| The ocean is the origin of life,which contains unlimited potential.Actively developing marine resources and vigorously developing the blue economy are important aspects of promoting a strong country strategy.The ocean contains a large number of available marine polysaccharides,and agar is one of the important components,which has broad application prospects.Agar-degrading bacteria can produce agarase,which provides a powerful tool for realizing green and efficient biodegradation of agar,and has received extensive attention.In this paper,agar-degrading bacteria are the main research objects,and a series of research work is carried out.Firstly,the marine microorganisms in Weihai and Rizhao coastal samples were screened and separated,and 14 potential novel species and 7 agar-degrading bacteria were obtained.Then,2 novel marine species(0448T and F02T)and a novel agar-degrading bacterium RQJ05T were classified and identified by polyphasic taxonomy.After identification,Cells of strain O448T are Gram-stain-negative,aerobic,non-motile and oxidase-and catalase-positive.The major respiratory quinone is Q-10.The dominant fatty acid is C18:?7c.The major polar lipids are phosphatidylethanolamine,phosphatidylcholine and phosphatidylglycerol.On the basis of phylogenetic,genome and phenotypic data,strai O448T was identified as a novel genus of family Rhodobacteraceae,named Oceanibium sediminis gen.nov.,sp.nov.is proposed.Cells of strain F02T are Gram-stain-negative,strictly aerobic,motile and oxidase-and catalase-positive.The major respiratory quinone is Q-8.The dominant fatty acids are C1s:1?7c,C16:1?7c,C15:0 and C18:1?9c.The major polar lipids are phosphatidylglycerol,phosphatidylethanolamine and one unidentified aminolipid.On the basis of phylogenetic,genome and phenotypic data,strain F02T was identified as a novel genus in the ?-proteobacteria,named Maribactrum sediminis gen.nov.,sp.nov.is proposed.Cells of strain RQJ05T are Gram-stain-negative,aerobic,motile,oxidase-positive and catalase-negative.The major respiratory quinone is Q-8.The dominant fatty acids are summed feature 3(C16:1 ?7c and/or C16:1 c.o6c),C16:0 and Summed Feature 8(C18:1 ?7c and/or C18:1 co6c).The major polar lipids are phosphatidylethanolamine,phosphatidylglycerol and two aminophospholipids.On the basis of phylogenetic,genome and phenotypic data,strain RQJ05T was identified as a novel genus of the family Alteromonadaceae,named Algilactibacter agarilytica gen.nov.,sp.nov.is proposed.Then,the genome sequencing of agar-degrading bacterium QM202T and RQJ05T and the agarase gene included were predicted and analyzed.A total of 28 potential agarase genes were predicted,among which 4 were in strain QM202T and 24 in strain RQJ05T.All the agarases predicted were exogenously expressed.After enzyme activity assay,a total of 11 agarases were found to have agar degradation activity,among which 4 were in starin QM202T and 7 in strain RQJ05T.Try to purify the active agarases and successfully purify the recombinant agarase QM202-1 and RQJ05-21 by affinity chromatography.Finally,the structure and enzymatic properties of the purified recombinant agarases were investigated.The results showed that the agarase QM202-1 belongs to the GH50 family,and the optimum reaction temperature is 30-40 ?.The optimum pH is 7.0.The enzyme activity can be greatly increased by DTT.The degradation product is neoagarotetraose and the specific enzyme activity is 34.7 U/mg.The agarase RQJ05-21 belongs to the GH16 family.The optimum reaction temperature is 50 °C and the optimum pH is 8.0.Co2+,Fe2+ and DTT can greatly increase the enzyme activity.It can also degrade starch,alginate and carrageenan to some extent.The specific enzyme activity was 66.5 U/mg when the agar was degraded,and the products were neoagarotetraose and neoagarohexaose.In this study,multiple agarose-degrading bacteria and multiple agarase proteins were isolated and provided,which provided a powerful tool for realizing green and efficient biodegradation of agarose polysaccharide. |
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