Expression Of Alkaline Xylanase Gene XynA In Escherichia Coli And Characterization Of Its 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 expression vector;3. Expression of xylA gene in E.coli DH5á;4. Studies on properties of the recombinant xylanase.The detailed results and conclusions were as follows:1. The gene fragment encoding alkaline xylanase(xylA) was cloned successfully from pCT124 plasmid by PCR method.Sequence analysis showed that the xylA gene cloned was identical with the gene reported.2. The expression vector pGEX-xylA was constructed by ligating the ORF of xylA gene cloned by PCR and 3'-terminal ofα-factor signal sequence on the pGEX-4T-1 with T4 DNA ligase in EcoRâ… and Xholâ… site.The recombinant plasmid pGEX - xylA(with the xylA ORF) introduced into E.coli DH5áwas identified by colony PCR and two-enzyme digestion. Enzymatic activity was measured in the cytoplasmic and periplasmic fractions and in the extracellular supernatant. The xylanase activity was found mainly in the periplasmic fraction, indicating that the secretion signals of this protein must be recognized by the secretion system of E. coli.3. The isoelectric point(pI) of the xylanase was 5.0, the molecular mass was 40.0kDa,this was almost the same as the molecular mass of the xylanase calculated from the deduced amino acid sequence(39.13kDa).The optimum reaction temperature of the xylanase was 40℃,and it still retained most of the original activity(50%) between 25-50℃,heat-resistant.The optimum pH was 8.0,and xylanase showed to have a good activity over a broad pH range from 6.0 to 11.0,quiet suitable to serve for the pulp paper industry. . 4. The xylanase activity was improved by Mg²⁺,Fe²⁺,K⁺,Ca²⁺ ions,was inhibited by Hg²⁺,Ag+,Mn²⁺,Cu²⁺,Fe³⁺,I₂ considerably; and Co²⁺,Na⁺,Ni²⁺,Zn²⁺,I^-,Cl^- 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 xylanase was alkaline,had a good activity over a broad pH range,its optimum reaction temperature was 40℃.So the xylanase, which was secreted from the Genetically engineered bacteria constructed by our lab,had a wide foreground in exploitation and utilization of Biological agent Bleaching.