Expression of a novel α-glucosidase from Aspergillus neoniger in Pichia pastoris and its efficient recovery for synthesis of isomaltooligosaccharides.

A gene conferring α-glucosidase (AG) with high transglycosylation activity from Aspergillus neoniger (a non-niger strain belonging to section Nigri) was cloned and expressed in Pichia pastoris. As the cDNA construction retained intronic portions due to alternative splicing, the exonic portions of the gene were stitched using restriction digestion and overlap extension PCR. Pre-determined open-loop exponential feeding strategies were evaluated for methanol dosage to improve the recombinant enzyme synthesis during high-cell density cultivation in 5 L bioreactor. Specific growth rate of 0.1 h-1 resulted in the highest enzyme activity of 182.3 mU/mL in the supernatant, whereas the activity of 3.8 U/g dry cell weight was obtained in the cell pellet. There was negligible enzyme activity in the cell lysate, indicating that approximately 80 % accumulation of total enzyme is in the periplasm. Later, this unreleased fraction was extracted to 90 % yield using 25 mM cysteine. The enzyme was purified and validated using western blot analysis and MS/MS profile. The SDS PAGE analysis revealed three bands corresponding to 80, 38, and 33 kDa indicating the multimeric nature of the enzyme. Thus, obtained enzyme was utilized in synthesis of a potential prebiotic molecule, isomaltooligosaccharides (IMOs), which can be used as a sweetener and bulk filler in the food industry. This is the first report to demonstrate challenges in cloning and expression of transglycosylating α-glucosidase from Aspergillus neoniger.

Identification and characterization of novel transglycosylating α-glucosidase from Aspergillus neoniger.

AIM: Aspergillus niger is well established for secreting α-glucosidase having transglycosylation activity, which is used as processing aid for synthesis of isomaltooligosaccharides. The present study focuses on identification and characterization of a non-niger Aspergillus isolate and its gene conferring strong transglycosylation activity. METHODS AND RESULTS: The soil isolate was identified as Aspergillus neoniger belonging to Aspergillus section Nigri using ITS (internal transcribed spacer) and ß-tubulin analysis. The sequence analysis of gene responsible for α-glucosidase synthesis revealed significant nucleotide variations when compared to other Aspergillus species. Molecular docking studies using the homology model revealed the presence of threonine at 694 subsite position instead of asparagine as in case of A. niger's α-glucosidase. The enzyme was purified to several fold using DEAE Sepharose-CL6B column and on SDS-PAGE analysis, it was found to be 145 kDa. MS/MS analysis of the purified enzyme validated the presence of threonine at 694 position. Commercial α-glucosidase (Transglucosidase L 'Amano') derived from A. niger and the α-glucosidase from isolate were compared for transglycosylation activity using constant test conditions. α-glucosidase from the isolate produced 27·4% higher panose when compared to that of commercial enzyme. Moreover, the rate of secondary hydrolysis of panose is much lower in case of the isolate's enzyme. CONCLUSIONS: Fungal isolate A. neoniger was characterized, and its gene conferring α-glucosidase activity was established for strong transglycosylation activity having higher panose yields. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report to establish a variant of α-glucosidase having strong transglycosylation activity from A. neoniger strain. We have demonstrated that this enzyme when used as processing aid could improve panose significantly, which is a potential prebiotic. Also, the sequence analysis established in our studies could provide pointers for directed evolution of this enzyme to further improve transglycosylation activity.

Feasibility Study for Bedside Production of Recombinant Human Acid α-Glucosidase: Technical and Financial Considerations.

OBJECTIVE: The high cost of orphan drugs limits their access by many patients, especially in low- and middle-income countries. Many orphan drugs are off-patent without alternative generic or biosimilar versions available. Production of these drugs at the point-of-care, when feasible, could be a cost-effective alternative. METHODS: The financial feasibility of this approach was estimated by setting up a small-scale production of recombinant human acid alpha-glucosidase (rhGAA). The commercial version of rhGAA is Myozyme™, and Lumizyme™ in the United States, which is used to treat Pompe disease. The rhGAA was produced in CHO-K1 mammalian cells and purified using multiple purification steps to obtain a protein profile comparable to Myozyme™. RESULTS: The established small-scale production of rhGAA was used to obtain a realistic cost estimation for the magistral production of this biological drug. The treatment cost of rhGAA using bedside production was estimated at $3,484/gram, which is 71% lower than the commercial price of Myozyme ™. CONCLUSION: This study shows that bedside production might be a cost-effective approach to increase the access of patients to particular life-saving drugs.

