NDLI: Significantly enhanced stability of glucose dehydrogenase by directed evolution

Content Provider	Springer Nature Link
Author	Baik, S. H. Ide, T. Yoshida, H. Kagami, O. Harayama, S.
Copyright Year	2003
Abstract	An NaCl-independent stability-enhanced mutant of glucose dehydrogenase (GlcDH) was obtained by using in vitro directed evolution. The family shuffling method was applied for in vitro directed evolution to construct a mutant library of GlcDH genes. Three GlcDH-coding genes from Bacillus licheniformis IFO 12200, Bacillus megaterium IFO 15308 and Bacillus subtilis IFO 13719 were each cloned by direct PCR amplification into the pTrc99A expression vector and expressed in the host, Escherichia coli. In addition to these three GlcDH genes, a gene encoding a previously obtained GlcDH mutant, F20 (Q252L), derived from B. megaterium IWG3, was also subjected to directed evolution by the family shuffling method. A highly thermostable mutant, GlcDH DN-46, was isolated in the presence or absence of NaCl after the second round of family shuffling and filter-based screening of the mutant libraries. This mutant had only one novel additional amino acid residue exchange (E170K) compared to F20, even though DN-46 was obtained by family shuffling of four different GlcDH genes. The effect of temperature and pH on the stability of the GlcDH mutants F20 and DN46 was investigated with purified enzymes in the presence or absence of NaCl. In the absence of NaCl, F20 showed very poor thermostability (half-life =1.3 min at 66°C), while the half-life of isolated mutant DN-46 was 540 min at 66°C, i.e., 415-fold more thermostable than mutant F20. The activity of the wild-type and F20 enzymes dropped critically when the pH value was changed to the alkaline range in the absence of NaCl, but no such decrease was apparent with the DN-46 enzyme in the absence of NaCl.
Starting Page	329
Ending Page	335
Page Count	7
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	61
Issue Number	4
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2003-03-05
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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