Purification and characterization of a novel chitinase from burkholderia cepacia strain KH2 isolated from the bed log of lentinus edodes,shiitake mushroom

摘要

One of the chitinases secreted in the culture filtrate of a gram-negative bacteria,Burkholderia cepacia strain KH2,which was isolated from the bed log of Lentinus edodes,Shiitake mush-rooms,was purified b y DEAE Sepharose CL-6B chromatography,followed by Sephacryl S-100 HR gel filtration.The purified enzyme was homogenous,determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),with an estimated molecular weight of 34,000 and an isoelectric point (pl) of 5.9.The enzyme was stable at pH values of 4.0-6.0,aand at temperatures up to 50degC;the optimum pH and temperature were 4.5 and 50degC,respectivley.The enzyme exhibited higher activities toward chitosan 7B,a 62% deacetylated chitosan,than toward the highly deacetylated chitosan substrates.The enzyme was observed to drastically hydorlyze partially deacetylated chitin substrates,with the subsequent formation of N-acetylchitooligosaccharides [(GIcNAc)_n=2-7].Separation and quantification of the hydrolysis porducts of (GIcNAc)_n,n=2-6,byHPLC showd the spliting into (GIcNAc)_n,n=3-6.Activity toward N-acetylchitobiose was not detected.Oligomers with a higher nubmer of units than the starting substrate wee also detected,whcih indicate transglycosylation activity.