Insight into the glycosylation and hydrolysis kinetics of alpha-glucosidase in the synthesis of glycosides.

α-Glucosidase, Agl2, from Xanthomonas campestris was successfully overexpressed in Escherichia coli BL21(DE3) cells and purified with Ni columns. The enzyme exhibits glycosylation abilities towards a wide range of phenolic substrates, including phenol, vanillin, and ethyl vanillin, with maltose as the glycosyl donor. The catalytic properties of the purified enzyme were further investigated. It was observed that the synthesized glycosides started to degrade with prolonged catalytic time, giving an "n"-shaped kinetic profile. To understand such catalytic behavior, the Agl2-catalyzed glycosylation process was investigated kinetically. Based on the obtained parameters, it was concluded that although the substrate conversions are thermodynamically restricted in a batch system, the glycosylation efficiency can be kinetically controlled by the glycosylation/hydrolysis selectivity. Glucose was produced by both glycosylation and hydrolysis, significantly impacting the glycosylation efficiency. This study provides a mechanistic understanding of the α-glucosidase-catalyzed glycosylation process in a water-based system. The developed kinetic model was successful in explaining and analyzing the catalytic process. It is suggested that when α-glucosidase is employed for glycosylation in a water-enriched environment, the catalytic efficiency is mainly impacted by the enzyme's glycosylation/hydrolysis selectivity and glucose content in the catalytic environment.

Chemical characterization of the main bioactive polyphenols from the roots of Morus australis (mulberry).

Morus species, commonly known as mulberry, is widely distributed in China. The mulberry tree is a high-value plant in agriculture. Morus australis is one of the major Morus species growing in Northern China. However, the biological properties of the main constituents of M. australis roots were not well studied. In the present study, through extensive chromatographic and spectral analysis, 12 phenolic compounds were isolated and identified from the M. australis roots. Compounds 1, 2, 8, 9 and 12 were isolated from M. australis roots for the first time. Antitumor activities of these polyphenols were studied on the A549 cell line. Compounds 1, 5 and 6 exhibited cytotoxicity on A549 cells and induced apoptosis in A549 cells via the intrinsic mitochondrial pathway. They also mediated inhibition of autophagic flux contributed cell death via the PI3k/Akt/mTOR pathway. In order to explore more potential bioactivities of these isolates, α-glucosidase, acetylcholinesterase and tyrosinase inhibitory activities were studied, and the results demonstrated that the inhibitory activity of these polyphenols on enzymes was not defined by their basic structural skeletons, but by the substituted position.

Synthesis of Fucose-Containing Disaccharides by Glycosylhydrolases from Various Origins.

Glycosylhydrolases of various origins were used to produce fucose-containing disaccharides with prebiotic potential using different donor substrates and L-fucose as the acceptor substrate. Eight different disaccharides were synthesized as follows: three ß-D-galactosyl-L-fucosides with glycosidase CloneZyme Gly-001-02 using D-lactose as a donor substrate, two with a structure similar to prebiotics; one ß-D-galactosyl-L-fucose with ß-D-galactosidase from Aspergillus oryzae using D-lactose as a substrate donor; and four α-D-glucosyl-L-fucosides with α-D-glucosidase from Saccharomyces cerevisiae using D-maltose as a donor substrate. All disaccharides were purified and hydrolyzed. In all cases, an L-fucose moiety was present, and it was confirmed for ß-D-galactosyl-L-fucose by mass spectrometry. High concentrations of L-fucose as the acceptor substrate enhanced the synthesis of the oligosaccharides in all cases. The three enzymes were able to synthesize fucose-containing disaccharides when L-fucose was used as the acceptor substrate, and the highest yield was 20% using ß-D-galactosidase from Aspergillus oryzae.

LC-MS guided isolation of diterpenoids from Sapium insigne with α-glucosidase inhibitory activities.

