Novel α-1,3/α-1,4-Glucosidase from Aspergillus niger Exhibits Unique Transglucosylation to Generate High Levels of Nigerose and Kojibiose

Abstract

α-Glucosidase from Aspergillus niger (AgdA; typical α-1,4-glucosidase) is known to industrially produce α-(1→ 6)-glucooligosaccharides. This fungus also has another α-glucosidase-like protein, AgdB. To learn its function, wild-type AgdB was expressed in Pichia pastoris. However, the enzyme displayed two electrophoretic forms due to heterogeneity of Nglycosylation at Asn354. The deglycosylation mutant N354D shared the same properties with wild-type AgdB. N354D demonstrated hydrolytic specificity toward α-(1→3)and α-(1→4)-glucosidic linkages, indicating that AgdB is an α-1,3-/α-1,4glucosidase. N354D-catalyzed transglucosylation from maltose was analyzed in short-and long-term reactions, enabling us to learn the transglucosylation specificity and product accumulation, respectively. A short-term reaction (<15 min) synthesized 3 II -O-α-glucosyl-maltose and maltotriose, indicating α-1,3-/α-1,4-transferring specificity. A long-term reaction (<24 h) accumulated kojibiose and nigerose using formed glucose as an acceptor substrate. AgdA and AgdB are distinct α-glucosidases. At a high concentration of glucose added exogenously, AgdB largely generated the rare sugars kojibiose and nigerose (exhibiting beneficial physiological functions) with 19% and 24% yields from maltose, respectively.

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

“…Although rAgdB expressed in P. pastoris was previously collected from culture supernatants, 7) within our study enzyme activity was not detected in the supernatant but rather in cell extracts.…”

“…We found that α-1,2-, α-1,3-, and small amount of α-1,1-β-linked oligosaccharides are present throughout the transglucosylation products of AgdB from maltose and maltopentaose. Similarly, Ma et al 7) detected four oligosaccharides consisting of α-1,2-and α-1,3-glucosidic linkages, namely, kojibiose, nigerose, 3 2 -O-α-D-glucosyl-maltose, and centose in the transglucosylation products from maltose. However, these products constituted a portion of the observed transglucosylation products of rAgdB.…”

Extracellular expression and characterization of an α-glucosidase from Oryza sativa and its transglycosylation for synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid

“…The reaction was initiated by adding maltose, and allowed to proceed for another 10 min. At different time intervals, samples were collected and the residual activity was assayed under the standard assay conditions [28,37] .…”

“…The supernatant was collected and stored at -20°C waiting for high performance liquid chromatography (HPLC) analysis. On the basis of the initial reaction condition, that is, the reaction last for 6 h at 37°C, pH 4.0, with the molar ratio of maltose to AA•Na set to 3:2, the influence of reaction temperature (30,37,40,45, and 50 degC), pH (100 mM sodium citrate buffer at pH 3.0-3.5 and 100 mM sodium acetate buffer at pH 4.0-5.5), and maltose/AA*Na molar ratio (1:1, 3:1 and 3:2) on the synthesis of AA-2G catalyzed by rAGL was investigated. Besides, the influence of enzyme concentrations on AA-2G synthesis was also investigated.…”

“…Pichia pastoris is the excellent expression host with well-established genetic tools and cultivation strategies for scale-up producing heterologous proteins, particularly industrial enzymes and biopharmaceuticals [35] . Several AGL fromAspergillus species [28,36,37] andApis cerana indica [38] have been successfully expressed in P. pastoris . The inducible alcohol oxidase I promoter (P AOX1 ) and the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (P GAP ) are commonly used in genetic engineering of P. pastoris .…”