title: Purification and characterization of an intracellular α-glucosidase with high transglycosylation activity from A. niger M-1.
abstract: An intracellular α-glucosidase with high transglycosylation activity was purified from a mutant strain of Aspergillus niger M-1 by sequential chromatography using a DEAE-cellulose 52 column, a DEAE-Sepharose CL-6B column, and a Sephadex G-100 column. The molecular mass of the purified enzyme was determined to be 116 kD with no subunits and a pI of 5.23. Maximal α-glucosidase activity occurred at pH 6.0 and 50°C. The N-terminal amino acid sequences were identified as N-SVPGTEYVV-. The presence of Ca(2+) enhanced the enzyme activity by 20%, while the α-glucosidase activity was strongly inhibited by p-chloromercuribenzoate, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, monochloroacetic acid, and 2-mercaptoethanol. In addition, Ag(+), n-bromosuccinimide, and acetylacetone inhibited enzyme activity by 70%, 50%, and 22%, respectively. K(m) values of 4.32 m mol L(-1) and V(max) of 3.10 × 10(-2) mol L(-1) min(-1) were found for methyl-α-D-glucopyranoside (α-MG). Maltose was identified as the preferred substrate. The high-performance liquid chromatography (HPLC) analysis indicated that the oligosaccharide products contained 10.54% of isomaltose, 8.08% of panose, and 9.29% of isomaltotriose, and the amount of glucose, maltose, maltotriose, and maltotetrose was dropped from 22.21% to 15.80% using the purified enzyme in the solution of 25% maltose and 3% glucose. This intracellular α-glucosidase has potential applications in the synthesis of sugar derivatives and the investigation of associated mechanisms.
authors: College of Life Science and Technology, Guangxi University, Nanning, Guangxi, P.R. China.
authors: Yun-kai
authors: YK
authors: Zhang
authors: Wei
authors: W
authors: Li
authors: Kong-yang
authors: KY
authors: Wu
authors: Gui-guang
authors: GG
authors: Chen
authors: Zhi-qun
authors: ZQ
authors: Liang
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000737:chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004791:Enzyme Inhibitors / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D000520:alpha-Glucosidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D008670:Metals; D009844:Oligosaccharides; D000520:alpha-Glucosidases