Ten new (1-10) and ten known (11-20) diterpenoids involving ent-atisane, ent-seco-atisane, ent-kaurane and ent-seco-kaurane types were isolated from Sapium insigne under the guidance of LCMS-IT-TOF analyses. Their structures were characterized by extensive spectroscopic analyses (HRESIMS, UV, IR, 1D and 2D NMR). A putative biosynthetic pathway was proposed for ent-seco-atisane diterpenoids. Their inhibitory activities on α-glucosidase in vitro were tested for the first time. Compound 4 showed moderate inhibitory effect on α-glucosidase with an IC50 value of 0.34 mM via a noncompetitive inhibition mechanism (Ki = 0.27 mM). The preliminary structure-activity relationships of the ent-atisane diterpenoids inhibiting α-glucosidase were discussed.

Hetarylcoumarins: Synthesis and biological evaluation as potent α-glucosidase inhibitors.

In search of better α-glucosidase inhibitors, a series of novel hetarylcoumarins (3a-3j) were designed and synthesized through a facile multicomponent route where p-toluenesulfonic acid (PTSA) was explored as an efficient catalyst. These new scaffolds were further evaluated for their α-glucosidase inhibition potentials. All the derivatives exhibited good to excellent results which were comparable or even better than of standard drug acarbose. Of these compounds, a dihalogenated compound 3f was found to be the most effective one with IC50: 2.53±0.002µM. Molecular docking has predicted the plausible binding interactions of compounds 3f, 3g and 3j with α-glucosidase.

Production of recombinant human acid α-glucosidase with high-mannose glycans in gnt1 rice for the treatment of Pompe disease.

Lysosomal storage diseases are a group of inherited metabolic disorders. Patients are treated with enzyme replacement therapy (ERT), in which the replacement enzymes are required to carry terminal mannose or mannose 6-phosphate residues to allow efficient uptake into target cells and tissues. N-acetylglucosaminyltransferase-I (GnTI) mediates N-glycosylation in the cis cisternae of the Golgi apparatus by adding N-acetylglucosamine to the exposed terminal mannose residue of core N-glycan structures for further processing. Mutant rice lacking GnTI produces only high mannosylated glycoproteins. In this study, we introduced a gene encoding recombinant human acid α-glucosidase (rhGAA), which is used in ERT for Pompe disease, into gnt1 rice callus by particle bombardment. Integration of the target gene into the genome of the gnt1 rice line and its mRNA expression were confirmed by PCR and Northern blot, respectively. Western blot analysis was performed to confirm secretion of the target proteins into the culture media. Using an indirect enzyme linked immunosorbent assay, we determined the maximum expression of rhGAA to be approximately 45mg/L, 13days after induction. To assay the enzymatic activity and determine the N-glycan profile of rhGAA, we purified the protein using a 6×histidine tag. The in vitro α-glucosidase activity of rhGAA from gnt1 rice callus (gnt1-GAA) was 3.092U/mg, similar to the activity of the Chinese hamster ovary cell-derived GAA (3.154U/mg). N-glycan analysis revealed the presence of high-mannose N-glycans on gnt1-GAA. In addition, the production of high-mannose GAA using gnt1 rice calli as an expression host was characterized, which may aid the future development of therapeutic enzymes for the treatment of Pompe disease.

Characterization of novel thermophilic alpha-glucosidase from Bifidobacterium longum.

In this study, the gene encoding α-glucosidase from Bifidobacterium longum subsp. longum JCM1217 (BLAG) was cloned and expressed in Escherichia coli. The amino acid sequence alignment demonstrated that BLAG belongs to glycoside hydrolase (GH) family 13. The optimal temperature for enzyme activity was 75°C; about 80% of the catalytic activity was lost at 50°C, which is very unusual for enzymes from the Bifidobacterium genus. In the presence of 5mM of Co2+ and Ca2+, enzyme activity was reduced to 47% and 48%, respectively. Furthermore, BLAG lost catalytic activity following the addition of 5mM of Fe2+ ion. The BLAG enzyme was able to hydrolyze α-1,2, α-1,3, α-1,4, and α-1,6 glycosidic O-linkages and liberated glucose from the non-reducing end of substrates. The kinetic study revealed that among the maltooligosaccharides, BLAG showed the highest kcat/Km value to maltotriose (G3), and had relatively low kcat/Km values on long-chain maltooligosaccharides. This is the first report describing the production of a thermophilic α-glucosidase from the Bifidobacterium genus.

