In this study, an endolytic alginate lyase, named Al163, was identified, cloned, and characterized from the Antarctic bacterium Pseudoalteromonas sp. NJ-21. Comparative sequence analysis showed that the predicted amin...In this study, an endolytic alginate lyase, named Al163, was identified, cloned, and characterized from the Antarctic bacterium Pseudoalteromonas sp. NJ-21. Comparative sequence analysis showed that the predicted amino acid sequence encoded by al163 belongs to the polysaccharide lyase 6(PL-6) family and has a molecular mass of about 80 kDa. Recombinant enzyme was purified by Ni-Sepharose affinity chromatography. Recombinant Al163 exhibited maximum activity(258 U/mg) at pH 7.0 and 40℃, and thermal stability assays showed retention of almost 90% activity after incubation at 30℃ for 30 min. Al163 activity was stimulated by Cd^(2+), Ca^(2+), Fe^(3+), and Mn^(2+), but inhibited by Cu^(2+), Si^(2+), Fe^(2+), and Ni^(2+). Thin-layer chromatographic analysis indicated that Al163 degraded sodium alginate, poly M, and poly G, generating disaccharides and trisaccharides as the final products. Only a few bacterial strains that produce a bifunctional alginate lyase have been reported. Our results indicate that recombinant Al163 exhibits broad substrate specificity and its products exhibit low degrees of polymerization. Both properties imply high potential for use of the enzyme in several industrial fields, including cosmetics and pharmaceuticals, based on the high demand for biologically active oligosaccharides.展开更多
A novel chitinolytic and chitosanolytic bacterium, Sphingomonas sp. CJ-5, has been isolated and characterized. It secretes both chitinase and chitosanase into surrounding medium in response to chitin or chitosan induc...A novel chitinolytic and chitosanolytic bacterium, Sphingomonas sp. CJ-5, has been isolated and characterized. It secretes both chitinase and chitosanase into surrounding medium in response to chitin or chitosan induction. To characterize the enzymes, both chitinase and chitosanase were purified by ammonium sulfate precipitation, Sephadex G-200 gel filtration and DEAE-Sepharose Fast Flow. SDS-PAGE analysis demonstrated molecular masses of chitinase and chitosanase were 230 kDa and 45 kDa respectively. The optimum hydrolysis conditions for chitinase were about pH 7.0 and 36 ℃, and these for chitosanase were pH 6.5 and 56 ℃, respectively. Both enzymes were quite stable up to 45 ℃ for one hour at pH 5-8. These results show that CJ-5 may have potential for industrial application particularly in recycling of chitin wastes.展开更多
基金Supported by the Public Science and Technology Research Funds Project of Ocean(No.201505026-4)the Chinese Polar Environment Comprehensive Investigation&Assessment Programs(No.CHINARE2016-01-05)+1 种基金the Basic Scientific Research Funds of First Institute of Oceanography,State Oceanic Administration(SOA)(No.2014T09)the Qingdao Applied Basic Research Project(No.14-2-4-14-jch)
文摘In this study, an endolytic alginate lyase, named Al163, was identified, cloned, and characterized from the Antarctic bacterium Pseudoalteromonas sp. NJ-21. Comparative sequence analysis showed that the predicted amino acid sequence encoded by al163 belongs to the polysaccharide lyase 6(PL-6) family and has a molecular mass of about 80 kDa. Recombinant enzyme was purified by Ni-Sepharose affinity chromatography. Recombinant Al163 exhibited maximum activity(258 U/mg) at pH 7.0 and 40℃, and thermal stability assays showed retention of almost 90% activity after incubation at 30℃ for 30 min. Al163 activity was stimulated by Cd^(2+), Ca^(2+), Fe^(3+), and Mn^(2+), but inhibited by Cu^(2+), Si^(2+), Fe^(2+), and Ni^(2+). Thin-layer chromatographic analysis indicated that Al163 degraded sodium alginate, poly M, and poly G, generating disaccharides and trisaccharides as the final products. Only a few bacterial strains that produce a bifunctional alginate lyase have been reported. Our results indicate that recombinant Al163 exhibits broad substrate specificity and its products exhibit low degrees of polymerization. Both properties imply high potential for use of the enzyme in several industrial fields, including cosmetics and pharmaceuticals, based on the high demand for biologically active oligosaccharides.
基金Project supported by the Marine Bureau of Zhejiang Province, China
文摘A novel chitinolytic and chitosanolytic bacterium, Sphingomonas sp. CJ-5, has been isolated and characterized. It secretes both chitinase and chitosanase into surrounding medium in response to chitin or chitosan induction. To characterize the enzymes, both chitinase and chitosanase were purified by ammonium sulfate precipitation, Sephadex G-200 gel filtration and DEAE-Sepharose Fast Flow. SDS-PAGE analysis demonstrated molecular masses of chitinase and chitosanase were 230 kDa and 45 kDa respectively. The optimum hydrolysis conditions for chitinase were about pH 7.0 and 36 ℃, and these for chitosanase were pH 6.5 and 56 ℃, respectively. Both enzymes were quite stable up to 45 ℃ for one hour at pH 5-8. These results show that CJ-5 may have potential for industrial application particularly in recycling of chitin wastes.
