| Xylan is the major component of hemicellulose, and the second most abundant bioresource in the nature. Xylanolytic enzymes are a system of hydrolytic enzymes, which act cooperatively to hydrolysis xylan to its constituent sugars. Among all the enzymes, endo-β-1,4-D-xylanase [EC3.2.1.8] plays a crucial role. It has great potential applications in feed industry, food industry, pulp and paper industry and textile industry. Since the development of using xylanase to bleach alki-treated wood pulp instead of using usual environmentally damaging chemical reagents, the research for alkaline xylanase has been extensively studied. In this research, we constructed a genomic library of a strain of actinomyce (strain 3-3) capable of producing xylanase (Kxp) and screened the gene (Kxg) encoding Kxp. Thus we determined the structure of the gene, and expressed it in the E.coli expression system. The properties of the xylanase was determined after purification.1) Determine the classification of the strain 3-3 by means of morphologic observation, performing physiological and biochemical tests and anlysis ofphylogenetic tress;2) In order to construct the genomic library of 3-3, the genomic DNA was extracted and cut. The fragments within the range of 4-9 kb was ligated to vectors and transfered into competence E.coli. A positive clon carrying Kxg, named X-27, was screened and Kxg was sequenced. The sequence of Kxp was determined by translating the sequence of Kxg. The analysis of Kxg shows that it has 389 amino acid residues and its molecular weight is 43146.4. Kxg is a member of hydrolase F/10.3) Kxg was obtained from by PCR amplification, using the plasmid extracted from X-27 as the template. After several steps of treatments, it was ligated into the E.coli expression vector pET-Duet with the correct open reading frame, transferred into E.coli rosetta (DE3), and finally the recombinant R32 was obtained. After induced by IPTG, Kxg was successfully expressed in R32 and Kxp was found in the supernatant.4) The Kxp crude enzyme was well purified by means of anion exchange. Then its properties were studied. Its optimal pH was 8.5, and it sustained above 50%of the enzyme activity between pH 7-10. Kxp showed high pH stability in 4℃overnight between pH 7.5-11.5, with more than 80%of the enzyme activity remained. Its optimal temperature was 55℃, and it showed low enzyme activity below 30℃Cor over 60℃. Kxp had high thermal stability, with 30%of the enzyme activity remaining after incubated at 70℃for 1 h. The research on the effect of 5 mM metallic ions on the enzyme activity showed that the enzyme activity was greatly affected by heavy metallic ions, while light metallic ions almost had no effects on the enzyme activity.5) Nine sites (H131, W135, E176, N179, H259, E288, W332, W340 and H102) were chosen for site-directed mutagenesis, and the properties of the mutanted proteins were studied. The results showed that the former eight sites palyed important roles in maintaining enzyme activity, while H131, W135 and H259 also effected the combination between the enzyme and xylan. In order to enhance the thermostability of Kxp, H102 was replaced by cysteine. The thermostability of KxpH102C was studied, and the resulted showed that the thermostability of KxpH102C wasn't enhanced compared to Kxp. |
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