In Vitro Evolution Of LipL41 Peroxidase

Peroxidase is a type of oxidase widely present in various animals,plants and microorganisms,and its structure mostly contains a heme prosthetic group(iron protoporphyrin).The most intensive peroxidase studied so far is horseradish peroxidase(HRP),which has the advantages of strong catalytic activity and stable enzyme activity,and is widely used in biosensors and clinical diagnosis.However,due to its high price and harsh use conditions,its application in production practice is restricted.LipL41 is the major lipoprotein of Leptospira,which has peroxidase activity in combination with heme,and can be expressed in fusion with Protein G and other functional proteins.Enzyme-linked immunosorbent assay,electron microscopy staining and semi-solid membrane immunosignal conversion Other aspects have great application potential.Due to the low activity of LipL41 peroxidase,its application is limited.Therefore,this experiment utilizes directed evolution technology to randomly mutate and recombine the target gene to construct a high-capacity mutation library,and to improve the enzymatic activity of LipL41 by high-throughput screening method based on clonal in situ enzyme activity assay.An enzyme for soluble labeling is provided in the field of detection and the like.In this experiment,the LipL41 gene was randomly mutated by error-prone PCR technology.The mutated gene was transformed into Rosetta E.coli,and the mutated gene library was constructed.After culturing on LB plate,the enzyme chromogenic substrate was sprayed,and the suspected color was selected according to the color depth.A total of 9 mutants with higher secretase activity were obtained from the mutants by enzyme activity assay.By detecting their enzyme activities,the enzyme activity improvement rate was found to be about 10%-25%.In order to further improve the LipL41 enzyme activity,this experiment used DNA shuffling technology to recombine 9 strains of secreted high-enzyme live mutant genes in vitro.A high-enzyme live mutant LipL41-P101 was obtained by screening,which was 1.9-fold higher than that of wild-type enzyme.Finally,analysis of wild-type and mutant enzymatic properties revealed that the wild-type peroxidase was more stable than the mutant enzyme LipL41-P101.The optimum reaction temperature of wild-type peroxidase and mutant enzyme LipL41-P101 was 30 °C.In terms of pH stability,the wild enzyme was more stable than the mutant enzyme LipL41-P101 at pH 5-9.The wild enzyme and mutant enzyme LipL41-P101 had the highest enzyme stability at pH=5.The optimum pH of the wild enzyme optimum and mutant enzyme LipL41-P101 was 6.In this experiment,a mutant enzyme with significantly increased enzyme activity was selected by directed evolution technology to lay the foundation for the application of the enzyme.