Molecular Engineering And Expression Optimization Of Streptomyces Griseus Trypsin

Trypsin（EC 3.4.21.4）is a serine protease widely existing in nature that can specifically hydrolyze the carboxyl terminus of arginine or lysine.Trypsin activates other proteases by participating in the enzymatic digestion cascade reaction.Streptomyces griseus trypsin（SGT）is a bacterial-derived trypsin.SGT has similar tertiary structure and catalytic mechanism to bovine（porcine）trypsin.Besides,the soluble expression of SGT mature enzyme in Pichia pastoris GS115 has been achieved in our laboratory in the early stage.It is considered to become a potnetial substitute of commercial trypsin.This paper focuses on the substrate specific modification of SGT and the improvement of the secretion level of SGT in P.pastoris.The main contents are as follows:（1）Expression optimization of SGT.Firstly,based on the secretomic data of Pichia pastoris GS115 under methanol-induced conditions,the pre region of theαsignal peptide was modifed,and seven proteases with high secretion levels were chosen:FLO10,SCW11,GAS1,MSB2,DAN4,PHO5 and EXG1.Another two pre-sequences（SP1 and SP4）previously verified by our laboratory were also selected.All these pre-sequences were used to replace the pre-sequence of theαsignal peptide.The screening results indicate that FL010 has the best effect on increasing enzyme activity from 37.8 U·m L^-1 to 59.8 U·m L^-1.By truncating amino acids 57-70 in the pro region of theαsignal peptide,followed by fusion at the C-terminus of the HL28 short peptide,the enzyme activity was further increased to 62.9 U·m L^-1.Finally,by co-expressing the signal peptide processing proteins Kex2 and Ste13,the secretion efficiency of SGT was further improved,and the final enzyme activity reached 68 U m L^-1,the secretion level was 1.8 times higher than that of theαsignal peptide.（2）Evolution-guided strategy improves SGT substrate specificity and catalytic efficiency.Sixty trypsin sequences from different sources were selected for multiple sequence alignment,and the occurrence frequency of amino acids at each position was analyzed.Potential"hot spots"in the SGT sequences were selected and mutated to amino acids with an occurrence frequency greater than 40.Among these single-point mutants,the enzyme activities of mutants A45S,V177I,and E180M were increased to 75.3 U·m L^-1,74.83 U·m L^-1,and 77.2 U·m L^-1,respectively.Then we performed saturation mutation screening and molecular docking at the T190 site.The results of saturation mutation showed that T190S was the most effective mutant.The molecular docking results showed that the interaction between S189 and the substrate was enhanced,and the interaction between S190 and the substrate was weakened,thus making it easier for arginine substrates with longer side chains to bind to mutant proteins.Eventually,the above change resulted in an increase in the preference of T190S for arginine substrates.（3）Performance characterization of combinatorial mutants.We carried out combined Iterative transformation of SGT based on evolution-guided strategy and scale-up culture.We carried out combined iterative mutagenesis of the above-mentioned single-point mutants with excellent performance.Among them,the combinatorial mutant A45S/V177I/E180M has the most obvious increase in enzyme activity,reaching 98 U·m L^-1.The enzyme activities of the combinatorial mutants introduced into T190S all decreased to varying degrees,but the substrate catalytic ratio（R/K）was increased to a certain extent.Among them,the combinatorial mutant V177I/E180M/T190S has the highest substrate catalytic ratio of 4.6,which is 1.1 times that of the control strain FLO10.At the same time,we also characterized the specific enzyme activity and substrate kinetics-related parameters of the combined mutants.The overall specific enzyme activity change trend is basically consistent with the change trend of amidase enzyme activity;the combined mutant A45S/V177I/E180M has the highest catalytic efficiency,the k_cat/K_m is1.21×106 s^-1·m M^-1,which is 13.7 times that of the control strain.Another combinatorial mutant V177I/E180M/T190S with the highest substrate catalytic ratio（R/K）had a specific enzyme activity of 1698 U·mg^-1.However,the K_m value increased and the binding ability to the substrate decreased,but the catalytic efficiency of the substrate increased,resulting in a slightly higher overall catalytic ability than the control strain FLO10.In the structural analysis of the combined mutant A45S/V177I/E180M,we found that the distance between H57 and D102 in its catalytic triad was shortened from 7.0（?）to 6.4（?）,which accelerated the rate of proton transfer during its catalytic process,which may be a performance factor One of the reasons for the improvement.In the analysis of its Ca²⁺binding interaction force,the introduction of two single point mutations V177I and E180M strengthened the binding force of the mutant with Ca²⁺,which may be the second reason for the improved performance.Finally,we performed fed-batch fermentation on these two combined mutants at the 3-L fermenter level,in which the combined mutant V177I/E180M/T190 extracellular amidase enzyme activity finally reached 460 U·m L^-1.The extracellular amidase activity of mutant A45S/V177I/E180M was further increased to 2506U·m L^-1,which is the highest level reported so far.In conclusion,the research has laid a good foundation for the industrial production of SGT and the preparation of insulin.