The Cloning And Overexpressing Of Marine Microbial Polysaccharide-Degrading Enzymes

The ocean has been regarded as the origin of life on Earth. The ocean includesthe largest range of habitats and the most life-forms. Competition amongstmicroorganisms for space and nutrients in the marine environment is a powerfulselective pressure which has led to evolution. The evolution prompts the marinemicroorganisms to generate multifarious enzyme systems to adapt to the complicatedmarine environments. Therefore, marine microbial enzymes can offer novelbiocatalysts with extraordinary properties. Marine microbialpolysaccharide-degrading enzymes are also multifarious, and have importantapplications in fermentation and exploitation of biomass energy. In this study, marinemicrobial polysaccharide-degrading enzymes such as alginate lyases, κ-carrageenasesand inulinase were characterized, cloned and overexpressed.The alginate lyase gene was amplified from the marine bacterium Vibriosp.QY101which was a pathogen of Laminaria sp. The alginate lyase gene was clonedinto the multiple cloning site of the surface display vector pINA1317-YlCWP110andexpressed in cells of Yarrowia lipolytica Po1h. The cells displaying the alginate lyasecould form clear zone on the plate containing sodium alginate, and the alginate lyaseactivity of transformant His7was208.0±5.3U/g. The cells displaying alginate lyasecould be used to hydrolyze poly-β-D-mannuronate and poly-α-L-guluronate andsodium alginate to produce different lengths of oligosaccharides. This was the firstreport that the yeast cells displaying alginate lyasewere used to produce differentlengths of oligosaccharidesfrom alginate.A bacterial strain LL1producing κ-carrageenase was isolated from the decayedseaweed collected from Yellow Sea, China and identified as Pseudoalteromonasporphyrae. The extracellular κ-carrageenase in the supernatant of cell culture of the marine bacterium P. porphyrae LL1was purified to homogeneity with a202.6-foldincrease in specific κ-carrageenase activity as compared to that in the supernatantbyultrafiltration, anion-exchange chromatography, and gel filtration chromatography.According to the data from sodium dodecyl sulfatepolyacrylamide gel electrophoresis,the molecular mass of the purified enzyme was estimated to be40.0kDa. The optimalpH and temperature of the purified enzyme were8.0and55℃. The purified enzymecould hydrolyze κ-carrageenan into κ-neocarrabiose (DP2) and κ-neocarratetraose(DP4) sulfate，which were characterized by ESI-MS.After that, the gene encoding κ-carrageenase and its transcriptional regulator in P.porphyae were cloned by reverse PCR and genome walking, and then characterized.The κ-carrageenase gene (CGK gene, accession number: GU386342) had an openreading frame of1224bp encoding a407amino acids protein with calculatedmolecular weight of42.5kDa. The deduced protein belonged to the family GH16.The catalytic residues of the deduced protein were constituted by the characteristicsequence pattern E(162)xD(164)x(x)E(167). Another gene encoding κ-carrageenasetranscriptional regulator had an open reading frame of318bp encoding a105aminoacids protein with calculated molecular weight of12.1kDa. The deduced proteinbelonged to the family AraC with two helix-turn-helix DNA binding domain. TheCGK gene was over-expressed in Y.lipolytica Po1h by constructing double CGK genecopies vector, and all the recombinant CGK was retained by the recombinant Y.lipolytica Po1h. The results of western blotting show that the molecular weight of therCGK was60.0kDa. The optimal pH and temperature of the rCGK were8.0and54°C, respectively. The rCGK had high κ-carrageenase activity and κ-carrageenan washydrolyzed into κ-neocarrabiose and κ-neocarratetraose sulfate. The optimalconditions for hydrolysis of κ-carrageenan were that the amount of rCGK was7.5U/mg of κ-carrageenan, κ-carrageenan concentration was2.0mg/mL, reaction timewas40min and temperature was55℃. Under such conditions,71.5±0.2%of addedκ-carrageenan was hydrolyzed. Therefore, the recombinant κ-carrageenase may have highly potential applications in biotechnology.Inulin and inulin-containing materials are good substrates for biofuel production.In order to construct genetically stable recombinant Pichia guilliermondii yeast cellscarrying the additional inulinase gene for inulinase production, a new rDNAintegration vector pMD-rDNA-HPT-INU was constructed in this study. After the INUgene encoding exo-inulinase was ligated into the rDNA integration vector, the vectorwas transformed into P. guilliermondii which was susceptible to hygromycin B andintegrated into its chromosomes. The transformant R3obtained could produce53.2±2.1U/mL of inulinase activity within72h，while the inulinase activity of native strainwas32.5±0.7U/mL. In the1.5L Fed-batch fermentation，the inulinase activity ofthe cultures was84.1±0.9U/mL.In order to improve the inulinase activity, error prone PCR was used to generaterandom point mutations of the native inulinase gene with the template INUM whichwas the directed mutagenesis inulinase gene from P. guilliermondii. Then the errorprone PCR products were ligased to expression vector Ycplac33and expreesed in S.cerevisiae W12d in order to screen and obtain high inulinase activity transformantswith mutated gene. The transformant carrying mutated gene INUDV82was indicatedas the highest inulinase activity, and the mutated gene INUDV82was found that fivebases and four amino acids were changed. Finally, expression vectorpMIRSC11-INUDV82was constructed and transformed into S. cerevisiae W12d,which was integrated into its chromosomes. The transformant INUDV5obtainedcould produce25.0±1.8U/mL of inulinase activity within72h, which increased49%than the transformant carrying unmutated gene INUM.