title: Heterologous Expression and Characterization of a Thermostable Exo-β-D-Glucosaminidase from Aspergillus oryzae.
abstract: An exo-β-D-glucosaminidase (AorCsxA) from Aspergillus oryzae FL402 was heterologously expressed and purified. The deduced amino acid sequence indicated that AorCsxA belonged to glycoside hydrolase family 2. AorCsxA digested colloid chitosan into glucosamine but not into chitosan oligosaccharides, demonstrating exo-β-D-glucosaminidase (CsxA) activity. AorCsxA exhibited optimal activity at pH 5.5 and 50°C; however, the enzyme expressed in Pichia pastoris (PpAorCsxA) showed much stronger thermostability at 50°C than that expressed in Escherichia coli (EcAorCsxA), which may be related to glycosylation. AorCsxA activity was inhibited by EDTA and most of the tested metal ions. A single amino acid mutation (F769W) in AorCsxA significantly enhanced the specific activity and hydrolysis velocity as revealed by comparison of Vmax and kcat values with those of the wild-type enzyme. The three-dimensional structure suggested the tightened pocket at the active site of F769W enabled efficient substrate binding. The AorCsxA gene was heterologously expressed in P. pastoris, and one transformant was found to produce 222 U/ml activity during the high-cell-density fermentation. This AorCsxA-overexpressing P. pastoris strain is feasible for large-scale production of AorCsxA.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China.
authors.forename: Dingxin
authors.initials: D
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China.
authors.forename: Linchun
authors.initials: L
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China.
authors.forename: Yuwei
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China.
authors.forename: Shumiao
authors.initials: S
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China.|Hubei Collaborative Innovation Center for Industrial Fermentation, Wuhan 430068, P.R. China.
authors.forename: Nan
authors.initials: N
authors.lastname: Peng
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China.|Hubei Collaborative Innovation Center for Industrial Fermentation, Wuhan 430068, P.R. China.
authors.forename: Yunxiang
authors.initials: Y
authors.lastname: Liang
mesh_terms: D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics*; D048271:Chitosan / Q000378:metabolism; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005285:Fermentation; D015870:Gene Expression; D006031:Glycosylation; D006596:Hexosaminidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D010843:Pichia / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D048271:Chitosan; D004492:Edetic Acid; D006596:Hexosaminidases; C064499:exo-beta-D-glucosaminidase
keywords: Aspergillus oryzae; Exo-β-D-glucosaminidase; Glucosamine; High-cell density fermentation; Thermostability