digest the parental (i.e., transglycosylation products through the modification of nonmutated) double-stranded DNA. Without ligation, digested PCR the enzyme regioselectivity. It also acts to improve the products were transformed into E. coli MC1061 by the heat-shock yield of the transglycosylation products of glycosidases. method. To select for transformants, 100 to 200 µl of the transformation mixture was spread on LB agar plates with ampicillin at a final concentration of 100 µg/ml. The double mutant, N291T/F412S, was MATERIALS AND METHODS constructed by three-piece ligation. The bglA genes of mutants N291T and F412S were digested by NdeI/NcoI and NcoI/HindIII Strain and Growth Conditions restriction enzymes, respectively. The two digested fragments were Thermotoga neapolitana KCCM 41025 (corresponding to NS-E, ligated with the p6xhis119 vector. DNA sequencing was performed ATCC 49049, DSM 4359) was cultivated under anaerobic conditions on a DNA auto-sequencer (ABI PRISM 377; Perkin Elmer) at in a 308 medium as described previously [21]. E. coli DH5α and Greengene Biotech (Suwon, Republic of Korea). Oligonucleotides MC1061, which were used as hosts for cloning and expression for sequencing were synthesized by Bionics (Seoul, Republic of studies, were grown in Luria-Bertani (LB) medium containing 1% Korea). Computer analyses of the DNA sequence data and the deduced (w/v) bacto-tryptone, 0.5% (w/v) yeast extract, and 0.5% (w/v) amino acid sequence were performed with programs available on NaCl, and supplemented with ampicillin (100 µg/ml). the Expasy Molecular Biology server (http://www.expasy.ch) on the World Wide Web. Enzymes and Chemicals Restriction endonucleases and modifying enzymes were purchased Purification of the Mutant Enzymes from Kosco (Sungnam, Republic of Korea) or Takara (Shiga, Japan). All purification steps of recombinant His6-tagged BglA mutants A QuikChange II Site-Directed Mutagenesis Kit for mutagenic PCR were performed at room temperature. E. coli harboring the amplification was purchased from Stratagene (La Jolla, U.S.A.). The p6xHis119 vector containing a mutated bglA gene was cultured at QIAquick Gel Extraction Kit, a purification kit for PCR products or 37oC in a shaking incubator for 18 h, and was then harvested by DNA restriction fragments, was obtained from Qiagen (Hilden, centrifugation at 10,000 ×g for 30 min at 4oC. The cell pellet was Germany). A silica gel 60 F254 thin-layer chromatography plate thoroughly resuspended in 100 mM Tris-HCl buffer (pH 7.5) and from Merck (Haar, Germany) was used for sugar analysis. The disrupted by sonication on an ice bath. The cell lysate was centrifuged determination of β-glucosidase activity was performed with p- at 16,000 ×g for 30 min at 4oC to pellet down the cellular debris. nitrophenyl-β-D-glucopyranoside

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