Genome Mining,Heterologous Expression And Enzymatic Properties Characterization Of Trypsin

Trypsin（EC 3.4.21.4）is a serine protein that specifically cleaves arginine or lysine residues in the peptide chains.It is widely used in leather bating,food processing,clinical diagnostics and biochemical assays.The commercial production of trypsin currently relies on extraction from bovine pancreas,which has several drawbacks such as limited source of raw materials,high isolation costs and immunogenicity hazards.The production of microbial trypsins is convenient,fast and low cost,therefore receiving much attention in recent years.In this thesis,novel trypsin genes were obtained through genome mining and their enzymatic properties were characterized after heterologous expression in Komagataella phaffiias.Moreover,mutants with better enzymatic properties were obtained through propeptide engineering,semi-rational modification of autodegradation sites.Finally,the fermentation condition of trypsin was optimized.The main findings of the thesis were as follows:（1）Four novel trypsins from H.spanies,B.taurus,P.vannamei and S.fradiae ATCC14544were screened by genome mining and solubly expressed in K.phaffiias GS115.The enzymatic properties of the four recombinant enzymes were characterized.Though the enzymatic activity of SFT was not the highest,its optimum temperature and p H values were 45^oC and p H9,respectively;Moreover,it had better stability at p H9-11,indicating that SFT had better tolerance towards high temperature and alkaline environment.This indicated this enzyme might have potential application value in leather bating.（2）To improve the expression level of SFT,the initial propeptide of SFT was substituted by the fusion tag Trx A,the unnatural peptide FVEF and the bovine-derived trypsin propeptide VDDDK.The enzymatic activity and specific activity of the obtained a SFT were 33.9 U·m L^-1and 20.6 U·mg^-1,which were 59.29%and 40.29%higher than those of wild-type.The K_m of a SFT was 0.0715 m M,which was lower than that of wild-type（0.0788 m M）,indicating that the substitution of propeptide led to better substrate affinity.（3）Through analysis of solvent accessibility of arginine and lysine in SFT structure and comparison of conserved amino acid sequences,five amino acids with potential self-degradation potential were inferred and mutated into alanine.The results showed that mutation of R124A,R135A and K295A significantly increased the catalytic properties.Among them,the enzyme activity of R295A mutant reached 39.39 U·mg^-1,which was 47.53%higher than that of WT.In addition,R295A,R315A and K333A mutants had fewer self-degradation bands.（4）Optimisation of fermentation at shake flask level.The optimum nitrogen source was tryptone at 40 g·L^-1,the optimum inorganic salt source was Ca Cl₂ at 2.5%,the optimum fermentation temperature was 30℃,the optimum fermentation p H was p H7,the optimum inoculum was 1%and the optimum loading volume was 30 m L.The fermentation enzyme activity after optimisation was 92.8 U·m L^-1,which was 2.55 times higher than that before optimisation.