Purification and Characterization of a Novel Chitinase from Bacillus brevis

Purification
and Characterization of a Novel Chitinase from Bacillus brevis

LI
Sheng, ZHAO Zhi-An, LI Ming, GU Zhen-Rong, BAI Chen, HUANG Wei-Da*

(
Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai
200433, China)

Abstract
An extracellular chitinase secreted by Bacillus brevis was purified
to homogeneity by a combination of ammonium sulfate precipitation, Phenyl-Sepharose
hydrophobic-interaction chromatography and DEAE anion-exchange chromatography.
On SDS-polyacrylamide gel electrophoresis analysis, the purified enzyme showed
a mass of 85 kD even in the presence of β-mercaptoethanol, but shifted to 48
kD when heated in boiling water or treated with 8 mol/L urea at 50 ℃ for 10
min. The depolymerization of subunits was accompanied with the loss of chitinase
activity, and removing denaturing factors by dialysis could restore the dimer
structure and enzymatic activity. The enzyme had an isoelectric point of 5.5
and an optimal temperature of 60 ℃, and was most active at pH 8.0. The enzymatic
activity was stable at pH 6-10, and inhibited by Ag⁺. Ten N-terminal
amino acids were determined to be AVSNSKIIGY, demonstrating that the purified
enzyme was a novel one. The hydrolysis pattern of the purified enzyme indicated
that the chitinase was an endochitinase. The extraordinary thermo-stability
and high resistance to proteolysis provide the enzyme with a good prospect to
be used as a new tool for biocontrol.