Properties and Inhibition Kinetics of Recombinant Bowman-Birk Trypsin Inhibitor

Bowman-Birk soybean trypsin inhibitor (BBTI), extracted from soybean (Glycine max L.) seeds, possesses insect resistance and anti-tumor properties. However, its specific mechanisms of action are still unknown. An efficient method to produce recombinant BBTI (rBBTI) in E. coli was reported. Some biochemical properties of rBBTI were revealed and the inhibition mechanism of BBTI was discussed. The rBBTI was successfully expressed with E. coli (BL21) expression system, and was further purified by Ni affinity chromatography and DEAE-FF column efficiently. The BBTI and rBBTI showed similar biochemical properties. The optimum conditions for inhibiting trypsin were pH 8 and 25 ℃, and the optimum conditions for inhibiting chymotrypsin were pH 9 and 16 ℃. BBTI and rBBTI were stable below 37 ℃. The inhibition kinetics assay of BBTI and rBBTI against trypsin as Lineweaver-Burk plots analysis showed an increased Michaelis constant (K_m) and a decreased maximum reaction rate of enzyme (V_max) with N-benzoyl-L-arginine ethyl ester(BAEE) as substrate. It suggested that BBTI and rBBTI were anti-competitive inhibitors interacted with trypsin. Both the inhibition kinetics assay of BBTI and rBBTI against chymotrypsin as Lineweaver-Burk plots analysis showed an unchanged K_m and a decreased V_max with N-acetyl-L-tyrosine ethyl ester(ATEE) as substrate. It suggested that BBTI and rBBTI were anti-competitive inhibitors interacted with chymotrypsin. Molecular modeling showed that LYS-16 of BBTI (trypsin active site of BBTI) interacted with residues of trypsin, forming effective hydrogen bonding interactions. However, there were hydrophobic residues at chymotrypsin activity domain of BBTI, and forming hydrophobic interactions with residues of chymotrypsin. These provide a reference for understanding the inhibition mechanism of BBTI, and the different inhibition rate of BBTI against trypsin or chymotrypsin.