Purification And Characterization Of An Extreme-thermostable Xylanase B From Thermotoga Maritima Expressed In E. Coli

Second only to cellulose in natural abundance, xylan is a major component of hemicellulose fraction of the plant cell walls. Depending on the sources, its backbone may carry various substitutents like arabinose and glucuronic acid. Typically, backbone depolymerization is accomplished by the action of endo-xylanases and ~3-xylosidases. Xylanase can hydrolyze P-1,4-glycosidic linkages of the xylan backbone to produce short chain xylo-oligosaccharides of various length, hence Endo-~3-xylanase is the crucial enzyme components of microbial xylanolytic systems.The hyperthermophilic bacterium The rmotoga maritima is able to gain metabolic energy utilizing xylan. It possesses two different xylanase genes which encode xylanase A and B, respectively. In the present dissertation, a second xylanase gene (xynB) from genome DNA of T maritima MSB8 was isolated, cloned and expressed in E. co/i, and purification and characterization of the translated xylanase were investigated. XynB may be attractive to industrial applications; therefore an effort was as well as made to study the action mode of XynB on the artificial substrates and effect of organic solvents on the activity of XynB.Cloning and expression of xynB gene in E. coiiThe xynB gene was isolated from a genome DNA of T maritima MSB8, cloned and expressed in F. coil. Two primers were designed on the basis of the nucleotide sequence of xynB gene. xynB gene fragments were successftilly amplified by PCR reactions under the suitable conditions. F. coil BL21 competent cells were transformed by electroporation with the ligated xynB -pET28. E co/i BL21 (DE3) containing Nco I -HindJII site. The translated protein was soluble with xylanase activity.XynB gene consists of 1044 bp, according to the homology search of the deduced amino acid sequence, the XynB is most closely to XynA from T sp. strain FjSS3-B. 1 (85% identity), and shows the second highest degree of similarity (82% identity) with XynB from T neapolitana. The rest of xylanases showed the identity less than 43%. The sequence of the gene shows that it encodes a family 10 xylanase with a single domain.Purification of XynBHeat treatment was used as convenient primary enrichment step in purifying XynB. Heat treatment of crude lysate at 70C for 10 mm provided a simple means forIllAbstracteliminating 23% of host proteins by precipation.After heat treatment, Ni-NTA agarose slurry was employed to trap the His-tagged enzyme. The purity of its fraction was more than 90% by the followingSDS-PAGE.XynB was purified to homogeneity by heat treatment, affinity chromatography and anion exchange. XynB was purified 44.4-fold with a recover yield of 11%. The purified enzyme showed as a single protein band on SDS-PAGE with a molecular mass of 42 kDa, this is in good agreement with the molecular mass of enzyme deduced from the DNA sequence, 42, 333 Da.Enzymic properties of XynBThe optimum pH was pH 6.14 (at 50擟), Thermostability is a desirable property for xylanases used in industrial processes, it is apparent that the cloned XyriB was extremely thermophilic and thermostable like other xylanases from the Thermotoga species. Besides, it was of interest that XynB was apt to be stable in the neutral to alkaline region and relatively less stable in the acid pH region, moreover, and it was quite stable over the pH range of pH 5.5 41.5 at 70C, was still stable up to 100Cat the pH ranging from pH 6.5 to 8.5. XynB was optimally active at 90~扖 (at optimum pH6. 14).The cloned xylanase B exhibited a broad substrate specificity, Km and kcat of the purified enzyme for p-nitrophenol-13-D-xylobioside were 0.0095mM and 16.4 s4 respectively. The synthetic substrates with pNP-derivatives were used to carry out the kinetic measurements at pH 6.14 at 30 C. It was expected that the recombinant XynB exhibited broad substrate specificity. The Km value of the purified XynB for pNP-J3-D-xylobioside was very small (0.0095?.0006). Km values for pNP-43-D-xylopyranoside, pNP...