Cloning And Overexpression Of Gene Encoding Acidic Endo-β-1, 4-Xylanase From Aspergillus Niger (N402)in Pichia Pastoris And Analysis Of Properties Of The Recombinant Xylanase

Plant cell walls contain three major polymers: cellulose, hemicellulose and lignin. Xylan is the major hemicellulose in plants. Second to cellulose, xylan is the most abundant renewable polysaccharides in nature. Because xylan is complex in structure, its biodegradation involves the actions of several xylanolytic enzymes, of which the endo-β-1,4-xylanase (for short: xylanase, EC 3.2.1.8) is the most important one.This thesis consisted of four parts:1. Cloning and sequencing of xylA gene;2. Construction of Pichia pastoris expression vector;3. Expression of xylA gene in Pichia pastoris;4. Studies on properties of the recombinant xylanase.The detailed results and conclusions were as follows:1. The gene fragment encoding A.niger xylanase(xylA) was cloned successfully from Aspergillus niger var niger strain N402 genome DNA by PCR method. Sequence analysis showed that the xylA gene cloned was identical with the gene reported.2. The expression vector pPIC9K-xylA was constructed by ligating the ORF of xylA gene cloned by PCR and 3'-terminal of α-factor signal sequence on the pPIC9K with T4 DNA ligase in EcoR I site. The xylA gene(no promoter sequence) was under the controll of the strong AOX1 gene promoter.3. The recombinant plasmid pPIC9K-xylA(with the xylA ORF) was introduced intoPichia pastoris SMD1168 by electroporation, then the transformants were screened on MD-G418 plate, and 102 transformants were obtained. After cultrue in FA,FB medium and enzyme activity detection, a strain(NO.2) was obtained, named as SMD1168-xylA-2(S2), which secreted recombinant xylanase with the highest enzyme activity of 453.6U/ml, 11.3 times higher than that of the original strain (40.2U/ml). The experiment of fermentation and PCR method show that S2 had a good genetic stability.4. The isoelectric point (pI) of the xylanase was 5.0, the molecular mass was 34.4kDa, this was almost the same as the molecular mass of the xylanase calculated from the deduced amino acid sequence(34.3kDa). Optimum reaction temperature of the xylanase was 37℃, and it still retained most of the original activity( ≥50%) between 25-50℃, heat-resistant. Optimum pH was 4.0, and xylanase showed to have a good activity over a broad pH range from 3.0 to 5.0, quiet suitable to serve as the animal feedstuff additive.5. the xylanase activity was improved by Mg2+,Fe2+,Ni2+ ions, was inhibited by Hg2+,Ag+,Mn2+,Cu2+,Fe3+,I2 considerably, this was accordance with the experimental result of Chen Hongge; wherease Co2+,K+,Na+,I- just had slight influence on it. The effect of EDTA on xylA activity was slightly, only 2% inhibition, this results suggested that no metal ion was needed in enzymatic reaction. The addition of 5mmol/L SDS inhibited xylanase activity by almost all, this may be because that the xylanase molecular had less disulfide-bond.According to above, this acidic xylanase was acid-resistant, had a good activity over a broad pH range, its optimum reaction temperature was 37℃. So the xylanase, which was secreted from the geneticengineering yeast constructed by our lab, had a wide foreground in exploitation and utilization of animal feedstuff resources.