Study On Substrate Specificity Of Myofibril-bound Serine Proteinase From Crucian Carp

Myofibril-bound serine proteinase(MBSP)is a trypsin analogue commonly found in animal muscles.Since there is a lacks in-depth research on the role of structure and function of MBSP,therefore,this paper probes into the substrate specificity of MBSP,and explores the relationship between structure and function,in order to expand its application value.In this study,the yeast expression strains SMD1168-pPICZ?A-tMBSP were successfully constructed and induced expression by genetic recombination technology.Expression produces named as D170 S,D170T,D170 K,S171A,G193 A,G203A,D170T/G203 A,G193A/G203 A,which were identified by western blot using rabbit anti crucian carp MBSP polyclonal antibody.The enzymatic activity was determined by the fluorescence substrates Boc-Phe-Ser-Arg-MCA(BFSR),Boc-Gln-Arg-Arg-Arg-MCA(BQRR),Boc-Glu-Lys-Lys-MCA(BVLK)and Boc-Val-Leu-Lys-MCA(BEKK),and the results showed that only 4 mutants(S171A,G193 A,G203A and G193A/G203A)have the ability to hydrolyze substrates.Substrate specificity of enzyme was further analyzed,and the results suggested that BQRR was the optimal substrate of wild type,compared with it: the optimal substrate of mutants G193 A and G193A/G203 A did not alter;the optimal substrate of mutant S171 A did not change,but it demonstrated a unique preference for only hydrolyzing substrates containing Arg residue;the optimal substrate of mutant G203 A was changed into BEKK,which was obviously biased towards the hydrolyzing of substrates with the Lys residue.Substrate character of mutant S171 A were specific than other mutants,so it was chosen to analyze in depth.The results of circular dichroism spectra and homology modeling revealed that Ser171 mutation has little effect on the structural stability of MBSP;Molecular docking analysis results showed that compared with wild-type,the docked energy values of BVLK,BEKK and mutant S171 A were changed from negative to positive,in addition,the active regine of BVLK,BEKK completely deviate from the catalytic triad and substrate-binding pocket of MBSP.Due to the key area of substrates combine with mutant enzyme changed,results in mutant S171 A only selective cutting Arg residues,which illustrating catalytic triad and substrate-binding pocket of MBSP plays an important role during enzymatic hydrolysis substrate.MBSP can degrade myofibrillar proteins efficiently,which was responsible for the modori phenomenon during surimi production.Lina bean trypsin inhibitor(LBTI)and soybean trypsininhibitor(STI)were selected used in the inhibitory kinetics analysis.For MBSP(wild-type and S171A),LBTI and STI were belong to competitive inhibitory types in reversible inhibition,which can compete with substrate for the binding sites of enzyme molecules.Therefore,MBSP can be inhibited by trypsin inhibitor,which remission the modori phenomenon.Furthermore,the enzymatic hydrolysis of whole protein(using MBSP and trypsin)was analyzed.The results showed the major allergens of Scylla paramosain,especially the arginine kinase had the resistance to digestion by trypsin,can be degraded by MBSP.Compared with their digestiblity rate,S171 A is the fastest,and arginine kinase can be degraded into small molecular fragments during 1 minute.Therefore,structure stability of MBSP is constant by protease molecule modification.Due to the role area of substrates with mutant enzyme change eventually led to mutant S171 A only selective cutting Arg residues by molecular docking technology for insight into the combined mechanism.In this paper,MBSP has some advantages,such as high-yield production,higher thermal stability and easier preparation,makes it expected to replace trypsin.