Construction Of Gene Engineered Bacteria Of Xylanase And Its Recombined Enzymatic Properties

Xylan is the principal type of hemicellulose, a polymer ofβ-1,4-linked xylose residues. Xylan may carry many different substitutents such as arabinose and glucuronic acid, et.al. Xylanase are a class of enzyme that thydrolyzes theβ-1,4 intemal bonds of the xylan backbone polymer. Soβ-xylosidases are the crucial enzyme in the hydrolytic enzymatic complex. Recently, xylanases deserves particular attention due to widespread potential application: for the manufacture of food and drinks, as a supplement in animal feed, bleaching of cellulose pulp and production of ethanol and xylitol. Owing to the great potential value of xylanase, many researches have been carried out and plentiful important progresses have been made at home and abroad. At present, there are diverse xylanase gene siquences that have been cloned from various microorganisms, and lots of genes have been overexpressed in suitable hosts. In this study, xynB (encoding forβ-xylosidases) from A.niger C71 had been cloned and had been expressed in E.coli BL21, and then the gene engineered bacteria G81 of xylanase was constructed. Fermentation conditons and characteristics of the recombinant xylanase were systematically analyzed. Subsequently, small-scale bioflo fermentation was tested. The main results obtained were as follows:XynB from A.niger C71 were cloned with an ORFof 678 bp and could encode for a protein of 225 amino acids. The molecular weight and pI of XynB were 24.127 kDa and 5.23, respectively. According to the homology research, the deduced amino acid sequence of xynB from A.niger C71 had the highest similarity with xyn(225aa) from A.usamii (97%), and the lowest similarity with xyn(231aa) from C.carbonum (60%). A 3D model of xynB from A.niger C71 was built and the 3D structure 1TE1 chain 'B' was the template by using modeling program SWISS-MODEL and ESyPred3D Web Server 1.0. This template shares 57.8% identities with xynB from A.niger C71 (using the ALIGN program). Compared with the template, the 3D model of xynB had the same second structure that included 15β-sheets and 1α-helix, and its composition of 15β-sheets was more than three amino acids.The xynB from A.niger C71 were cloned into plasmid pET32a(+) which had double restriction site of KpnI and EcoR I, and then the xynB in pET32a(+) was transformed into the host E.coli BL21(Engineered Bacteria G81 of xylanase). After recovered the expression products from engineered bacteria G81, we found xylanase activity and obtained a specific band approximately 30 kDa by using SDS-PAGE electrophoresis analysising and coomassie brilliant blue staining.Based on the pET-xynB recombinant, subsequently shake flask fermentation and small-scale bioflo fermentation of engineered bacteria G81 were tested. The influences of fermentation conditions on the production of xylanase B(Y ) was probed into by rotary orthogonal experimental design. In this paper, the tested fermention condions included fermentation temperature(X₁), inducer concentration (X₂) and fermentation time(X3). We established the mathematic model of engineered bacteria G81 producing xylanase which was influenced by these determination conditions: Y=234.38+113.70X₁+24.76X₂+0.01 X₃+30.55 X₁X₂-2.52X₁X₃-2.99 X₂X₃+49.34X12-42.02X₂²-42.85X₃².These results indicated that the fermentation conditions optimization of G81 were as follow: fermentation temperature 28.6℃, inducer concentration 0.8 mmol/L and fermentation time 15h. Fermentation temperature influenced the production of xylanase B significantly. Engineered bacteria G81 fermentation was carried out in the 10L fermentor at 28.6℃, 0.8 mmol/L of IPTG concentration. After adding inducer, the xylanase B produced slowly within 6h. increased rapidly from 6h to 8h, and achieved maximum at 8 h(86100.21U).Temperature had significant influence on the activity of xylanase B produced by engineered bacteria G81. The xylanase B retained most of the original activity(98.62%) at 40℃in 1 hour, but enzyme activity declined quickly (21.28%) at 50℃in 1 hour.The activity of xylanase B of G81wasn't sensitive to pH Its optimum pH was 6.0 and had a good activity from pH 3.0 to 5.0. The activity of xylanase B of G81was improved by Mg2+ and Fe2+ ions and inhibited by Ca²⁺,Mn²⁺,Cu²⁺ and Zn²⁺ , espectively.