Tannic acid enhancing insecticidal activity of protoxin produced in Bacillus thuringiensis subsp. Kurstaki KB100 Strain Against Spodoptera exigua

Insecticidal activity was enhanced when Bacillus thuringiensis subsp. kurstaki KB100 strain containing insecticidal activity against Spodoptera exigua was mixed with tannic acid, a protease inhibitor. To investigate the cause of this result, inhibition rate of tannic acid against protease activity in midgut juice of S. exigua was measured. As a result, it was found that protease activity in midgut juice of 5. exigua was about 83.1%, 77.6%, 68.0%, and 40.1% at concentrations of 10, 20, 40 and 80 mM, respectively. Such reduction of protease activity was measured with increasing concentration. Analysis of substrate reaction in several kinds of proteases was performed. Trypsin exhibited 91.4% and 89.4% of proteolytic activity in BApNA and BPVApNA substrates, respectively. Thus, when Trypsin was treated with tannic acid, proteolytic activity was 62.2% and 54.5% in BApNA and BPVApNA substrates, respectively. Trypsin's proteolytic activity was inhibited by 29.2% and 34.9% when mixed with tannic acid. However, proteolytic activity of chymotrypsin and elastase was not inhibited by tannic acid. Digestion of protoxin produced in B. thuringiensis KB100 strain by Trypsin was analyzed using SDS-PAGE. As a result, a band appeared at about 60 kDa-70 kDa and it seemed to be inhibited by tannic acid. Digestion patterns of protoxin were measured over time. As it was over-digested in a group of Trypsin treatment, a band of protoxin at 60 kDa completely disappeared. On the other hand, when it was treated with tannic acid, a digestion inhibitor, protoxin was maintained for up to 24 hours.