PCR: polymerase chain reaction MES: 2-morpholinoethanesulfonic acid monohydrate MOPS: 3-morpholinopropanesulfonic acid CAPS: N-cyclohexyl-3-aminopropanesulfonic acid DS: dissolved substrate HPLC: high performance liquid chromatography DP: degree of polymerization HSQC: heteronuclear single quantum correlation MALDI-TOF MS: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Kawano et al.: Transglucosylation of Aspergillus niger α-Glucosidase 3 According to whole-genome sequencing, Aspergillus niger produces multiple enzymes of glycoside hydrolases (GH) 31. Here we focus on a GH31 α-glucosidase, AgdB, from A. niger. AgdB has also previously been reported as being expressed in the yeast species, Pichia pastoris; while the recombinant enzyme (rAgdB) has been shown to catalyze tranglycosylation via a complex mechanism. We constructed an expression system for A. niger AgdB using Aspergillus nidulans. To better elucidate the complicated mechanism employed by AgdB for transglucosylation, we also established a method to quantify glucosidic linkages in the transglucosylation products using 2D NMR spectroscopy. Results from the enzyme activity analysis indicated that the optimum temperature was 65 °C and optimum pH range was 6.0-7.0. Further, the NMR results showed that when maltose or maltopentaose served as the substrate, α-1,2-, α-1,3-, and small amount of α-1,1-β-linked oligosaccharides are present throughout the transglucosylation products of AgdB. These results suggest that AgdB is an α-glucosidase that serves as a transglucosylase capable of effectively producing oligosaccharides with α-1,2-, α-1,3-glucosidic linkages. Aspergillus; α-glucosidase; glycoside hydrolase family 31; transglucosylation J. Appl. Glycosci.: Advance Publication 4 INTRODUCTION The enzyme α-glucosidase (EC 3.2.1.20; α-D-glucoside glucohydrolase) is a typical exo-type glycosidase that hydrolyzes α-1,4-glucosidic linkages and releases α-D-glucose from the non-reducing end. The α-glucosidases are primarily classified into either the glycoside hydrolase (GH) 13 or GH31 families. 1) The GH31 family is comprised of various types of enzymes, including α-xylosidases (EC 3.2.1.177), isomaltosyltransferases (EC 2.4.1.-), sulfoquinovosidases (EC 3.2.1.199) and α-glucosidases. 2) Aspergillus species are reported to possess multiple GH31 enzymes; for instance, Aspergillus nidulans and Aspergillus oryzae have ten GH31 proteins. 2) Moreover, within the Aspergillus niger genome, seven GH31 proteins are encoded, namely, AgdA, AgdB, AgdE, AgdF, AgdG, AxlA, and AxlB. 3) AgdA is the α-glucosidase that produces oligosaccharides containing α-1,6-glucosidic linkages; 4) 5) while AxlA is an α-xylosidase. 6) Additionally, Ma et al. recently cloned an AgdB gene from A. niger strain K1 and successfully expressed recombinant AgdB in Pichia pastoris (rAgdB). This recombinant protein was reported to have high hydrolytic specificity toward α-1,3-and α-1,4-glucosidic linkages and produced kojibiose and nigerose via transglucosyl...