Directed Evolutoin Of Phosphlipase A1 From Serratia Marcescens

Phospholipase A1 is a class of enzymes that can hydrolyze phospho-lipids, participate in a variety of regulation of physiological function in vivo and widely present in a variety of organisms.Phospholipase A1ean hydrolyse phospholipids to generate lysophosphatide which can be used as food additives and emulsifiers and widelyused in food processing, oil degumming, phospholipids modified, pharmaceutical and other industries. Phospholipase A1 has a huge industrial application view. Currently phospholipase A1 used by the industry is imported from abroad. Therefore, Itis a meaningful job to transform phospholipase Al gene by directed evolution to solve the problem such as poor enzymatic activity, low yields and unstable and so on.In this paper, the gene of phospholipase Al plaB from Serratia marcescens PL-06 were directed evolution by error-prone PCR. The error-prone PCR product was obtained by introducing various concentrations of Mg2+, Mn2+ and was cloned into pET28a (+) expression vector. And a mutant library was constructed. After the first and secondary screening, one mutant which showed significant elevated activity was screened by hydrolysis ring, and was named M8. The protein was expressed by the gene of phospholipase A1 with helper protein and was Exocytosis protein after SDS-PAGE and sequencing analysis.We found that the geneplaBhas 11 basesmutationresulting in 6 amino acid mutations.And phospholipase A1 protein revealed three amino acid substitutions:Y97C and P98S,and auxiliary protein revealed four amino acid substitutions: P46L,Q58L,N136D,H233R.Optimized fermentation condition by shake flask fermentation. The results showed that the mutant M8 has the highest enzyme activity when optimal induction time is 2h, the optimal induction temperature is 37℃, the optimum concentration of IPTG induction of 0.3 mmol/L. The wild enzyme’s optimal induction time is 4h, the mutant M8 hasn shorten the induction time. The enzyme activity of strain BP28 was 16 U/mL and The enzyme activity of the mutants was 19.6 U/mL and22.5% higher than that of the wild-type control. The enzyme activity of the mutants was22.5 U/mL and 14.8% increased after optimal Fermentation conditions.Subsequently, biochemical properties of the mutants were investigated. The optimum pH of mutant M8 is 7.0. Compered with the wild-type control, Mutant M8 exhibits alkali resistance and its optimum reaction temperature decreased to 40℃.The analysis of bioinformatics demonstrated that there is no significant changes in the structure of the mutant enzyme. And the activity of phospholipase A1 maybe affected by the 98th amino acid.In this paper, the gene of phospholipase A1 (plaB) has been directed evolution in vitro to obtain a mutant strain wuich has higher activity. And the relationship between the structure and function of phospholipase A1 has also been discussed. These results have provided a base for the study ofthe relationship between structure and function of phospholipase A1and provides a theoretical basis for the development of high-yield phospholipase A1 with excellent catalytic properties of phospholipase A1 provides a theoretical basis.