Evolution Of Phytase For Increased Thermostability Guided By Rational Parameters

Phytase(myo-inositol hexakisphosphate phosphohydrolase)has been applied for the efficient hydrolysis of phytate and stepwise liberation of the bound phosphate as well as the various chelated metal ions.As an environmentally-friendly enzyme,phytase has been used in the feed and food industries as well as agriculture.Monogastric animals such as swine,poultry and fish are unable to digest phytate.Therefore,phytase is supplemented to their diet to increase the available phosphate content and ease the environmental pollution stemming from the phosphate released as non-digested phytate.However,the enzyme is unstable during the high-temperature palletization process,which greatly limits its application range and value.In view of this,enhancing the enzyme's thermostability and elevating its industrial robustness have become some of the most urgent demands to resolve in current agricultural biotechnology.Semi-rational design of protein molecules is a common method to modify or modify enzyme molecules.Semi-rational design method is based on the spatial structure or function of proteins to design mutation libraries that may contain positive mutations,and then screen and verify them in the mutation library.In this study,Bvalue calculation was applied to rationally evolve the heat stability of Escherichia coli phytase(GenBank: DQ513832).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.The temperature factor(B-value)is used to reflect the blurring of atomic electron densities and ambiguity of the atomic spatial state in the crystal structure.Consequently,thirteen potential amino acid mutation sites were obtained and categorized into six domains to construct mutant libraries.After screening,five mutants with good thermal stability were obtained,named BLP11-BLP16.Then 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 55% after incubation at 90°C for 5 min.The mutant phytase BLP56214 was cloned into a expression vector and introduced into P.pastoris GS115,yielding the engineered strain ZLA365.The high-efficient heterogenous expression of phytase was successfully verified by using Western Blot analysis and thermal stability measurement.The residual enzyme activity of the mutant ZLA365 increased 32.7% after incubation at 90°C for 5 min.In this study,we obtained ZLA365 mutant with good thermal stability in P.pastoris by using semi-rational design and iterative mutation method.Compared with the original wild-type phytase,the activity of the enzyme was decreased but the thermal stability was improved.Therefore,this study provides a good research basis for further improving the thermal stability and enzyme activity of phytases as green catalysts.