2019 ; Vol.29-3: 419~428
|Author||Jiadi Li, Xinli Li, Yuanming Gai, Yumei Sun, Dawei Zhang|
|Place of duty||Dalian Polytechnic University, P.R. China,Chinese Academy of Sciences, P.R. China,Chinese Academy of Sciences, P.R. China|
|Title||Evolution of E. coli Phytase for Increased Thermostability Guided by Rational Parameters|
J. Microbiol. Biotechnol.2019 ;
|Abstract||Phytases are enzymes that can hydrolyze phytate and its salts into inositol and phosphoric
acid, and have been utilized to increase the availability of nutrients in animal feed and
mitigate environmental pollution. However, the enzymes’ low thermostability has limited
their application during the feed palletization process. In this study, a combination of B-value
calculation and protein surface engineering was applied to rationally evolve the heat stability
of Escherichia coli phytase. After systematic alignment and mining for homologs of the original
phytase from the histidine acid phosphatase family, the two models 1DKL and 1DKQ were
chosen and used to identify the B-values and spatial distribution of key amino acid residues.
Consequently, thirteen potential amino acid mutation sites were obtained and categorized into
six domains to construct mutant libraries. After five rounds of iterative mutation screening,
the thermophilic phytase mutant P56214 was finally yielded. Compared with the wild-type,
the residual enzyme activity of the mutant increased from 20% to 75% after incubation at 90°C
for 5 min. Compared with traditional methods, the rational engineering approach used in this
study reduces the screening workload and provides a reference for future applications of
phytases as green catalysts.|
|Key_word||Phytase, B-value, protein surface engineering, thermostability|
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