Optimization Of Oligosaccharide From A Deep Sea Bacterium Flammeovirga Pacifica By Degrading Asparagus And Cloning, Expression Of Related Gene

A deep-sea bacterium Flammeovirga pacifica which was isolated from sediment samplesin the west Pacific Ocean can degrade Gracilaria lemaneiformis to produce algaeoligosaccharides. The optimal enzymolysis conditions of strain Flammeovirga pacifica toproduce oligosaccharides were studied. One sulfatase gene playing a role in the process ofenzymolysis was cloned and expressed in E.coli.(1) The enzymolysis conditions of Flammeovirga pacifica degrading asparagus wereestablished. Firstly, the enzymolysis time, enzymolysis temperature, initial pH, inoculum size,medium volume, concentration of asparagus, nitrogen sources and concentrations, salts andconcentrations and metal ions and concentrations were used in the single-factor test. The resultsshowed that the single-factor conditions of the highest oligosaccharide yield were: enzymolysistime42h, enzymolysis temperature37℃, initial pH7, inoculum5%, liquid volume50mL/250mL, concentration of asparagus30g/L, peptone concentration5g/L, KCl concentration15g/L,Ca2+concentration0.1g/L. Secondly, the significance of these factors was evaluated on theFlammeovirga pacifica degrading asparagus by Plackeet-Burman. We found that the qualitywas influenced significantly by liquid volume, inoculum and initial pH. Finally, the maximumwas determined by steepest ascent method and further optimization with central compositedesign. Response surface analysis predicted that the final oligosaccharide yield could reach3.36g/L when liquid volume was53.75mL, inoculum was5.45%and initial pH was6.74. And theshake flask experiments were also used to verify the results which showed the actualexperimental data and the predict value fitting can reach99%. The oligosaccharide yield ofoptimization conditions increased by25.3%than the initial conditions. In accordance with theoptimization conditions, we used the5L fermentor to expand experiment. Eventually, theoligosaccharide yield reached3.06g/L at42hours.(2) A sulfatase gene of Flammeovirga pacifica was cloned and expressed in E.coli.Agarase and eighty-seven sulfatase gene were found in the genome sequence by analyzing thegenome sequences of Flammeovirga pacifica. We conjectured the strain may use sulfatase toremove sulfate of agar, and then reuse agarase to degrade agar. A sulfatase gene ofFlammeovirga pacifica was researched in this thesis in order to verify the correctness of thismechanism. The highest homology gene ary1671was chosen to clone and express from the complete genome sequence of this strain. The total length of the encoding gene was1611bp. Itencoded a protein of536amino acids with a calculated molecular mass of62.9KDa. pColdΙ-ary1671in E.coli BL21was constructed, induced by IPTG and expressed. SDS-PAGEshowed that target protein was inclusion.