Abstract

The upregulation in expression of endogenous proteases of Bacillus thuringiensis kurstaki (Btk) HD-73 could be achieved by anti- Nitrogen metabolite repression (anti-nmr). In pursuance of this, a new medium was developed limiting N₂-source to detect the induced proteases. Rice Gruel (RG) medium composed of leftover liquid containing starch and minimal proteins upon cooking devoid of any external protein or amino acid supplement was evaluated for δ- endotoxin and protease synthesis. Efficiency of four varieties of rice (Gutisharna, Najirshail, 28 and 29) were assessed along with Trypticase Soy Broth (TSB). The experiment was carried out in shake flask culture incubating at 30 °C and 200 rpm up to 7 days. Following (NH4)2SO4 precipitation, desalting and partial purification by size exclusion chromatography (SEC) through Sephadex G-75 column, extracellular enzymes were recovered. Estimation of alkali soluble protein and enzyme activity was performed by Bradford method and azo-casein digest method respectively. Statistical analysis was performed by Tukey HSD test. TSB medium with 2.4-fold higher δ- endotoxin yield (0.3±0.1 mg/ml) was rendered superior to RG media and the differences among the RG media were not significant in this case. Hence, higher protease activity was observed in RG-G medium (3263 u/ml) followed by RG-N (2147), RG-28 (1772), RG-29 (1285) and TSB (462) media. A 7-fold increase in protease activity was observed in RG-G to TSB medium with significant differences among all except RG-N and RG-28. The highest protease activity was attained at 96 hrs in RG-G medium. A positive correlation (+0.508) between the protease activity and the δ- endotoxin was observed. Multiple discrete fractions (4, 10, 16 and 18), obtained through SEC, demonstrated high protease activity which were characterized by LC-MS and expression of multiple protease enzymes were evidenced. Due to anti-nmr degradation of complex proteins occurred to make N₂-source available which facilitated the synthesis of δ- endotoxins with times.

Keywords: Protease, RG media, Bacillus thruingiensis, δ- endotoxins, correlation.