| As discovering of marine biological resources,a variety of physiological activity of alginate oligosaccharides was widely attention.Alginate lyase,especially endo-algi nate lyase,which was researched widely as tool to degredate alginate efficiently and environmentally.At present,alginate was used as marerial to produce ethanol,so,it was researched widely about screening of alginate-degradating bacteria,metabolic pathway of alginate and ethanol engineering bacteria and so on.However,the research of preparation of alginate monosacchride is less.The resherch used Sunxiuqiniasp.SH-52 which was isolated by our laboratory and three alginate lyases which is expresses in E.coil to research mechanism of degradation of alginate,metabolic pathway of alginate and characteristics of SHA-?.Results are as following:1.The synergy of recombinational alginate lyases of ZHO-?,ZHO-? and ZHO-?V on alginateRecombinant alginate lyases ZHO-?,ZHO-? and ZHO-IV were used to reaserch the synergy of alginate degradation.When alginate is as substrate,ZHO-? and ZHO-? have synergy.When molar ratio of ZHO-? and ZHO-? is 4:1,synergy degree)nf is 1.3 which is the highest.When PM is as substrate,with as the increase of ZHO-?concentration keeping mole percentage of ZHO-IV the same,the synergy is increasing.When 30%ZHO-IV and 70%ZHO-? are added,synergy degree is 1.7 which is the highest.When PG is as substrate,the synergy between ZHO-IV and ZHO-? is stronger than ZHO-? and ZHO-?.When molar ratio of ZHO-? and ZHO-? is 1:1,synergy degree is 1.5 which is the highest.2.Whole genome sequencing and analysis of related genes of alginate metabolis m of Sunxiuqinia sp.SH-52Sequencing results showed that the genome length is 4593397 bp,GC content is 46.56%,3904 ORFs was found,52 tRNA and 3 rRNA were found.Five alginate lyases were analysised by system evolution.Alignment analysis of Sunxiuqinia sp.SH-52 and amino acid sequence of others alginate lyases,it is showed that SHA-? and SHA-? are PL7;SHA-?,SHA-? and SHA-? are PL6.3.The research of metabolic pathway of Sunxiuqinia sp.SH-52The metabolic pathway of Sunxiuqinia sp.SH-52 was reaserched by adding inhibitors and transcriptomics.When iodoacetic acid and sodium fluoride were added,the growth of SH-52 was inhibited with glucose and alginate as substrate.It is demonstated that glyceraldehyde-3-phosphoroc acid dehydrogenase and pruvate kinase are involved in alginate metabolism.Transcriptome shows that several transporter gene(periplasmic transporter,ABC-transporter,MSF transporter),five alginate lyases gene,KdgK gene and KDPG aldolase gene are higher eapression.Short-chain dehydrogenase/reductase SDR is confirmed to be DEH reductase.Enzymatic pathway for alginate assimilation into intracellular and for the complete degradation of alginate into the central metabolism was studied.Firstly,alginate is transported into intracellular by transporters.Then,alginate is degraded by alginate lyases,releasing unsaturated oligosaccharides that are further degraded down to the monosaccharide 4-deoxy-L-erythro-5-hexoseulose uronic acid(DEH).DEH reductase then convert DEH into 2-keto-3-deoxy-D-gluconate,which is further phosphorylated by KdgK.The final product is 2-keto-3-deoxy-6-phosphogluconate(KDPG)that can be catalysted by KDPG aldolase into Entner-Doudoroff pathway.Pyruvate metabo lism is mainly to produce acetic acid.4.Induction of alginate lyases of Sunxiuqinia sp.SH-52 on different carbons and characteristic analysisThe induced sources of SH-52 are narrow.Alginate lyase is strongly induced by alginate,PM and PG,and faintly induced by galactose.In order to further study the expression of alginate lyase,alginate,galactose and glucose were ued to induce the expressin of alginate.When alginate and glucose were added,expression of alginate lyase was restrained,but when glucose and galactose were added,the induction is usual.When alginate and galactose were added together,the expression of alginate lyase was delayed.Semi-quantified PCR was used to study the alginate lyases expression at transcriptional level in Sunxiuqinia sp.SH-52.SHA-? is mildly induced by galactose;SHA-?,SHA-? and SHA-? are strongly induced by PG;all of five alginate lyases are strongly induced by PM;SHA-? and SHA-V are strongly induced by alginate.SHA-? was expressed in E.coil,and characteristics were analysised.Research shows that total protein is 162mg before purification,and after is 3.2mg.The enzyme activity of purified reached 13U/mg,and enzyme activity of crude protein only is 2.6U/mg.SHA-I was polyM specificity of alginate lyase.Optimum temper ature is 50?,and the optimum pH is 7.0,It can remain stable below 35?. |
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