Gene Cloning And Characterization Of The Xylanolytic Enzymes From Streptomyces Spp.

Xylan is the major component of plant hemicellulose. Due to its complexity, complete hydrolysis of xylan requires cooperation of a large variety of enzymes. Among them, xylanase plays an important role in the xylanolytic enzyme system by hydrolyzing theβ-1,4-glycoside linkages in xylan backbone. The research on xylanolytic enzyme system is received increasing attention. And nowadays, screening enzyme-producing strains from special environments and obtaining good enzymes with superior properties has been one of the important researching methods.In this study, two xylanase-producing strains, designated S9 and S27, were isolated from the soil of Flaming Mountain in the Turpan Basin of Xinjiang, China, and were classified into the Streptomyces based on sequence comparison of the 16S ribosomal deoxyribonucleic acid (rDNA). To determine the effect of different carbon sources on the production of xylanolytic enzymes, mediums containing oat spelt xylan, birchwood xylan, wheat bran or corncob were used to induce Streptomyces sp. S9. The results indicated that all four carbon sources could induce Streptomyces sp. S9 to produce xylanase,β-xylosidase,α-L-arabinofuranosidase and acetylxylan esterase, and oat spelt xylan was the best carbon source for enzyme production.Streptomyces sp. strains S9 and S27 and Streptomyces fradiae k11 (a strain isolated from the feather deposits, with high keratinase-producing yield and ability to hydrolyze feathers and hair effectively) were selected for further gene cloning and expression. Based on alignment of known sequences from five glycoside hydrolase families and one carbohydrate esterase family, six degenerate primer sets were designed and nine xylanolytic enzyme genes belonging to six families were obtained. They are a xylanase gene (xyn10Sf) belonging to glycoside hydrolase family 10 (GH10) from Streptomyces fradiae k11; a GH10 xylanase gene (xynAS9), a GH11 xylanase gene (xynBS9), a GH39β-xylosidase gene (xyl39S9), a GH51α-L-arabinofuranosidase gene (abf51S9), a GH62α-L-arabinofuranosidase gene (abf62S9) and an acetylxylan esterase gene (axe7S9) belonging to carbohydrate esterase family 7 from Streptomyces sp. S9; a GH10 xylanase gene (xynAS27) and a GH11 xylanase gene (xynBS27) from Streptomyces sp. S27. By genomic library construction, eight full-length genes (except for abf62S9) were obtained. Based on homology searches in GenBank using BLAST and multiple alignments, the sequences of the nucleotides and amino acids of the eight genes shared highest identities of were 63.6~91.2% and 50.8~87.8% with known sequences, respectively. The results suggest that some of them are novel. Three-dimensional structures and catalytic sites of them were also analyzed.Six genes, i.e., xyn10Sf, xynAS9, xynBS9, abf51S9, xynAS27 and xynBS27, were expressed in Escherichia coli BL21 (DE3) or Pichia pastoris, respectively. The recombinant proteins were purified and characterized. The recombinant Xyn10Sf, which was expressed in Escherichia coli BL21 (DE3), showed the optimal pH of 7.8, optimal temperature of 60oC, and retained stable over pH4.0～10.0. Xyn10Sf was also highly resistant to various neutral and alkaline proteases, indicting its good application prospect in many industries requiring cooperation with proteases. XynAS9, XynBS9 and XynAS27 showed the same optimal pH and temperature were pH6.5 and 60oC, respectively. XynAS9 and XynBS9 showed good stability over the pH range of 4.0～12.0. Compared with most mesophilic xylanases, XynAS9 exhibited adaptability to a wider temperature range, retaining more than 65% of the maximum activity when assayed at 50～80oC. XynAS27 showed good stability over the pH range of 2.2～12.0, and 85% of its hydrolysis product of xylan was xylobiose. The optimal pH of Abf51S9 was pH6.0 and the optimal temperature was 60～65oC. It showed good stability over the pH range of 5.0～11.0. The synergistic interaction of Abf51S9 and XynAS9 to degrade substrates could increase the product of reducing sugar by 20%. The recombinant XynBS27 expressed in Pichia pastoris showed the optimal pH and temperature was pH6.5 and 65oC, respectively. The specific activity of XynBS27 was 3,272.0U/mg.The structure-function analysis of XynAS27 was also performed. The enzymatic properties of the intact and the carbohydrate-binding module (CBM) with/without linker-region-truncated versions of XynBS27 were determined and compared. The results indicated that CBM not only improved the binding ability of XynAS27 to insoluble substrates, but also improved its thermal and pH stabilities. Furthermore, the linker region connecting CBM and catalytic domain also plays an important role in the enzyme stability. These results provided more information for future studies of enzyme structure-function relationship.