title: High-level expression of a novel α-galactosidase gene from Rhizomucor miehei in Pichia pastoris and characterization of the recombinant enyzme.
abstract: The second α-galactosidase gene (designated as RmgalB) was cloned from the thermophilic fungus Rhizomucor miehei and expressed in Pichia pastoris. The gene belonging to glycoside hydrolase (GH) family 36 has an open reading frame (ORF) of 2241bp encoding 746 amino acids with two introns. The recombinant α-galactosidase (RmgalB) was secreted at high levels of 1953.9Uml(-1) in high cell density fermentor, which is the highest yield obtained for a α-galactosidase. The purified enzyme as a tetramer gave a single band corresponding to a molecular mass of 83.1kDa in SDS-PAGE. The enzyme exhibited a very high specific activity of 505.5Umg(-1). The optimum temperature and pH of RmgalB were determined to be 55°C and pH 5.5, respectively. It was stable within pH 5.5-9.5 and up to 55°C. RmgalB displayed specificity toward raffinose and stachyose, and completely hydrolyzed the anti-nutritive raffinose family oligosaccharides (RFOs). These properties make RmgalB useful in the food and feed industries.
authors.affiliation: Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Zhou
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, Beijing 100083, China. Electronic address: yanqj@cau.edu.cn.
authors.forename: Qiaojuan
authors.initials: Q
authors.lastname: Yan
authors.affiliation: Department of Biotechnology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China. Electronic address: zhqjiang@cau.edu.cn.
authors.forename: Zhengqiang
authors.initials: Z
authors.lastname: Jiang
authors.affiliation: Department of Biotechnology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Biotechnology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Yuchen
authors.initials: Y
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005285:Fermentation; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D016366:Open Reading Frames; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D055503:Protein Multimerization; D011887:Raffinose / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020103:Rhizomucor / Q000737:chemistry* / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature; D000519:alpha-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D005656:Fungal Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; C005695:stachyose; D000519:alpha-Galactosidase; D011887:Raffinose
keywords: Expression; Pichia pastoris; Rhizomucor miehei; α-Galactosidase