Cloning And Expression Of Aspergillus Niger XZ-3S Xylanase Gene And Characterization Of The Recombinant Xylanase

BackgroudXylanase is the main enzyme to catalyze the xylan,which is widely used in the fields of paper, food, feed, medicine and energy. Therefore, how to use modern biotechnology means to produce cheap and efficient xylanase products has become an urgent need for the development of various industries.Objective1.Cloning of Aspergillus niger XZ-3S strain xylanase gene, and its bioinformatic analysis;2.Building of E. coli engineered bacterias and optimizing its xylanase produced by fermentation conditions;3.Purification of recombinant xylanase and determination of its enzymatic properties provided a theoretical basis for molecular of xyanase, which thus was laid a solid foundation for the application of xylanase in industry.Methods1.The xylanase gene was cloned by using molecular biology and genetic engineering techniques from Aspergillus niger XZ-3S.2.Nucleotide,deduced amino acid sequences and homology of the sequence were analyzed using DNAMAN6.0. The amino acid sequence which was translated according to nucleotide was submitted to http://www.expasy.org/tools/website to analyze the amino acid sequence, predict the secondary structure and homology modeling of three-dimensional structure.3.The recombinant expression plamid was constructed successfully by the mature peptide coding region of PCR products with the vector pET-28a after double digestion with restriction endonucleasea and T4DNA ligase connection. The engineered bacteria was obtained by transforming the recombinant expression plamid into E. coli.4.The optimum fermentation conditions were determined by the single factor experiment and the orthogonal test from the inducer concentration, induction chance, induction time and induction temperature, respectively.5.The recombinant xylanase was purified by using nickel column affinity chromatography methods. And effect of temperature, pH and various metal ions on the enzyme activity or stability were studied and measured.Results1.Three kinds of xylanase gene were successfully cloned from the genome of the Aspergillus niger. The GenBank accession numbers were JQ700381(xynZF-1), JQ700382(xynZF-2) and JQ700383(xynZF-3), respectively.2.Sequence analysis showed that:xynZF-1had an ORF(open reading frame) of633bp that encoded a27-aa signal peptide and a184-aa mature peptide; xynZF-2had an ORF of675bp that encoded a18-aa signal peptide and a207-aa mature peptide; xynZF-3had an ORF of954bp that encoded a19-aa signal peptide and a299-aa mature peptide. Three kinds of xylanase were consist of a-helix, β-sheet and a small amount of coner. XynZF-1and xynZF-2belonged to GH11superfamily with a typical right half-grip shaped space structure, while xynZF-3has a5-bladed beta-propeller domain belonging to the GH43superfamily.3.Three kinds of xylanase engineered bacteria were constructed successfully and named BL21/xynZF-1, BL21/xynZF-2, BL21/xynZF-3, respectively. Induced by IPTG, SDS-PAGE analysis and DNS method determining enzyme actiivity confirmed that three kinds of xylanase achieved heterologous expression.4.The best optimization program of the engineered bacteria which produced xylanase was confirmed through the single factor experiment and orthogonal test. The optimum fermentation conditions of xynZF-1were optimized as follows:inducer concentration 0.95mmol/L, induction chance1.5h, induction time1h, induction temperature31℃. The optimum fermentation conditions of xynZF-2were optimized as follows:inducer concentration2.5mmol/L, induction chance2h, induction time2h, induction temperature31℃. The optimum fermentation conditions of xynZF-3were optimized as follows: inducer concentration1.5mmol/L, induction chance2h,induction time2.5h, induction temperature25℃.5.The electrophoresis pure grade recombinant xylanase was got successfully by nickel column affinity chromatography method, which basically could satisfy the characterization analysis. The enzymatic properties were as follows:the optimum temperature and pH of zynZF-1were45℃and4.6, and Fe3+, Mg2+, Mn2+, Pb2+, Ni2+had significant inhibitory effect on the enzyme, while the Ca2+had a certain role in promoting; the optimum temperature and pH of xynZF-2were40℃and5.0, and Fe2+, Cu2+, Mg2+,Pb2+, Li+, Ca2+had an inhibitory effect on the enzyme, while the Zn2+, Fe3+, Mn2+, Ni2+, EDTA had a certain role in promoting; the optimum temperature and pH of xynZF-3were45℃and5.0, and Fe2+, Mn2+, Pb2+, Li+, Ni2+had an obvious inhibitory effect on the enzyme, while Fe3+has a significant role in promoting. These three kinds of recombinant xylanase had a good stability at40℃and pH5.0, and xynZF-3was more stable than the others.The activity of xynZF-2decreased rapidly at pH3-4, but the had a good stability between pH5-9.ConclusionsThree kinds of xylanase gene were cloned and amplified from Aspergillus niger XZ-3S at the first time, and were studied on the bioinformatics analysis, the heterologoous expression of E. coli, the optimization of fermentation condition, the purification and enzymatic properties of the recombinant xylanase, which enriched the amount and type of xylanase, laid a solid foundation for the xylanase gene eukaryotic expression and enzymatic molecular structure transformation, so as to provide theoretical basis and guarantee of industrial production and application of the enzyme.