Study Of Dextranase From A Marine Bacterium Catenovulum SP. DP03

The experiments were carried out to study a dextranase-producing marine bacterium, including its screening and identification, its production conditions, dextranase purification and characterization, and the effect of dextranase on Streptococcus mutans biofilm inhibitory and reduction.1. A psychrotolerant dextranase-producing bacterium was isolated from the Gaogong island seacoast near Jiangsu, China. The bacterium was identified as Catenovulum sp. based on its phenotype, biochemical characteristics, and16S rRNA gene comparison. It is here called DP03. G+C mol%calculation and DNA hybridization study indicate that strain DP03is a new species which belongs to genera of Catenovulum. The optimal enzyme production time, initial pH, temperature, and aeration conditions of strain DP03were found to be28h, pH8.0,30℃, and25%volume of liquid in100-ml Erlenmeyer flasks, respectively. The ability of1%dextran T20to induce dextranase was investigated.2. With a four-step protocol, dextranase from a Catenovulum sp.(Cadex) was purified29.6-fold. A specific activity of2,309U/mg protein and a yield of16.9%were determined. The SDS-PAGE results showed Cadex to be a monomer with a molecular weight of75kDa. Cadex showed maximum activity at pH8.0and40℃and was stable at temperatures under30℃and at pH ranging from5.0-11.0. A metal ion and chemical dependency study showed Mn2+, Sr2+, ethanol, and carboxybenzene to exert a positive effect on Cadex, while Cu2+, Fe3+, Zn2+, Cd2+, Ni2+, Co2+, and sodium lauryl sulfate were found to be inhibitors. Substrate specificity study showed Cadex specifically cleaves the a-1,6glycosidic linkages, and thin layer chromatogram indicated that the main hydrolysis products of Cadex are isomaltotriose, isomaltotetraose, and isomaltopentaose.3. Base on the biofilm mass assay, the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm reduction concentration (MBRC) of Cadex made using S. mutans ATCC25175were measured. MBIC90was calculated when30U/ml Cadex was presented in the medium, in which Cadex effectively impeded the biofilm formation observed in the scanning electron microscopy study. MBRC50was also obtained by adding20U/ml Cadex into the medium, and effects of Cadex on the formed biofilms were studied by calculating the biofilms’ dry weight discrepancy. In conclusion, Cadex shows positive effects on both S. mutans biofilm inhibitory and reduction.