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Molecular Cloning, Heterologous Expression Of Xylanase Genes And Characterization Of Xylanases

Posted on:2007-11-20Degree:DoctorType:Dissertation
Country:ChinaCandidate:G M ZhangFull Text:PDF
GTID:1100360185495361Subject:Microbiology
Abstract/Summary:PDF Full Text Request
Endo-β-1, 4-xylanases (EC.3.2.1.8) catalyze the hydrolyzation of the xylan backbone to produce oligosaccharides. They have shown greatly potential application in the bleaching of kraft pulp, bakery, animal feed, production of alcohol, etc. However, when xylanase applied in industry, xylanases with excellent characters are favorable. Moreover, different kinds of xylanases are required in different applied fields. So, exploiting new xylanase and construction of engineered strain with high-yield xylanase have important significances. In this study, three xylanase genes were cloned from Bacillus pumilus, Plectosphaerella cucumerina and Verticillium dahliae. Ten xylanase gene fragments were ampilified from soil microorganisms. Three xylanase genes were expressed in Pichia pastoris. These results are as follows:1. Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastorisA gene xynHB encoding alkaline xylanase was cloned from Bacillus pumilus through shot-gun method. The gene was cloned into vector pHBM905 A, and expressed in Pichia pastoris GS115. Xylanase-secreting transformants were selected on plate containing RBB-xylan. Enzymatic activity in the culture supernatants was up to 644 U/mL and the optimal secreting time was 4 days at 25℃. SDS-PAGE showed two bands 32.2 kDa and 29.6 kDa larger than 22.4kDa of the protein mass deduced by Peptool program based on its amino acid sequences. Zymogram analysis demonstrated that both bands can hydrolyze xylan. Deglycosylation by Endoglycosidase H revealed that they are from the same gene but contain different glycosylation extent (30% and 25%). The optimal pH and temperature of the enzyme were pH6-9 and 50℃. Respectively. The three Asn residues in the glycosylation sites were replaced with Ala by stie-directied mutagensis. Mutated genes were respectively expressed in GS115. According to the SDS-PAGE, the three glycosylation sites Asn25-Thr-Ser, Asn65-Ile-Ser, Asnl88-Gly-Ser showed different opportunities of glycosylation, while the Asn25-Thr-Ser residues occurs highest opportunity, Asnl88-Gly-Ser residues lest. The extent of glycosylation on the Asnl88-Gly-Ser residues is very small. The band of 29.6 kDa was achieved if glycosylation on three sites individually. The 32.2 kDa band was gained if glycosylation on both sites. These suggest different glycosylation extent of xynHB gene products result from different frequency of glycosylation on glycosylation sites, instead of different chain length of oligosaccharide.Temperature sensitivity experiments of XynHB mutants demonstrated that the Asn25-Thr-Ser residues glycosylation site is vitally in improving the heat stability of recombinant enzyme.2. Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucurnerinaBy comparing the amino acid sequences of over 10 xylanases in 11 families from fungi through BLAST, two highly conserved regions, with a fragment of about 150 amino acids coding sequence in between, were identified. Degenerate primers complementary to the ends of these two conserved regions were designed to amplify the in-between sequence from nematophagous fungus P. cucumerina. The total gene encoding for a new xylanase with high specific activity, named xynZG, from the P. cucumerina was cloned by Genome-walking PCR method. It contains an open reading frame of 780 bp inserted by two intron of 50 and 46 bp. The cDNA of 684 bp was isolated via RT-PCR. This is the first report on the cloning of a functional gene from P. cucumerina. The cDNA was cloned into vector pHBM905B, and expressed in Pichia pastoris GS115. Xylanase-seereting transformants were selected on plate containing RBB-xylan. Enzymatic activities in the culture supernatants reached maximum level of 362 U/ml and the optimal secreting time was 3 days at 25℃. The molecular mass of the enzyme was estimated to be 19 kda by SDS-PAGE. The optimal pH and temperature of the purified enzyme were pH6 and 40℃. The purified enzyme was stable at room temperature for 10 h, and it also remained 90% residual activity for five months at 4℃. The enzyme had a specific activity of 363 U/mg, Km of 2.06 mg/ml and a Vmax of 0.49 mmol/min/mg for birchwood xylan. The enzyme activity was not affected by EDTA at concentration of 5 mmol, but was inhibited by many metal ions, especially Hg+, in concentration of 5 mmol.3. Molecular cloning and heterologous expression of a new xylanase gene from Verticillium dahliaeFungus V. dahliae caused greensickness of cotton. The in-between xylanase sequence was ampilied from V. dahIiae by degenerate primers mentioned above. The whole xylanse gene containing intron was achieved by Genome-walking PCR method. A 63 bp intron was found through BLAST, the whole eDNA xynG was cloned by PCR to splicing two exons. The deduced amino acid sequence showed 72% identity with endo-β-1, 4-xylanase from Cochliobolus carbonum and C. sativusin in the GenBank, which means xynG is a new xylanase gene. The eDNA was inserted into pHBM905A and expressed in GS115, Xylanase-secreting transformants were selected on plate containing RBB-xylan. The transformant with the largest halos was selected for study the character of xylanase. The optimal pH of the purified recombinant enzyme was pH6. It remains over 50% relative activity at pH5-9. The optimal temperature was 45℃. The most favorable substrate for XYNG is Beechwood xylan. Mg2+ and Ca2+ improve the enzyme activity by 33.7% and 16.6%, respecitively. EDTA,β-Mercaptoethano and NaN3 don't affected the enzyme activity. Tween-80 and DMSO activated enzyme activity by 28.4% and 12.8%. Hg+, in concentration of 5 mM, also inhibited the enzyme activity.4. Diversity of xylanase gene from soil microbial DNAA improved method was used to extract soil microbial DNA by indigenous silica gel instead of imported glass beads. A new degenerate PCR-primer pair P1/P2 was designed to amplify directly xylanase gene fragments from soil microbial DNA. PCR products were ligated with pMD18-T vector. Transformants were got by transforming ligation product into E.coli. Ten different recombinants that were confirmed by RFLP were sequenced. The sequence analysis shows that all of the ten fragments encode partial xylanase. The BLAST of deduced amino acid of fragments show that eight out of the ten were high in sequence homology with xylanase from Actinomycetes, two with Pseudomonas. Asn27 was found in all partial xylanase based on the alignment of deduced amino acid sequences, which means that all xylanase fragments were alkali-tolerant. Phylogenetic tree shows that all xylanase fragments were positive and over 70% of similarity.
Keywords/Search Tags:Xylanase, Bacillus pumilus, Plectosphaerella cucumerina, Verticillium dahliae, Pichia pastoris, Glycosylation, Soil microbial DNA, Gene diversity
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