title: Cloning and high-level expression of β-xylosidase from Selenomonas ruminantium in Pichia pastoris by optimizing of pH, methanol concentration and temperature conditions.
abstract: β-xylosidase and several other glycoside hydrolase family members, including xylanase, cooperate together to degrade hemicelluloses, a commonly found xylan polymer of plant-cell wall. β-d-xylosidase/α-l-arabinofuranosidase from the ruminal anaerobic bacterium Selenomonas ruminantium (SXA) has potential utility in industrial processes such as production of fuel ethanol and other bioproducts. The optimized synthetic SXA gene was overexpressed in methylotrophic Pichia pastoris under the control of alcohol oxidase I (AOX1) promoter and secreted into the medium. Recombinant protein showed an optimum pH 4.8 and optimum temperature 50 °C. Furthermore, optimization of growth and induction conditions in shake flask was carried out. Using the optimum expression condition (pH 6, temperature 20 °C and 1% methanol induction), protein production was increased by about three times in comparison to the control. The recombinant SXA we have expressed here showed higher turnover frequency using ρ-nitrophenyl β-xylopyranoside (PNPX) substrate, in contrast to most xylosidase experiments reported previously. This is the first report on the cloning and expression of a β-xylosidase gene from glycoside hydrolase (GH) family 43 in Pichia pastoris. Our results confirm that P. pastoris is an appropriate host for high level expression and production of SXA for industrial applications.
authors.affiliation: Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran.
authors.forename: Ehsan
authors.initials: E
authors.lastname: Dehnavi
authors.affiliation: Nanobiotechnology Engineering Laboratory, Faculty of Engineering and New Technologies, Shahid Beheshti University, GC, Tehran, Iran; Protein Engineering Laboratory, Protein Research Center (PRC), Shahid Beheshti University, GC, Tehran, Iran. Electronic address: o_ranaei@sbu.ac.ir.
authors.forename: Seyed Omid
authors.initials: SO
authors.lastname: Ranaei Siadat
authors.affiliation: National Institute of Genetic Engineering and Biotechnology [NIGEB], Tehran, Iran.
authors.forename: Mehrnoosh
authors.initials: M
authors.lastname: Fathi Roudsari
authors.affiliation: Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran. Electronic address: khajeh@modares.ac.ir.
authors.forename: Khosro
authors.initials: K
authors.lastname: Khajeh
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D015870:Gene Expression; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D000432:Methanol / Q000737:chemistry*; D010843:Pichia / Q000235:genetics / Q000254:growth & development*; D020357:Selenomonas / Q000201:enzymology / Q000235:genetics; D014995:Xylosidases / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; D000432:Methanol
keywords: Heterologous expression; Optimization; Pichia pastoris; β-xylosidase