Development Of New Anti-autolysis And High-activity Trypsin

Trypsin is an alkaline serine proteolytic enzyme,which is widely used in food processing,medicine and scientific research.Trypsin specifically hydrolyzes the peptide bond at the carboxyl end of arginine and lysine,and has strong amino acid site specificity.Therefore,it is used as a significant tool enzyme in the field of scientific research.Traditionally,trypsin is mainly extracted from the pancreas of animals,with limited raw materials and difficulty in separation and purification.In addition,trypsin will also attack its own lysine and arginine sites,causing its enzyme activity to decline rapidly during storage or use.In view of this,it is necessary to obtain novel enzyme molecules with better performance through microbial recombinant expression and molecular mutation modification research.In this study,pig-derived trypsin was used as the research object.First,the gene heterologous expression of trypsin was achieved by genetic recombination,and the autolysis sites of trypsin was determined by autolysis site analysis.Then,homologous sequence alignment was used to determine the type of naturally mutated amino acid at the autolysis site,and molecular simulation techniques were used to analyze the feasibility of the type of mutation.Finally,on the basis of verifying the positive single mutant,combined mutations were made on the autolysis site of trypsin,and finally the multi-point combination mutant with better anti-autolysis effect and better specific activity performance was obtained by screening,and the specificity to amino acid was evaluated.The research results are as follows,In this study,we used the pPIC9K plasmid as the expression vector and P.pastoris GS115 as the expression host to successfully achieve heterologous expression of pig-derived trypsin and obtain wild-type trypsin with a specific activity of approximately 16,500 U/mg(BAEE).The LC-ESI-Q-TOF MS/MS method was used to analyze the autolysis sites,and it was preliminarily determined that trypsin tends to cleave at K133,K157,R115,R107,K97,K77 and R57 sites.The homologous sequence alignment and molecular simulation techniques identified the types of anti-autolysis mutations,namely R107H,R107L,R115S,R115T,K133A,K133L,K147D,K157E,K208P and K210A.On this basis,through combination mutation and performance screening,we have obtained multi-point combination mutants R107L/R115S/K133A、R107L/R115T/K133A、R107L/R115S/K133A/K147D、R107L/R115S/K133A/K147D/K157ER107L/R115S/K 133A/K147D/K1 57E/K208P/K210A.Which are resistant to autolysis and has better specific activity performance.Its anti-autolysis effect higher than wild-type trypsin,which were improving 2.33 times,2.13 times,2.76 times,2.86 times and 3.07 times,respectively.Its initial specific activity alsohigher than wild-type trypsin,whichwere improving 1.74 times,1.46 times,1.34 times,1.12 times and 0.09 times,respectively.The anti-autolysis effect and enzyme activity of trypsin are improved to varying degrees,which has huge development value.Based on the LC-MS/MS method,the site specificity of the substrate was evaluated.The results showed that the type and relative abundance of the enzymatic hydrolysis products did not change significantly compared with the wild type,indicating that the site specificity of these trypsin mutants did not change significantly.The new trypsin obtained in this study has the advantages of good anti-autolysis effect,high specific activity and no obvious change in specificity.The highest expression of trypsinogen in pichia pastoris cells was 0.43 mg/mL.It can be used for large-scale industrial production and significantly improve the application effect.Applications in other fields are of great significance.