Heterologous Expression And Biochemical Characterization Of A Trypsin Inhibitor From Glycine Max

The protease inhibitor is a generic term of a class of substances that reduce or even eliminate protease activity but not denature it.The trypsin inhibitor is the small molecule polypeptide,belonging to serine protease inhibitor,which has extensive biological activity and potential application value in agricultural pest resistance and medicine anticancer.So far,the protease inhibitors of polypeptide properties have been mainly obtained through extraction and purification.For this reason,this study investigated the heterologous expression and biological characteristics of the trypsin inhibitor S2TI derived from soybean,which function is not clear.The main results is:(1)Through bioinformatics analysis,a 507 bp open reading frame(s2ti)was discovered tfrom the soybean genome,but the function was not clear;this open reading frame was analyzed and it has typical trypsin inhibitor structure characteristics;It consists of 168 amino acid residues and its predicted molecular mass is 19.2 kDa.Redesigned s2ti codon according to the preference of the Pichia pastoris codon and performed chemical synthesis;the recombinant yeast GS115-S2TI was constructed by molecular cloning techniques,and the inhibitory activity and the expression yield of extracellular S2TI.were 39.06 TIU/mg and 30 mg/L,respectively,at the shake flask level.(2)After the recombinant S2TI was prepared by fermentation,it was isolated and purified by ammonium sulfate fractionation and HiTrap anion ion chromatography.The specific activity of the purified S2TI to trypsin inhibitor was 803 TIU/mg increased by 26.71 times,which was higher than the crude extract specific activity.(3)The biochemical characteristics of recombinant S2TI were analyzed.Lineveaer-Burk plot showed that the inhibitor was a type of non-competitive inhibition;The inhibitory activity of S2TI is unaffected heating for 1 h at 80?;The inhibitory activity of S2TI was almost same in the range of pH 2.0-11.0.The chemical reducing agent DDTand the ultrasound can effectively remove its inhibitory effect.S2TI has a strong inhibitory effect on trypsin,the inhibition rate is up to 70%,and the inhibitory effect on chymotrypsin and other serine proteases is weak,the inhibition rate is about 30%,and there is no inhibitory effect on pepsin and papain;It has an inhibitory effect on Bacillus subtilis with a minimum inhibitory concentration of 58.59 mg/L.