Effects of lysosomal biotherapeutic recombinant protein expression on cell stress and protease and general host cell protein release in Chinese hamster ovary cells.

Recombinant human Acid Alpha Glucosidase (GAA) is the therapeutic enzyme used for the treatment of Pompe disease, a rare genetic disorder characterized by GAA deficiency in the cell lysosomes (Raben et al., Curr Mol Med. 2002; 2:145-166). The manufacturing process for GAA can be challenging, in part due to protease degradation. The overall goal of this study was to understand the effects of GAA overexpression on cell lysosomal phenotype and host cell protein (HCP) release, and any resultant consequences for protease levels and ease of manufacture. To do this we first generated a human recombinant GAA producing stable CHO cell line and designed the capture chromatographic step anion exchange (IEX). We then collected images of cell lysosomes via transmission electron microscopy (TEM) and compared the resulting data with that from a null CHO cell line. TEM imaging revealed 72% of all lysosomes in the GAA cell line were engorged indicating extensive cell stress; by comparison only 8% of lysosomes in the null CHO had a similar phenotype. Furthermore, comparison of the HCP profile among cell lines (GAA, mAb, and Null) capture eluates, showed that while most HCPs released were common across them, some were unique to the GAA producer, implying that cell stress caused by overexpression of GAA has a molecule specific effect on HCP release. Protease analysis via zymograms showed an overall reduction in proteolytic activity after the capture step but also revealed the presence of co-eluting proteases at approximately 80 KDa, which MS analysis putatively identified as dipeptidyl peptidase 3 and prolyl endopeptidase. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:666-676, 2017.

Production and characterization of recombinant human acid α-glucosidase in transgenic rice cell suspension culture.

Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid α-glucosidase (GAA), a glycogen-degrading lysosomal enzyme. In this study, the human GAA cDNA gene was synthesized from human placenta cells and cloned into a plant expression vector under the control of the rice α-amylase 3D (RAmy3D) promoter. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by Agrobacterium tumefaciens. Genomic DNA PCR and Northern blot analysis were used to determine the integration and mRNA expression of the hGAA gene in the putative transgenic rice cells. SDS-PAGE and Western blot analysis showed that the glycosylated precursor recombinant hGAA had a molecular mass of 110kDa due to the presence of seven N-glycosylation sites. The accumulation of hGAA protein in the culture medium was approximately 37mg/L after 11 days of culturing in a sugar depletion medium. The His tagged-hGAA protein was purified using an Ni-NTA column and confirmed as the precursor form of hGAA without the signal peptide encoded by the cDNA on the N-terminal amino acid sequence. The acid alpha-glucosidase activity of hGAA produced in transgenic rice cells gave results similar to those of the enzyme produced by CHO cells.

A cold-adapted and glucose-stimulated type II α-glucosidase from a deep-sea bacterium Pseudoalteromonas sp. K8.

OBJECTIVES: To express and characterize a putative α-glucosidase, Pagl, from Pseudoalteromonas sp. K8 obtained via genome mining approach. RESULTS: Pagl was expressed and purified to homogeneity, with a molecular mass of 60 kDa. It was optimally active at pH 8.5 and 30 °C, and showed cold-adapted activity. Pagl exhibited specific activity towards substrates with α-1,4-linkage, with the highest specific activity of 19.4 U/mg for maltose, followed by pNPαG and maltodextrins, suggesting that Pagl belongs to the type II α-glucosidase. Interestingly, the activity of Pagl is significantly enhanced (2.7 times) in the presence of 200 mM glucose. CONCLUSION: The unique catalytic properties of Pagl make it an attractive candidate for several industrial applications.

Enzymatic transglycosylation of ginsenoside Rg1 by rice seed α-glucosidase.

