Abstract
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°C; the optimum pH and temperature were 4.5 and 50°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, n=2–6, 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.
