Inhibition Mechanism Of Recombinant Trypsin Inhibitor

Trypsin inhibitors have been studied for their application value.Among them,Bowmanbirk trypsin inhibitor and Kunitz trypsin inhibitor have been studied most wildly.In this study,we focused on the Bowman-birk protease inhibitor from soybean(SBTI)and the Kunitz protease inhibitor from bovine pancreas(BPTI),and overexpressed them with E.coli(BL21)under an optimized induction conditions.Subsequently,the two recombinant protease inhibitors were purified and separated withNi column affinity chromatography,enzyme digestion,DEAEFF anion exchange chromatography and dialysis,respectively.Finally,the enzymatic properties and inhibitory mechanisms of two highly activated recombinant trypsin inhibitors were studied.The enzymatic properties and inhibition kinetics that recombinant SBTI(rSBTI)against trypsin(Lys16-Ser17)and chymotrypsin(Leu43-Ser44)were studied,respectively.it showed that the optimal pH and temperature of rSBTI inhibiting trypsin were 8.0,25℃,However,the optimal pH for inhibiting chymotrypsin was 9.0 and the optimal temperature was 16℃.The decrease of kinetic parameters(Vmax、Km)of rSBTI inhibiting trypsin suggested that rSBTI is an anti-competitive inhibitor,with IC50 and Ki values of 0.0125 mM/mL and 4.7×10-2 mol/L.Whereas,the Kinetic analysis,when rSBTI interacted with chymotrypsin,suggested a typical non-competitivemodel with the IC50 and Ki values were 0.1 mM/mL and 1.7×10-1 mol/L.These results showed that rSBTI exhibited a higher inhibitory capacity for trypsin,and that has been further illustrated by the analysis of the interaction between rSBTI and trypsin.As for rBPTI,the inhibition Kinetics indicated that rBPTI inhibits trypsin with IC50 0.0008 mM/mL and Ki values of 3.5×10-5 mol/L,and inhibition mechanism was the same as rSBTI inhibiting trypsin.The IC50 and Ki values of rBPTI inhibiting chymotrypsin were 0.011 mM/mL and 4.5×10-3 mol/L,and rBPTI changed into a characteristic of non-competitive inhibition.Comparably,the inhibition efficiency of rBPTI against trypsin was stronger because the interactions involves more hydrogen bonds between rBPTI and trypsin.Summary,rSBTI and rBPTI were anti-competitive inhibitor inhibiting trypsin,but non-competitive inhibitor when against chymotrypsin.