Purification and Characterization of an Antibacterial Substance from Aerococcus urinaeequi Strain HS36.

A bacterial strain inhibiting the growth of Vibrio anguillarum, the causative agent of vibriosis, was isolated from fish intestines. The isolated strain HS36 was identified as Aerococcus urinaeequi based on the characteristics of the genus according to Bergey's Manual of Systematic Bacteriology and by 16S rRNA sequencing. The growth rate and antibacterial activity of strain HS36 in shaking culture were higher than those in static culture, while the optimal pH and temperature for antibacterial activity were 7.0 and 30°C, respectively. The active antibacterial substance was purified from a culture broth of A. urinaeequi HS36 by Sephadex G-75 gel chromatography, Sephadex G-25 gel chromatography, and reverse-phase high-performance liquid chromatography. Its molecular weight, as estimated by Tricine SDS-polyacrylamide gel electrophoresis, was approximately 1,000 Da. The antibacterial substance produced by strain HS36 was stable after incubation for 1 h at 100°C. Although its antibacterial activity was optimal at pH 6-8, activity was retained at a pH range from 2 to 11. The purified antibacterial substance was inactivated by proteinase K, papain, and ß-amylase treatment. The newly purified antibacterial substance, classified as a class II bacteriocin, inhibited the growth of Klebsiella pneumoniae, Salmonella enterica, and Vibrio alginolyticus.

Characterization of Cryptopygus antarcticus endo-ß-1,4-glucanase from Bombyx mori expression systems.

Endo-ß-1,4-glucanase (CaCel) from Antarctic springtail, Cryptopygus antarcticus, a cellulase with high activity at low temperature, shows potential industrial use. To obtain sufficient active cellulase for characterization, CaCel gene was expressed in Bombyx mori-baculovirus expression systems. Recombinant CaCel (rCaCel) has been expressed in Escherichia coli (Ec-CaCel) at temperatures below 10°C, but the expression yield was low. Here, rCaCel with a silkworm secretion signal (Bm-CaCel) was successfully expressed and secreted into pupal hemolymph and purified to near 90% purity by Ni-affinity chromatography. The yield and specific activity of rCaCel purified from B. mori were estimated at 31 mg/l and 43.2 U/mg, respectively, which is significantly higher than the CaCel yield obtained from E. coli (0.46 mg/l and 35.8 U/mg). The optimal pH and temperature for the rCaCels purified from E. coli and B. mori were 3.5 and 50°C. Both rCaCels were active at a broad range of pH values and temperatures, and retained more than 30% of their maximal activity at 0°C. Oligosaccharide structural analysis revealed that Bm-CaCel contains elaborated N- and O-linked glycans, whereas Ec-CaCel contains putative O-linked glycans. Thermostability of Bm-CaCel from B. mori at 60°C was higher than that from E. coli, probably due to glycosylation.