Cellulolytic enzymes in Microbulbifer sp. Strain GL-2, a marine fish intestinal bacterium, with emphasis on endo-1,4-ß-glucanases Cel5A and Cel8.

Cellulose is an abundant biomass on the planet. Various cellulases from environmental microbes have been explored for industrial use of cellulose. Marine fish intestine is of interest as one source of new enzymes. Here, we report the discovery of genes encoding two ß-glucosidases (Bgl3A and Bgl3B) and four endo-1,4-ß-glucanases (Cel5A, Cel8, Cel5B, and Cel9) as part of the genome sequence of a cellulolytic marine bacterium, Microbulbifer sp. Strain GL-2. Five of these six enzymes (excepting Cel5B) are presumed to localize to the periplasm or outer membrane. Transcriptional analysis demonstrated that all six genes were highly expressed in stationary phase. The transcription was induced by cello-oligosaccharides rather than by glucose, suggesting that the cellulases are produced primarily for nutrient acquisition following initial growth, facilitating the secondary growth phase. We cloned the genes encoding two of the endo-1,4-ß-glucanases, Cel5A and Cel8, and purified the corresponding recombinant enzymes following expression in Escherichia coli. The activity of Cel5A was observed across a wide range of temperatures (10-40 ˚C) and pHs (6-8). This pattern differed from those of Cel8 and the commercial cellulase Enthiron, both of which exhibit decreased activities below 30 ˚C and at alkaline pHs. These characteristics suggest that Cel5A might find use in industrial applications. Overall, our results reinforce the hypothesis that marine bacteria remain a possible source of novel cellulolytic activities.

Isolation of cellulase-producing Microbulbifer sp. from marine teleost blackfish (Girella melanichthys) intestine and the enzyme characterization.

Most animals cannot digest cellulose but have symbiotic microbes that degrade the matrix polysaccharides of plant matter. Herbivorous and omnivorous marine fish are similarly expected to rely on symbiotic microbes, but reports to date on cellulase-producing bacteria in fish intestines are limited. Here, we report the isolation of new cellulase-producing bacteria from the marine omnivorous teleost, blackfish (Girella melanichthys), and the characterization of cellulase activity. Three strains of cellulase-producing bacteria sp. were isolated from the hindgut of wild G. melanichthys. The strains of cellulase-producing bacteria grew in medium with artificial seawater but not in NaCl alone. Growth was optimum at 20-35°C, but there was no growth at 40°C, suggesting adaptation in a marine environment at a low temperature. Isolates were identified to Microbulbifer sp., among which GL-2 strain produced a high enzyme activity. The GL-2 strain was further used for enzyme characterization with carboxymethyl cellulose (CMC) as the substrate. Maximum activity of the cellulase was observed at 60°C, and activity was more than 30% at 20°C, while commercial cellulase Enthiron showed an optimum activity at 50°C and 17% activity at 20°C. Hydrolytic products by GL-2 cellulase were cellobiose but not glucose, suggesting a deficiency of ß-glucosidase activity. Active gel electrophoresis containing CMC showed five bands, suggesting several cellulolytic enzymes. The GL-2 strain and its enzyme are potential probiotics for aquaculture fish and the industrial production of cellobiose.

Complete Genome Sequence of Cellulase-Producing Microbulbifer sp. Strain GL-2, Isolated from Marine Fish Intestine.

Microbulbifer sp. strain GL-2 was isolated from the intestine of a teleost, Girella melanichthys. Here, we report the complete genome sequence of this strain, which produces cellulase(s). Twelve cellulase candidate genes were found on the chromosome.