Screening And Characterization Of Low Temperature Enzymes From Deep-sea Cold Seep

The deep-sea environment is unique and contains a large number of special habitats,such as hydrothermal solution,cold seep,abyss,etc.In order to adapt to the environment,the microorganisms in these special habitats have evolved unique physiological and biochemical characteristics,such as producing enzymes with unique biological activities.In order to screen the enzymes with better biological activity at low temperature,the strains with enzyme producing ability were isolated from the cold seep sediments of South China Sea,and the strains with good enzyme activity at low temperature were isolated and identified,and corresponding enzymes were purified and their enzymatic properties were determined.In this study,the preferred 50 strains with protease activity were screened from deep-sea cold seep sediments.According to 16S r RNA sequence analysis,they belong to four categories:Bacillus,Pseudoalteromonas,Vibrio and Alteromonas.In addition,their ability to produce protease,amylase and lipase was tested,respectively.The strain gcc-1belonging to Bacillus had higher protease activity at low temperature,and the strain gcc21 belonging to Pseudomonas had higher lipase activity at low temperature.In order to further explore the cold-active protease produced by gcc-1,the protease was separated and purified by ammonium sulfate precipitation,anion exchange chromatography and gel column chromatography.SDS-PAGE detection showed that the protease molecular weight was around 28 k Da.Physiological and biochemical tests showed that the protease had high activity and stability in the range of 10?-80?and pH 6.5-11.5 with the optimum temperature of 40?and the optimum pH of 8.5,and it still maintained high activity at 10?.Meanwhile,the protease showed great stability in the presence of different surfactants,organic solvents,oxidant H₂O₂and commercial detergents,indicating that the protease has a wide application prospect in the detergent additive industry.According to 16S r RNA sequence analysis,strain gcc21 cold-active lipase activity was identified as a new species.In order to further identify gcc21,the polyphasic taxonomic identification was carried out by genome sequence analysis and physiological and biochemical properties determination.The results showed that the strain was a new species of Pseudomonas,named Pseudomonas marinensis.Because strain gcc21 showed strong cold-active lipase activity,and there were two cold-active lipases(Lipase 1 and Lipase 2)in the genome.In order to further examine the activities of two lipases,we heterologously expressed and purified Lipase 1 and Lipase 2in E.coli,and tested their physiological and biochemical properties.The results showed that the optimal catalytic temperature of both Lipase 1 and Lipase 2 were 4?,but the activity and stability of Lipase 2 were higher than that of Lipase 1,which indicated that Lipase 2 had a great application prospect.The catalytic triad of serine,aspartic acid and histidine in Lipase 1 and Lipase 2 is necessary to maintain lipase activity according to the amino acid mutation of lipase catalytic center,and Lipase 1 and Lipase 2 belong to lipase family III according to the phylogenetic tree analysis.In conclusion,the deep-sea cold seep strain gcc-1 isolated in this research has great cold-active protease activity,and gcc-21 is a new strain,and also exhibites good cold-active lipase activity.Therefore,the deep-sea cold seep is not only an important rich source for isolation and screening of potential unique cold-active enzyme,but also an important source for screening novel strains.