title: Molecular Characterization of Xylobiose- and Xylopentaose-Producing β-1,4-Endoxylanase SCO5931 from Streptomyces coelicolor A3(2).
abstract: 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.
authors.affiliation: Division of Bioscience and Bioinformatics, Myongji University, Yongin, 449-728, South Korea.|International Medical Center LLC, Intermed hospital, Ulaanbaatar, Mongolia.
authors.forename: Bolormaa
authors.initials: B
authors.lastname: Enkhbaatar
authors.affiliation: Division of Bioscience and Bioinformatics, Myongji University, Yongin, 449-728, South Korea.
authors.forename: Chang-Ro
authors.initials: CR
authors.lastname: Lee
authors.affiliation: Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology, Chungbuk, 363-883, South Korea.
authors.forename: Young-Soo
authors.initials: YS
authors.lastname: Hong
authors.affiliation: Division of Bioscience and Bioinformatics, Myongji University, Yongin, 449-728, South Korea. skhong@mju.ac.kr.
authors.forename: Soon-Kwang
authors.initials: SK
authors.lastname: Hong
mesh_terms: D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004187:Disaccharides / Q000096:biosynthesis*; D043364:Endo-1,4-beta Xylanases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D029964:Fagus / Q000737:chemistry; D005798:Genes, Bacterial; D006868:Hydrolysis; D009844:Oligosaccharides / Q000096:biosynthesis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D048372:Streptomyces coelicolor / Q000201:enzymology*; D013379:Substrate Specificity; D014994:Xylose / Q000096:biosynthesis*
chemical_list: D004187:Disaccharides; D009844:Oligosaccharides; D014994:Xylose; D043364:Endo-1,4-beta Xylanases; C004173:xylobiose
keywords: GH 10 family; SCO5931; Streptomyces coelicolor; XlnA; Xylanase A