Domain structure and proteolysis of bacillus subtilis endo-β-1,4-glucanase

Abstract

The proteolysis of endo-$\beta$-1,4-glucanse (endoglucanase) in Bacillus megaterium and the study of cellulose binding domain were performed in this study. An endoglucanase gene of Bacillus subtilis was cloned previously from B. subtilis BSE616 in E. coli in our laboratory. Also, we have transferred this gene in B. megaterium ATCC 14945 which was cellulolytic negative strain. This intact endoglucanase (52kD) secreted into the culture medium was gradually cleaved to truncated form (33 kD) by extracellular protease. The intact endoglucanase, truncated endoglucanase and extracellular protease were purified to certify the proteolysis and domain structure. The extracellular protease was purified by Q-Sepharose, Sephadex and Hydroxyapatite chromatography. The molecular weight of the purified extracellular protease was 38 kD. The protease showed maximal activity at 55$^\circ$C and at pH 7.5. The protease that requires $Ca^{++}$ for its activity was metal protease. The intact endoglucanase was purified by affinity towards insoluble cellulose and Mono-Q Chromatography. The truncated endoglucanase was purified by gel filtration and chromatofocusing. The cleavage of the intact endoglucanase was investigated by using immunoblotting and reaction with purified extracellular protease. The secreted form of endoglucanase was 52 kD at early log phase, and this enzyme was further turther into the smallest form (33kd) by the extracellular protease.silmilar results were obtained with chymotrypsin and trypsin.from the N-terminal sequencing data, it was concluded that direction of cieavage is from C-terminus. The molecular activities of these enzymes towards the soluble substrayes were identical. However, the insolube substrate, Avicel, was hydrolyzed slowly by the intact endgoglucanase on the other hand the truncated endolucanase did not hydrolyze the Avicel. Isoelectric points (pI) of the intact and truncated endoglucanase were 6.2 and 5.6, respectively. Optimum pH and temperature ...