Characterization of a thermostable phytase from Bacillus licheniformis WHU and further stabilization of the enzyme through disulfide bond engineering

•A thermostable phytase PhyBL from Bacillus licheniformis WHU was identified and characterized.•A variant G197C/A358C with enhanced thermostability and proteolytic resistance was obtained by disulfide bond engineering.•The stabilization mechanism in the G197C/A358C variant was revealed by molecular dynamics analyses.•The variant G197C/A358C released more inorganic phosphorous from feedstuff than the WT at high temperatures. Phytases are important industrial enzymes widely used as feed additives to hydrolyze phytate and release inorganic phosphate. In this study, a phytase gene PhyBL isolated from Bacillus licheniformis WHU was cloned and expressed in Escherichia coli. PhyBL showed the highest activity at pH 7.0 and retained more than 40 % of its activity at a wide temperature range from 35 to 65 °C. Ca2+ significantly affected the stability and activity of the enzyme. We further improved the stability of PhyBL through extensively disulfide engineering. After constructing and screening a series of variants, an enhanced stable G197C/A358C variant was obtained. The G197C/A358C variant had a half-life at 60℃ roughly 3.8-fold longer than the wild type. In addition, the G197C/A358C variant also showed enhanced proteolytic resistance to pepsin and trypsin. The potential mechanism underlying these improvements was investigated by molecular dynamics analysis. Our results suggest that the G197C/A358C variant may have potential application as an additive enzyme in aquaculture feed.