Introduction of a disulfide bridge enhances the thermostability of a <Emphasis Type="Italic">Streptomyces olivaceoviridis</Emphasis> xylanase mutant |
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Authors: | H M Yang B Yao K Meng Y R Wang Y G Bai N F Wu |
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Institution: | (1) Feed Research Institute, Chinese Academy of Agricultural Sciences, 100081 Beijing, China;(2) Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081 Beijing, China |
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Abstract: | Substitution of the N-terminus of Streptomyces olivaceoviridis xylanase XYNB to generate mutant TB has been previously shown to increase the thermostability of the enzyme. To further improve
the stability of this mutant, we introduced a disulfide bridge (C109–C153) into the TB mutant, generating TS. To assess the
effect of the disulfide bridge in the wild-type enzyme, the S109C-N153C mutation was also introduced into XYNB, resulting
in XS. The mutants were expressed in Pichia pastoris, the recombinant enzymes were purified, and the effect of temperature and pH on enzymatic activity was characterized. Introduction
of the disulfide bridge (C109–C153) into XYNB (XS variant) and TB (TS variant) increased the thermostability up to 2.8-fold
and 12.4-fold, respectively, relative to XYNB, after incubation at 70°C, pH 6.0, for 20 min. In addition, a synergistic effect
of the disulfide bridge and the N-terminus replacement was observed, which extended the half-life of XYNB from 3 to 150 min.
Moreover, XS and TS displayed better resistance to acidic conditions compared with the respective enzymes that did not contain
a disulfide bridge. |
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Keywords: | Xylanase Thermostability Disulfide bridge N-terminus replacement Synergistic effect |
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