Six α-monoglucosyl derivatives of ginsenoside Rg1 (G-Rg1) were synthesized by transglycosylation reaction of rice seed α-glucosidase in the reaction mixture containing maltose as a glucosyl donor and G-Rg1 as an acceptor. Their chemical structures were identified by spectroscopic analysis, and the effects of reaction time, pH, and glycosyl donors on transglycosylation reaction were investigated. The results showed that rice seed α-glucosidase transfers α-glucosyl group from maltose to G-Rg1 by forming either α-1,3 (α-nigerosyl)-, α-1,4 (α-maltosyl)-, or α-1,6 (α-isomaltosyl)-glucosidic linkages in ß-glucose moieties linked at the C6- and C20-position of protopanaxatriol (PPT)-type aglycone. The optimum pH range for the transglycosylation reaction was between 5.0 and 6.0. Rice seed α-glucosidase acted on maltose, soluble starch, and PNP α-D-glucopyranoside as glycosyl donors, but not on glucose, sucrose, or trehalose. These α-monoglucosyl derivatives of G-Rg1 were easily hydrolyzed to G-Rg1 by rat small intestinal and liver α-glucosidase in vitro.

Purification and characterization of a chloride ion-dependent α-glucosidase from the midgut gland of Japanese scallop (Patinopecten yessoensis).

Marine glycoside hydrolases hold enormous potential due to their habitat-related characteristics such as salt tolerance, barophilicity, and cold tolerance. We purified an α-glucosidase (PYG) from the midgut gland of the Japanese scallop (Patinopecten yessoensis) and found that this enzyme has unique characteristics. The use of acarbose affinity chromatography during the purification was particularly effective, increasing the specific activity 570-fold. PYG is an interesting chloride ion-dependent enzyme. Chloride ion causes distinctive changes in its enzymatic properties, increasing its hydrolysis rate, changing the pH profile of its enzyme activity, shifting the range of its pH stability to the alkaline region, and raising its optimal temperature from 37 to 55 °C. Furthermore, chloride ion altered PYG's substrate specificity. PYG exhibited the highest Vmax/Km value toward maltooctaose in the absence of chloride ion and toward maltotriose in the presence of chloride ion.

Absolute quantification of the total and antidrug antibody-bound concentrations of recombinant human α-glucosidase in human plasma using protein G extraction and LC-MS/MS.

The administration of protein-based pharmaceuticals can cause the in vivo formation of antidrug antibodies (ADAs), which may reduce the efficacy of the therapy by binding to the protein drug. An accurate determination of the total and ADA-bound concentrations of the drug gives information on the extent of this immune response and its consequences and may help develop improved therapeutic regimens. We present an absolute quantitative method to differentiate between total, free, and ADA-bound drug for recombinant human alpha acid glucosidase (rhGAA) in plasma from patients suffering from Pompe's disease. LC-MS/MS quantification of a signature peptide after trypsin digestion of plasma samples before and after an extraction of the total IgG content of plasma with protein G coated beads was used to determine the total and the ADA-bound fractions of rhGAA in samples from Pompe patients after enzyme infusion. The methods for total and ADA-bound rhGAA allow quantitation of the drug in the range of 0.5 to 500 µg/mL using 20 µL of plasma and met the regular bioanalytical validation requirements, both in the absence and presence of high levels of anti-rhGAA antibodies. This demonstrates that the ADA-bound rhGAA fraction can be accurately and precisely determined and is not influenced by sample dilution, repeated freezing and thawing, or extended benchtop or frozen storage. In samples from a patient with a reduced response to therapy due to ADAs, high ADA-bound concentrations of rhGAA were found, while in the samples from a patient lacking ADAs, no significant ADA-bound concentrations were found. Since protein G captures the complete IgG content of plasma, including all antidrug antibodies, the described extraction approach is universally applicable for the quantification of ADA-bound concentrations of all non-IgG-based biopharmaceuticals.

Functional expression and molecular characterization of Culex quinquefasciatus salivary α-glucosidase (MalI).

