| Marine sediment contains abundant microorganisms,enrichment culture method can effectively isolate new marine bacteria and special functional bacteria and microbial research of marine functional in-depth for the development and utilization of marine resources is of great significance.In this study,three strains of marine new bacteria were identified,the agglomeration conditions of a strain of agar degrading bacteria were optimized,and genomic bioinformatics analysis of two agar-degrading bacteria have been completed.Six agarase genes were exogenously expressed and the products were analyzed.As well as the purification and enzymatic properties of recombinant agarase Aga2293 was completed.A novel Bacteroidetes strains which were characterized using a polyphasic approach,designated FB208T,is considered to represent a novel species in the genus Marinifilum of the family Marinilabiliaceae for which the name Marinifilum albidiflavum sp.nov.is proposed.Another novel Bacteroidetes strains which were also characterized using a polyphasic approach,designated HF401T,is considered to represent a novel species in the genus geofilum of the family Marinilabiliaceae,with the name geofilum rhodophaea sp.nov.In addition,a novel Gammaproteobacteria strains which were characterized using a polyphasic approach,designated TS1T.The scientific name is Agarimarina marina gen.nov.,sp.nov..It is suggested to establish the level classification of new units named as Agarimarinaceae.The fermentation conditions of the Agantictica rhodophytacola 017T,a marine agar degradation bacterium,were optimized by single factor and response surface optimization experiments.The optimum fermentation conditions were as follows:agar powder content 1.5 g/L,salinity 29.6 g/L,temperature 33.17℃,tryptone 6 g/L,bottling volume 50 mL,inoculation amount 2%,initial pH of 6.5,shaker speed of 150 rpm/min,culture 36 h.Under this culture condition,the enzyme activity of the fermentation culture reached 2.46 U/mL.The genomic sequencing of TS1T and 017T were performed.The genome of bacterial TS1T does not contain plasmids and predicted that the chromosomal DNA contained 12 agarase genes.In addition to chromosomal DNA,Bacteria 017T also contained four plasmids,19 agarase genes were found in chromosomal DNA and 3 agarase genes were present on plasmid one.The bioinformatics studies were carried out on six genes that could encode agarase of strains TS1T and 017T,aga1766,aga2293,aga2578,aga1620,aga2943 and aga3830 belonged to the glycoside hydrolase GH96,GH16,GH39,GH96,GH16,GH50 family,respectively.These six genes were expressed and the results showed that the four recombinant agarases had agar degradation ability,named Aga2293,Aga1620,Aga2943 and Aga3830,except that Aga1766 andAga2578 were inactive.TLC showed that the hydrolyzate of Aga2293,Aga1620 and Aga3830 were neoagarotetraose and neoagarohexaose,the hydrolyzate of Aga2943 was neoagarooctaose and neoagarotetraose.Based on the results of bioinformatics analysis,aga2293,aga1620 and aga3830 were identified as new agarase genes.Recombinant agarase Aga2293 was purified and refolded using Ni NTA affinity chromatography and gradient dialysis.After its purity was detected by SDS-PAGE,the enzymatic properties were studied preliminarily.It was found that the optimum substrate concentration of Aga2293 was 2%,the optimum reaction temperature was 40℃,and the optimum activity was pH 7,Cu2+ has a great inhibitory effect on enzyme activity,Pb2+,Zn2+,Ca2+,Mn2+,Mg2+ have a certain inhibitory effect on the enzyme activity,Fe2+ and DTT can greatly promote enzyme activity.The specific activity of recombinant agarase Aga2293 was 83.3 U/mg under the conditions of substrate concentration of 2%,pH 7,reaction temperature of 40℃,and final concentration of 10mM Fe2+. |
