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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.

A novel high-throughput method was established for rapid screening of large numbers of Aspergillus niger mutants with high transglucosylation activity by exploiting that yeast can hardly hydrolyze isomaltooligosaccharides (IMO). Supernatants of A. niger fermentation were incubated with Saccharomyces cerevisiae to remove glucose and maltose, and the remaining non-reducing sugars, which is positively correlated with the amount of IMO, the products of transglucosylation reaction, were used as indicator of transglucosidase activity of A. niger and examined by dinitrosalicylic acid assay. Using this method, 15 stains that could convert liquefied cassava starch to IMO more efficiently were selected from 8721 A. niger mutants. Among them, mutant C-6181 strain had transglycosidase activity of 4.61 U/ml (increased by 122% compared to its parental strain) and IMO yield of 83.7%. Taking together, the method is easy, simple, efficient and cheap, and has great application potential in selection of transglucosidase-producing strains used in industrial IMO fermentation.

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