Salivary α-glucosidases (MalI) have been much less characterized when compared with midgut α-glucosidases, which have been studied in depth. Few studies have been reported on the partial characterization of MalI, but no clear function has been ascribed. The aim of this study is to purify and characterize the recombinant Culex quinquefasciatus (CQ) α-glucosidase expressed in Pichia pastoris. The cDNA encoding mature Cx. quinquefasciatus α-glucosidase gene with polyhistidine tag (rCQMalIHis) was successfully cloned into the expression vector, pPICZαB, designated as pPICZαB/CQMalIHis. The activity of recombinant rCQMalIHis expressed in P. pastoris could be detected at 3.75U/ml, under optimal culture conditions. The purified rCQMalIHis showed a single band of molecular weight of approximately 92kDa on SDS-PAGE. After Endoglycosidase H digestion, a single band at 69kDa was found on SDS-PAGE analysis, suggesting that rCQMalIHis is a glycoprotein. Additionally, tryptic digestion and LC-MALDI MS/MS analysis suggested that the 69kDa band corresponds to the Cx. quinquefasciatus α-glucosidase. Thus, rCQMalIHis is a glycoprotein. The rCQMalIHis exhibited optimum pH and temperature at 5.5 and 35°C, respectively. The catalytic efficiency (kcat/Km) of the purified rCQMalIHis for maltotriose is higher than those for sucrose, maltotetraose, maltose and p-nitrophenyl-α-glucoside, indicating that the enzyme prefers maltotriose. Additionally, the rCQMalIHis is significantly inhibited by d-gluconic acid δ-lactone, but not by Mg(2+), Ca(2+) and EDTA. The rCQMalIHis is strongly inhibited by acarbose with IC50 67.8±5.6nM, but weakly inhibited by glucose with IC50 115.9±7.3mM.

Purification and characterization of a novel α-glucosidase from Malbranchea cinnamomea.

OBJECTIVES: To characterize a novel α-glucosidase from the thermophilic fungus Malbranchea cinnamomea. RESULTS: The enzyme was purified to homogeneity with purification fold of 40 and a recovery of 7.2 %. It was a monomer with molecular mass of 65.7 kDa on SDS-PAGE. It was optimally active at pH 6 and 50 °C (measured over 10 min) and exhibited a wide range of substrate specificity with the highest specific activity of 47.4 U mg(-1) for p-nitrophenyl α-D-glucopyranoside (pNPGlu) followed by isomaltose, panose and sucrose, suggesting that the enzyme belongs to the type I α-glucosidases. The K m values of the α-glucosidase for pNPGlu and isomaltose were 1.1 and 19.3 mM, respectively. CONCLUSION: Because of its unique properties, the α-glucosidase may have a potential in several industrial applications.

Biochemical properties and substrate recognition mechanism of GH31 α-glucosidase from Bacillus sp. AHU 2001 with broad substrate specificity.

α-Glucosidases are ubiquitous enzymes that hydrolyze the α-glucosidic linkage at the non-reducing end of substrates. In this study, we characterized an α-glucosidase (BspAG31A) belonging to glycoside hydrolase family 31 from Bacillus sp. AHU 2001. Recombinant BspAG31A, produced in Escherichia coli, had high hydrolytic activity toward maltooligosaccharides, kojibiose, nigerose, and neotrehalose. This is the first report of an α-glucosidase with high activity toward neotrehalose. The transglucosylation products, nigerose, kojibiose, isomaltose, and neotrehalose, were generated from 440 mm maltose. Substitution of Tyr268, situated on the ߠ→ α loop 1 of BspAG31A, with Trp increased hydrolytic activity toward isomaltose. This mutation reduced the hydrolytic activity toward maltooligosaccharides more than toward kojibiose, nigerose, and neotrehalose. Analysis of the Y173A mutant of BspAG31A showed that Tyr173, situated on the N-terminal domain loop, is associated with the formation of subsite +2. In Y173A, the kcat/Km for maltooligosaccharides slightly decreased with an increasing degree of polymerization compared with wild type. Among the amino acid residues surrounding the substrate binding site, Val543 and Glu545 of BspAG31A were different from the corresponding residues of Bacillus thermoamyloliquefaciens α-glucosidase II, which has higher activity toward isomaltose than BspAG31A. The E545G mutation slightly enhanced isomaltase activity without a large reduction of hydrolytic activities toward other substrates. V543A showed 1.8-3.5-fold higher hydrolytic activities toward all substrates other than neotrehalose compared with wild type, although its preference for isomaltose was unchanged.

Production of 1,5-anhydro-d-fructose by an α-glucosidase belonging to glycoside hydrolase family 31.

α-1,4-Glucan lyases [glycoside hydrolase family (GH) 31] catalyze an elimination reaction to form 1,5-anhydro-d-fructose (AF), while GH31 α-glucosidases normally catalyze a hydrolytic reaction. We determined that a small amount of AF was produced by GH31 Aspergillus niger α-glucosidase from maltooligosaccharides by elimination reaction, likely via an oxocarbenium ion intermediate.