Gene Cloning,Expression And Characterization Of Agarase From Microorganisms Isolated From Various Environments

Agar is a viscous polysaccharide present in the cell wall of agarophyte red algae,and is one of the world's most widely used three largest seaweed gel.Agarase is the floorboard of polysaccharide degrading enzymes,which can degrade agar.Agarases have great applications in marine aquaculture,food,medicine,molecular biology,and so on.40 producing-agarase microorganisms from different environments were screened and 16 agarase gene fragments were got from these microorganisms in this research.And a full-length gene cloning and expression is realized in E.coli,then the enzymology properties were studied.Detailed results were as follows:(1)Samples were used to screened,and 40 producing-agarase microorganisms were purified,which were collected from Nanri Island abalone farms,Guangxi mangrove,Zhoushan Islands and other environments.By morphological observation and molecular biology identification,these enzyme producing strains were mainly distributed in Pseudoalteromonas sp.,Vibiro sp.and Streptomyces sp.and other species.(2)A total of 16 different agarase gene fragments,9 GH 50 and 7 GH16,were obtained from 25 producing-agarase strains using 6 pairs of degenerate primers.1 GH50 agarase gene fragment from Vibrio sp.ZC-1 was used to amplify the full-length gene.Then it was amplified by TAIL-PCR using chromosomal DNA as a template which was from strain Vibrio sp.ZC-1.Gene agaZC-1 was 2853 bp in length and encodes a protein of 950 amino acids among which 1-25 amino acids were predicted signal peptide.(3)The pET-agaZC-1 expression vector was successfully constructed,and the recombinant expression was realized in E.coli BL21.The recombinant AgaZC-1(rAgaZC-1)was purified using affinity chromatography,and then enzymology properties were studied.The results showed that:The optimum pH is 7.0,and was stable over pH 6.0?8.0.The optimum temperature is 40?,while the enzyme is not stable at 45?.Processing more than 10 min was completely inactivated,but was stable below 35?.Ca2+,Mg2+ and ?-ME have a obvious promotion effect on the enzyme activity.The Km,Vmax and kcat for the rAgaZC-1 on agar were 2.428 mg/ml,36.90 ?mol/(mg·min)and 62.847 s-1,respectively.The study result of substrate specificity showed that it has a strong specificity to agar.And the main products of agar degradation are neoagarobiose and neoagarotetraose.The research found new agarase genes and laid a foundation for the development and applications of agarases through the screening of producing-agarase microorganisms from different environments,gene cloning and expression,and enzymology characteristics of research.