Purification and characterization of a novel chitinase from Burkholderia cepacia strain KH2 isolated from the bed log of Lentinus edodes, Shiitake mushroom.
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 mushrooms, was purified by 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 (pI) of 5.9. The enzyme was stable at pH values of 4.0-6.0, and at temperatures up to 50 degrees C; the optimum pH and temperature were 4.5 and 50 degrees C, respectively. 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 hydrolyze partially deacetylated chitin substrates, with the subsequent formation of N-acetylchitooligosaccharides [(GlcNAc) (n), n=2-7]. Separation and quantification of the hydrolysis products of (GlcNAc) (n), n52-6, by HPLC showed the splitting into (GlcNAc)(n), n=3-6. Activity toward N-acetylchitobiose was not detected. Oligomers with a higher number of units than the starting substrate were also detected, which indicate transglycosylation activity.
Ogawa
Kihachiro
K
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, Miyazaki University, Miyazaki 889-2192, Japan.
Yoshida
Naoto
N
Kariya
Kunichi
K
Ohnishi
Chikako
C
Ikeda
Ryuichiro
R
D000595:Amino Acid Sequence; D016956:Burkholderia cepacia / Q000201:enzymology*; D002686:Chitin / Q000031:analogs & derivatives* / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D048271:Chitosan; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020942:Shiitake Mushrooms; D013696:Temperature
D002686:Chitin; D048271:Chitosan; D002688:Chitinases