title: Purification and characterization of a novel chitinase from Bacillus brevis.
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 beta mercaptoethanol, but shifted to 48 kD when heated in boiling water or treated with 8 mol/L urea at 50 degrees 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 degrees, 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.
authors.affiliation: Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Sheng
authors.initials: S
authors.lastname: Li
authors.forename: Zhi-An
authors.initials: ZA
authors.lastname: Zhao
authors.forename: Ming
authors.initials: M
authors.lastname: Li
authors.forename: Zhen-Rong
authors.initials: ZR
authors.lastname: Gu
authors.forename: Chen
authors.initials: C
authors.lastname: Bai
authors.forename: Wei-Da
authors.initials: WD
authors.lastname: Huang
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D002686:Chitin / Q000031:analogs & derivatives* / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D048271:Chitosan; D002851:Chromatography, High Pressure Liquid; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008970:Molecular Weight; D020539:Sequence Analysis, Protein; D013696:Temperature
chemical_list: D002686:Chitin; D048271:Chitosan; D002688:Chitinases