Molecular Characterization of Xylobiose- and Xylopentaose-Producing β-1,4-Endoxylanase SCO5931 from Streptomyces coelicolor A3(2)

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• 作者：Bolormaa Enkhbaatar ; Chang-Ro Lee ; Young-Soo Hong…
• 关键词：Streptomyces coelicolor ; SCO5931 ; Xylanase A ; XlnA ; GH 10 family
• 刊名：Applied Biochemistry and Biotechnology
• 出版年：2016
• 出版时间：September 2016
• 年：2016
• 卷：180
• 期：2
• 页码：349-360
• 全文大小：2,413 KB
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Biochemistry
  
• 出版者：Humana Press Inc.
• ISSN：1559-0291
• 卷排序：180

文摘

Streptomyces coelicolor A3(2) sco5931 gene was predicted to encode a putative xylanase A, a 477 amino acid protein belonging to glycoside hydrolase family 10. The entire sco5931 coding region was cloned and overexpressed in Streptomyces lividans TK24. Mature SCO5931 protein comprising 436 amino acids (47 kDa) was purified by single-step gel filtration chromatography from culture broth after ammonium sulfate precipitation, with 25.8-fold purification and yield of 30.6 %. The purified protein displayed a pronounced activity toward beechwood xylan as a substrate, but no activity was detected toward carboxymethylcellulose, Avicel, galactan, barley β-glucan, and xyloglucan, demonstrating that SCO5931 is a substrate-specific xylanase. Optimal xylanase activity was observed at 60 °C and pH 6.0. The addition of metal ions or EDTA did not affect the xylanase activity, while 4 mM MnCl₂ severely inhibited the enzyme, reducing its activity by 87 %. Kinetic parameters of SCO5931 toward beechwood xylan were determined (K_m = 0.24 mg/mL, V_max = 6.86 μM/min). Thin layer chromatography and mass spectrometry analyses of the beechwood xylan SCO5931 hydrolysis products were conducted. Product masses corresponded to sodium adducts of xylobiose (m/z 305.24) and xylopentaose (m/z 701.59), indicating that SCO5931 specifically cleaves the β-1,4 linkage of xylan to yield xylobiose and xylopentaose.