Exploring The Stabilization Mechanism And Molecular Improvement Of Serine Protease

Due to the properties of efficiently hydrolyzing peptide bonds in proteins,broad substrate spectrum,and high p H stability,fungal serine proteases are widely used in various industrial applications.However,the characteristic of self-cleavage leads to poor stability,which limits the effectiveness of its application.Therefore,it is important to explore serine proteases with excellent enzymatic properties,investigate their self-cleavage and stability mechanisms,and improve their stability through enzyme engineering.First,serine protease mining was performed.Forty-one serine protease genes were screened and cloned from the GenBank database and laboratory-preserved strains,12 of which were successfully heterologous expressed in Pichia pastoris.These recombinant proteases were purified,and the properties were determined.Recombinant serine proteases have an optimum p H of 6.5-9.5,which are neutral or alkaline proteases.Among them,alkaline proteases exhibit a wider p H stability（p H 4.0-11.0）.All recombinant proteases have an optimum temperature mainly concentrated in the range between 55 and60℃with large differences in thermal stability.Their specific activities are distributed in the range between 400 and 4,000 U/mg.In addition,all proteases exhibited different degrees of self-degradation,most severely in ThSPT3,MaSPT16,and Pl SPT19.Based on the excellent enzymatic properties of ThSPT3,it was selected as the target for molecular improvement in the study.Secondly,the self-degradation mechanism of serine protease was investigated.Large differences existed in the degree of self-degradation of proteases from different fungi,but similarities existed in the composition and molecular weight of the autolysis products.ThSPT3 and MaSPT16 were used to investigate the self-degradation mechanism of the serine protease S8 family.Concentration,temperature,and catalytic activity were found to affect the autolysis rate of serine protease,and showed a positive relationship.A primary self-degradation behavior,which is not dependent on high activity,was observed in the early stages of self-degradation by controlling protease activity.Based on N-terminal and C-terminal protein sequencing techniques,key sites(Leu²³⁸-Met²³⁹ or Tyr²³⁹-Leu²⁴⁰)for the primary self-degradation behavior of ThSPT3 and MaSPT16 were identified,respectively.Subsequently,the higher frequency region where primary self-degradation occurs（more than 40 residues after the C-terminus ofα-helix₇）was identified.The region appears to be poorly conserved in the S8 family.In addition,two general self-cleavage sites of ThSPT3(Asp¹⁶⁶-Ala¹⁶⁷ and Ala¹⁷³-Ser¹⁷⁴)were identified.Rational design for the stability of the sensitive region of primary self-degradation of ThSPT3 was carried out.A combination of loop transplantation,energy calculation,surface cavity optimization,and loop anchoring strategy was used to design and optimize the secondary structure(loop₁₈,α-helix₈,and loop₁₉)of the region,respectively.The corresponding three mutants with improved enzymatic properties and self-cleavage resistance,ThSPT3-M2,M4,and M5,were obtained.By superimposition,the mutant ThSPT3-M9(M²³⁹GKDGAVAAGLC²⁵⁰→M²³⁹TLNRTTAANAC²⁵⁰/A251E/A254Q/R259L/A267E/S280N)with substantially increased self-cleavage resistance was obtained.It has a 62%increase in specific activity（4,985 U/mg vs.3,078 U/mg）,a 5°C increase in optimal temperature（65°C vs.60°C）,a7.5°C increase in melting temperature（T_m）（75.7°C vs.68.2°C）and a 38-fold increase in half-life(t_1/2)at60°C（62.4 min vs.1.6 min）.Its t_1/2 at 60°C is 12 times longer than that of commercial Pro K.Therefore,improving stability in the sensitive region of primary self-degradation is essential for optimizing protease stability and catalytic activity.The rational design strategy applied to self-degradation alleviation provides a novel idea for molecular improvement of other proteases.Further rational design was performed for the surface loop region of ThSPT3.Multiple potential self-cleavage sites exist in the surface loop region,and the probability of self-degradation behavior can be reduced by improving the stability of the loops.Glycosylation modifications and consensus sequence strategies resulted in mutants V189N and S131N/M132L with improved stability and catalytic activity,respectively.Combination with ThSPT3-M9 resulted in mutant ThSPT3-M12(S131N/M132L/V189N/M²³⁹GKDGAVAAGLC²⁵⁰→M²³⁹TLNRTTAANAC²⁵⁰/A251E/A254Q/R259L/A267E/S280N)that further improved enzymatic properties and self-cleavage resistance.Compared with ThSPT3-M9,specific activity increased from 4,985 U/mg to 6,273 U/mg,optimum temperature increased from 65°C to 70°C,T_m increased from 75.7°C to 78.6°C,and t_1/2 at 60°C increased from 62.4 min to 111.8 min.Its t_1/2 at 60°C is 22 times longer than that of commercial Pro K,providing better thermal resistance.The application experiment of enzymatic deproteinization of shrimp shell has proved that ThSPT3-M12 with high catalytic activity and stability has great application potential.It is apromising competitor for industrial protease preparations.