Abstract:
:GH11 xylanase XynJ from Bacillus sp. strain 41M-1 has a β-jellyroll fold composed of eight β strands with a deep active-site cleft. We hypothesized that the thermostability of XynJ will increase if the flexibility of the β strands in the jellyroll structure is decreased without impairing activity. To verify this hypothesis, we introduced random mutations into Tyr13-Arg104 and Gly169-Tyr194, both of which are located in the β-jellyroll fold of XynJ, to construct a site saturation mutagenesis library. By screening 576 clones followed by site saturation mutation analysis of Thr82, T82A was selected as the most thermostable variant. In the hydrolysis of beechwood xylan at pH 7.8, the temperatures required to reduce initial activity by 50% in 15 min were 61 °C for the wild-type XynJ (WT) and 65 °C for T82A. The optimum hydrolysis temperatures were 60 °C for WT and 65 °C for T82A. There was little difference in the kcat and Km values and the pH dependence of activity between WT and T82A. Crystallographic analysis of WT and T82A revealed that thermostabilization by the T82A mutation might result from the removal of unfavorable van der Waals interactions. Thus, a highly thermostable XynJ variant was generated without impairing activity using this mutation strategy.
journal_name
Enzyme Microb Technoljournal_title
Enzyme and microbial technologyauthors
Takita T,Nakatani K,Katano Y,Suzuki M,Kojima K,Saka N,Mikami B,Yatsunami R,Nakamura S,Yasukawa Kdoi
10.1016/j.enzmictec.2019.109363subject
Has Abstractpub_date
2019-11-01 00:00:00pages
109363eissn
0141-0229issn
1879-0909pii
S0141-0229(19)30100-0journal_volume
130pub_type
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