Research On The New Inhibitory Peptide Aldehyde Of Alkaline Protease Savinase

Subtilisins are extracellular serine proteases from Bacillus,and highly efficient in protein hydrolysis under alkaline conditions.Savianse is one kind of Subtilisin.Savianse has an extensive application in multiple industries such as washing,food,leather,and so on,of which the washing industry accounts for about 2/3 of the global demand for alkaline proteases.Proteases can be inactivated due to self-cutting,so the liquid detergent containing alkaline proteases needs to add inhibitors in the process of preservation to avoid the reduction of washing performance due to its self-cutting.At present,boric acid inhibitors are widely used,which are not friendly to the environment and human health,therefore,it is urgent to develop green,environmentally-friendly and safe inhibitors for alkaline proteases.Nowadays,the development of peptide inhibitors is a major research direction.The previous research of our group found that the aldehyde-modified product of a small peptide(peptide A),peptide aldehyde A,exhibited good inhibitory activity against Savinase,and had the potential as an inhibitor to maintain the stability of Savinase in liquid preparations and detergents.In order to further verify the stabilization effect of the novel peptide aldehyde on Savinase preservation,this study investigated the effect of peptide aldehyde A in stabilizing Savinase in liquid enzyme system and detergent matrix system respectively,and on this basis,the composition of liquid enzyme stabilizer was investigated.Finally,the biosynthesis method of peptide aldehyde A was also explored.The specific research results are as follows:First,in the liquid enzyme system and detergent matrix system,the effect of peptide aldehyde A in stabilizing Savinase was investigated and verified by comparison with the mainstream inhibitor 4-FPBA and domestic and foreign commercial enzymes.Stored under accelerated conditions at 37°C for several months,and compared the enzyme activity retention rate at different time points to investigate the effect of peptide aldehyde A on stabilizing Savinase.In the liquid enzyme system,16 mM peptide aldehyde A showed an obvious stabilization effect,and the stabilization effect was similar to that of 4-FPBA.Peptide A showed little stabilizing effect.In the detergent matrix system,16 mM peptide aldehyde A still showed an obvious stabilizing effect on Savinase,and the enzyme activity retention rate of peptide aldehyde A group was 77.0 % on the 45 th day.The concentration of peptide aldehyde A in the detergent matrix was further optimized,and the stabilization effect was relatively good when the concentration was greater than 0.004%.Secondly,the compound research on the formulation of liquid enzyme stabilizer was carried out.The main components used in compounding are glycerin,propylene glycol,water,inhibitor and surfactant.The results showed that the stabilizer with the formula of 30 %glycerol,50 % water and 20 % propylene glycol had the best effect,and the enzyme activity retention rate reached 75% after 90 days of storage under accelerated conditions at 37 ℃.Finally,methods for the biosynthesis of peptide A and peptide aldehyde A were explored.Different combinations of peptide A multiple sequences were designed and synthesized by Pichia pastoris yeast expression system and Escherichia coli expression system.The product was specifically cut by chymotrypsin,and finally monomeric peptide A was successfully prepared.Carboxyl-terminal aldolylation We explored two methods,one is biological method,heterologous expression of carboxylate reductase,using this enzyme to modify the carboxylterminus of peptide A,but the results are not as expected,Pichia pastoris expressed Carboxylic acid reductase did not show activity;the second is chemical method,and the aldehyde modification of peptide A was successfully carried out in cooperation with other research groups,but the yield is still relatively low.This study investigated and verified a novel peptide inhibitor of Savinase,developed its liquid enzyme compound formulation,and explored the biosynthesis method of this inhibitory peptide.This research is of great significance for improving the application level and competitiveness of protease in my country’s washing industry.