Study On The Characterization, Molecular Cloning, And Expression Of A Chitosanase From Microbacterium Sp.OU01

A strain with high chitosanase activity was isolated from soil samples and identified as Microbacterium sp. It is the only Microbacterium strain that can degrade chitosan reported so far. The optimal compositions of the medium producing chitosanase were as follows (g/L): chitosan powder 10, (NH4)2SO4 19.0, glucose 1, MgSO4·7H2O 1.3, K2HPO4·3H2O 1.4, KH2PO4 0.3, NaCl 5.0 and yeast extract 3.0. After sterilization at 121 oC for 20min, main culture medium was inoculated with 4% inoculum (V/V). The main culture was carried out in 500 mL Erlenmeyer flasks containing 100 mL of medium. The flasks were kept at 30°C at 150 r/min for 96h.The chitosanase activity of fermentation liquid could reach 118U/mL when OU01 was incubated in flask under the optimal cultivition conditions. The chitosanase from OU01 strain was an inducible and secreting extracellar enzyme. The chitosanase of the strain was extracted by ammonium sulfate precipitation(40-90% saturation), and purified by ion-exchange chromatography (Q-Sepharose Fast Flow column) and gel filtration (Sephadex G-75 column), SDS-PAGE showed single band for the purified chitosanases and the molecular weight was estimated to be 30 kD(ChiN),81kD(ChiX),respectively. Apparent Michaelis contents Km and Vmax were 5 mg chitosan/mL and 1260 U/mg protein(ChiN), 1.9 mg chitosan/mL and 154 U/mg protein(ChiX), respectively. Studies on characterization of ChiN and ChiX showed the optimal temperature and pH for hydrolysis of the chitosanase were 50℃and 6.2(ChiN),60℃and 6.6 (ChiX), respectively. The chitosanase activity was stable in the pH range of 6.6-7.4(ChiN)and 5.2-7.8 (ChiX), respectively under 50℃; Mn2+ could enhance the enzyme activity of ChiN and ChiX significantly, whereas Ag+ and Hg2+ could inhibite the enzyme activity; ChiN and ChiX showed activity for hydrolysis of chitosan and carboxymethyl chitosan. ChiN degraded 95%-100% deacetylated chitosan, and ChiX degraded 86%-100% deacetylated chitosan most effectively. Through in-gel digestion and identification using LC-ESI-MS, two peptide fragments (VYFDPAVAQGK and DAFGGIR) of ChiN and ChiX were matched with Pseudomonas sp. A-01. The enzymatic hydrolysis process for preparing chitooligosaccharides was studied. Experiment results demonstrated the optimal temperature, pH value, concentration of substrate, enzyme quantity were 50℃, 6.2, 5%, 30 U/g chitosanase respectively, 12 hours later the reaction reached balance. The components of the hydrolysis product were analyzed by acetylacetone method, and the average molecular weight of the chitosan hydrolyates was estimated as about 1200. A gene (mschito) encoding chitosanase was cloned, sequenced, and compared with other functionally related genes. The deduced amino acid sequence of MSCHITO was similar to those of the classical chitosanase belonging to glycoside hydrolase family 46. Chitosanase was successfully expressed in E. coli and purified with Ni-NTA affinity chromatography. After refolded and cleaved the fusion tag, the recombinant MSCHITO with chitoanase activity was obtained.