Cloning, overexpression and characterization of a xylanase gene from a novel Streptomyces rameus L2001 in Pichia pastoris

• In this study, we successfully cloned a novel xylanase gene from Streptomyces rameus L2001 by hiTAIL-PCR.
• We illustrated an effective use of a high-cell-density culture to generate a low cost xylanase in BSM medium.
• Presumed glycosylation of the recombinant xylanase was confirmed.

A novel GC-rich xylanase gene named xynA from Streptomyces rameus L2001 was cloned by high-efficiency thermal asymmetric interlaced PCR. The open reading frame of the cloned gene contained 672 bp and encoded 223 amino acid residues with a calculated molecular mass of 24.5 kDa. The recombinant xylanase (XynA) was scaled up in a 2.5 L fermenter using BSM and BMMY medium; the highest enzyme activity, 13626 U/mL, was obtained from BSM. This is the first report of high-cell-density production of a xylanase from S. rameus in Pichia pastoris. The xylanase showed a single band at about 25.0 kDa on SDS-PAGE after being treated with endo-β-N-acetylglucosaminidase H. Studies of enzymatic properties showed that the optimal temperature and pH of XynA were 55 °C and 4.5, respectively. XynA was very stable in a broad pH range (3.0–11.0), and the residual activity was >70% after incubation at 50 °C for 30 min. Thin-layer chromatography analysis showed that the recombinant xylanase was able to digest xylan, forming xylotriose and xylobiose as the main products; hence XynA from S. rameus L2001 could potentially be useful for preparation of xylooligosaccharides.

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