Improve The Catalytic Properties Of Phospholipase A1by DNA Shuffling

Phospholipase A1(PLA1, EC3.1.1.32) is an enzyme that hydrolyzes phospholipids and produces2-acyl-lysophospholipids and fatty acids.It has wide applications in many fields such as Vegetable oil degumming, baked goods industry, dairy processing industry and egg yolk industry. We started late development than the lag in the development of the enzyme. Currently, industrial phospholipase A1also rely on imported from abroad. Therefore, it is a rewarding job to develop the phospholipase A1strains by various breeding methods.The strain W22which have phospholipase activity was identified by observed strains lecithin as the sole carbon source. Gas chromatography identified the type of enzyme production phospholipase A1. Identification of the genus Bacillus Bacillus cereus by means of the strains of morphological, physiological and biochemical analysis and molecular biological identification. Application GenBank accession number: KC178715.Optimized Fermentation conditions by shake flask fermentation. Strains with the highest enzyme activity when initial pH8.0, culture temperature of32℃, shaking speed200r/min fermentation12h. Enzyme activity reached9.12U/mL, relative to the initial enzyme activity of6.74U/mL increased35.3%.Wild mushroom W22was breeded by UV mutagenesis and plasma mutagenesis (Atmospheric and Room Temperature Plasma,ARTP).Gain several strains with enzyme improved and genetic stability. Based on UV mutagenesis W22-a W22-g and Plasma mutagenesis W22-5, protoplast fusion breeding.Investigated the conditions of protoplast preparation and inactivated.Finally,WR-2was screened from many fusants. Colonies is small than W22and activity reached17.78U/mL.Based on engineering bacteria BPU (plaB-pUC19) which containing phospholipase Al gene. Combined with error-prone PCR and DNA Shuffling technology,we constructed rearrangement phospholipase Al expression library.M2was gain in the library through twice DNA shuffling. The mutant enzyme has good heat tolerance,while maintain70%enzyme activity after50℃for half an hour. Under same induction conditions for enzyme production the enzyme activity of E. coli expression system of plaB gene BP28(plaB-pET-28a (+)) was16U/mL, while which of M2could reach23.7U/mL, increased by48.13%.Sequencing and simulation of protein three-dimensional structure showed that total of six amino acid mutations occurred.And four mutations near the predicted enzyme active center. The amino acid changes may enhance the rigidity of the catalytic center, thus to some extent exhibit on the thermal stability of the mutant enzymes. The mutations of Glyl66lead to changes in the structure of the a-helix structure.This paper has done a lot of work in phospholipase A1breeding, tried emerging mutagenesis techniques of plasma mutagenesis,also gain higher enzyme activity of strain by protoplast fusion. Has done useful work for phospholipase breeding. Further, since the defect of the experimental material itself,gene reorganization failed to get more than the parental chimeric mutant gene. This paper lays the foundation for industrial application of the phospholipase A1, and also provides theoretical basis for the development of phospholipase A1which is high-yield and perform excellent catalytic property.