Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Modulation of the Regioselectivity of a Thermotoga neapolitana $\beta$-Glucosidase by Site-Directed Mutagenesis

  • Choi, Ki-Won (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Park, Kyung-Min (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Jun, So-Young (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Park, Cheon-Seok (Department of Food Science and Technology, Graduate School of Biotechnology and Institute of Life Sciences and Resources, KyungHee University) ;
  • Park, Kwan-Hwa (Department of Food Science and Biotechnology, School of Agricultural Biotechnology, Seoul National University) ;
  • Cha, Jae-Ho (Department of Microbiology, College of Natural Sciences, Pusan National University)
  • Published : 2008.05.31
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Abstract

Thermotoga neapolitana $\beta$-glucosidase (BglA) was subjected to site-directed mutagenesis in an effort to increase its ability to synthesize arbutin derivatives by transglycosylation. The transglycosylation reaction of the wild-type enzyme displays major ${\beta}(1,6)$ and minor ${\beta}(1,3)$ or ${\beta}(1,4)$ regioselectivity. The three mutants, N291T, F412S, and N291T/F412S, increased the ratio of transglycosylation/hydrolysis compared with the wild-type enzyme when pNPG and arbutin were used as a substrate and an acceptor, respectively. N291T and N219T/F412S had transglycosylation/hydrolysis ratios about 3- and 8-fold higher, respectively, than that of the wild-type enzyme. This is due to the decreased hydrolytic activity of the mutant rather than increased transglycosylation activity. Interestingly, N291T showed altered regioselectivity, as well as increased transglycosylation products. TLC analysis of the transglycosylation products indicated that N291T retained its ${\beta}(1,3)$ regioselectivity, but lost its ${\beta}(1,4)$ and ${\beta}(1,6)$ regioselectivity. The altered regioselectivity of N291T using two other acceptors, esculin and salicin, was also confirmed by TLC. The major transglycosylation products of the wild type and N291T mutant were clearly different. This result suggests that Asn-291 is highly involved in the catalytic mechanism by controlling the transglycosylation reaction.

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References

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