title: Expression of an endo-β-1,4-glucanase gene from orpinomyces PC-2 in Pichia pastoris.
abstract: The endo-β-1,4-glucanase gene celE from the anaerobic fungus Orpinomyces PC-2 was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPIC9K, and integrated into the genome of a methylotrophic yeast P. pastoris GS115 by electroporation. The strain with highest endo-β-1,4-glucanase activity was selected and designed as P. pastoris egE, and cultivated in shaking flasks. The culture supernatant was assayed by SDS-polyacrylamide gel electrophoresis and showed a single band at about 52 kDa. Furthermore, the recombinant P. pastoris egE was proved to possess the ability to utilize sodium carboxymethyl cellulose as a carbon source. The recombinant endoglucanase produced by P. pastoris showed maximum activity at pH 6.0 and temperature 45 °C, indicating it was a mesophilic neutral endo-β-1,4-glucanase, suitable for denim biofinishing/washing. Further research was carried out in suitable fermentation medium in shaking flasks. The most favorable methanol addition concentration was discussed and given as 1.0%. After methanol induction for 96 h, the endo-β-1,4-glucanase activity reached 72.5 IU mL(-1). This is the first report on expression and characterization of endo-β-1,4-glucanase from Orpinomyces in P. pastoris. The endo-β-1,4-glucanase secreted by recombinant P. pastoris represents an attractive potential for both academic research and textile industry application.
authors.affiliation: Department of Chemical Engineering and Bioengineering, Zhejiang University, Hangzhou 310027, China; E-Mails: guyin018@gmail.com (X.J.); mengnan17@163.com (N.M.).
authors.forename: Xin
authors.initials: X
authors.lastname: Jin
authors.forename: Nan
authors.initials: N
authors.lastname: Meng
authors.forename: Li-Ming
authors.initials: LM
authors.lastname: Xia
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D020085:Neocallimastigales / Q000235:genetics*; D030781:Organisms, Genetically Modified / Q000378:metabolism; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D002480:Cellulase