Analysis of Transglucosylation Products of <i>Aspergillus niger</i> α-Glucosidase that Catalyzes the Formation of α-1,2- and α-1,3-Linked Oligosaccharides

Abstract

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 <Abstract> 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. <Key words> 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...

Biochemical characterization of glycoside hydrolase family 31 α-glucosidases from Myceliophthora thermophila for α-glucooligosaccharide synthesis

“…3). The two smaller bands (70 kDa and 55 kDa) may be proteolytically processed products of the full length protein during protein maturation, which has also been reported for α-glucosidases from A. nidulans (AgdB) [7], A. niger (AgdB) [9,22], A. implicatum (AiG) [20] and A. sojae (AsojAgdL) [15,43]. Native PAGE analysis of AgdB from A. niger and AsojAgdL from A. sojae revealed the presence of a single band, suggesting that these two products of proteolysis form a heterodimer for catalysis, which was confirmed by structural analysis of AsojAgdL [43].…”

“…In the MT31α2 product mixtures, an anomeric signal at δ 5.33 was detected (Fig. 7B), suggesting the Although the amino acid sequence identity of MT31α2 with AgdB (52.6 %) from A. nidulans and AgdB (58.5 %) from A. niger are comparable, the product linkage specificity of MT31α2 is similar to that of AgdB from A. niger [9,22] but not that of AgdB from A. nidulans [7]. Asn694 of AgdA in β→α Loop7 is close to the +1/+2 subsites and its mutations altered the activity and product profiles [11].…”

“…GH31 α-glucosidases from Aspergillus sojae (AsojAgdL) [15], Aspergillus neoniger (AG) [16,17], Xantophyllomyces dendrorhous [18], and Aspergillus oryzae ZL-1 (AgdA) [19] have also been reported to catalyze the synthesis of α-(1→6) linked glucooligosaccharides. Besides, GH31 α-glucosidases from Acremonium implicatum (AiG) [20], Paecilomyces lilacinus [21] and A. niger (AgdB) [9,22] α-glucooligosaccharides are reported to exhibit prebiotic effects [23][24][25][26]. Especially, IMOs are important prebiotic oligosaccharides [14,27,28], which stimulate the proliferation of intestinal beneficial bacteria and enhance human immunity [23,24,29].…”

Structural basis for the recognition of α‐1,6‐branched α‐glucan by GH13_47 α‐amylase from Rhodothermus marinus

“…Signals at approximately 5.4 and 4.95 ppm were identified as H‐1 protons of α‐1,4‐glucosidic and α‐1,6‐glucosidic linkages, respectively (Figure 2A). 35 A signal for an α‐1,6‐glucosidic linkage appeared in the enzymatic reaction, and the ratio of signal areas of α‐1,6‐glucosidic linkage/α‐1,4‐glucosidic linkage increased as the reaction progressed (Figure 2B).…”