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Three kinds of alpha-glucosidases, I, II, and III, were purified from European honeybees, Apis mellifera L. In addition, an alpha-glucosidase was also purified from honey. Some properties, including the substrate specificity of honey alpha-glucosidase, were almost the same as those of alpha-glucosidase III. Specific antisera against the alpha-glucosidases were prepared to examine the localization of alpha-glucosidases in the organs of honeybees. It was immunologically confirmed for the first time that alpha-glucosidase I was present in ventriculus, and alpha-glucosidase II, in ventriculus and haemolymph. alpha-Glucosidase III, which became apparent to be honey alpha-glucosidase, was present in the hypopharyngeal gland, from which the enzyme may be secreted into nectar gathered by honeybees. Honey may be finally made up through the process whereby sucrose in nectar, in which glucose and fructose also are naturally contained, is hydrolyzed by secreted alpha-glucosidase III.

Replacement of the catalytic nucleophile Asp481 by glycine in Schizosaccharomyces pombe alpha-glucosidase eliminated the hydrolytic activity. The mutant enzyme (D481G) was found to catalyze the formation of an alpha-glucosidic linkage from beta-glucosyl fluoride and 4-nitrophenyl (PNP) alpha-glucoside to produce two kinds of PNP alpha-diglucosides, alpha-isomaltoside and alpha-maltoside. The two products were not hydrolyzed by D481G, giving 41 and 29% yields of PNP alpha-isomaltoside and alpha-maltoside, respectively. PNP monoglycosides, such as alpha-xyloside, alpha-mannoside, or beta-glucoside, acted as the substrate, but PNP alpha-galactoside and maltose could not. No detectable product was observed in the combination of alpha-glucosyl fluoride and PNP alpha-glucoside. This study is the first report on an "alpha-glycosynthase"-type reaction to form an alpha-glycosidic linkage.

Trehalase (EC 3.2.1.28) of the bound type was purified as an electrophoretically homogeneous protein from adult honeybees by fractionation with ammonium sulfate, hydrophobic chromatography, and DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, butyl-Toyopearl 650M, and p-aminophenyl beta-glucoside Sepharose 4B column chromatographies. The enzyme preparation was confirmed to be a monomeric protein containing 3.1% carbohydrate. The molecular weight was estimated to be approximately 69,000, and the optimum pH was 6.7. The Michaelis constant (Km) was 0.66 mM, and the molecular activity (k0) was 86.2 s(-1). The enzyme was an "inverting" type which produced beta-glucose from alpha, alpha-trehalose. Dependence of the V and Km values on pH gave values for the ionization constants, pKe1 and pKe2, of essential ionizable groups 1 and 2 of the free enzyme of 5.3 and 8.5, respectively. When the dielectric constant of the reaction mixture was decreased, pKe1, and pKe2 were shifted to higher values of + 0.2 and + 0.5 pH unit, respectively. The ionization heat (deltaH) of ionizable group 1 was estimated to be + 1.8 kcal/mol, and the deltaH value of group 2 was + 1.5 kcal/mol. These findings strongly support the notion that the essential ionizable groups of honeybee trehalase are two kinds of carboxyl groups, one being a dissociated type (-COO(-), ionizable group 1) and the other a protonated type (-COOH, ionizable group 2), although the pKe2 value is high.

The gene encoding an extracellular isomaltotrio-dextranase (IMTD), designed dexT, was cloned from the chromosomal DNA of Brevibacterium fuscum var. dextranlyticum strain 0407, and expressed in Escherichia coli. A single open reading frame consisting of 1923 base pairs that encoded a polypeptide composed of a signal peptide of 37 amino acids and a mature protein of 604 amino acids (M(r), 68,300) was found. The primary structure had no significant similarity with the structure of two other reported exo-type dextranases (glucodextranase and isomalto-dextranase), but had high similarity with that of an endo-dextranase isolated from Arthrobacter sp. Transformed E. coli cells carrying the gene encoding mature protein of IMTD overproduced IMTD under the control of the T7 phage promoter induced by IPTG. The purified recombinant enzyme showed the same optimum pH, lower specific activity, and similar hydrolytic pattern, as to those of native IMTD.

The catalytic amino acid residue of Aspergillus niger alpha-glucosidase (ANGase) was identified by modification with conduritol B epoxide (CBE), a mechanism-based irreversible inactivator. The inactivation by CBE followed pseudo-first order kinetics. The interaction of CBE and ANGase conformed to a model with a reversible enzyme-inhibitor complex formed before covalent inactivation. A competitive inhibitor, Tris, decreased the inactivation rate. The incorporation of one mole of CBE per mole of ANGase was completely abolished the enzyme activity. A dissociated carboxyl group (-COO-) in the active site was suggested to attack the C-1 of CBE. ANGase was composed of two subunits (P1 and P2), of which P2 was modified by CBE. The labelled residue was included in a peptide (LY3) that was obtained from Lys-C protease digestion of CBE-bound P2. The sequence analysis of CBE-labelled LY3 showed that an Asp was the modified residue, that is, one of the catalytic amino acid residues of ANGase. The primary structure of LY3 was determined by analyzing the sequence of peptide fragments prepared by several proteases.

Asp. niger α-glucosidase was crystallized in ammonium sulfate solution after chromatographies on DEAE-Sepharose CL-6B and TOYOPEARL HW-55 columns. The crystalline α-glucosidase, which was a glycoprotein containing 25.5% carbohydrate as glucose, gave a single band on polyacrylamide disc gel electrophoresis. The molecular weight was estimated to be about 12.5 × 104 by SDS-disc gel electrophoresis. However, the crystalline enzyme consists of two components (M.W. about 3.3 × 104 and 9.8 × 104, respectively) separable only by reverse-phase HPLC causing irreversible inactivation. The optimum pH was 4.3. The enzyme also hydrolyzed α-glucans such as soluble starch and amylose. The ratios of V values (Km values, in parentheses, mm of nonreducing terminal) for maltose, kojibiose, nigerose, isomaltose, phenyl α-glucoside, phenyl α-maltoside, malto-triose, -tetraose, -pentaose, -hexaose, -heptaose and -octaose, maltodextrin (DP = 17), and soluble starch were estimated to be 100 (0.75): 23 (4.6): 62 (12): 35 (8.0): 1....

(1992). Complete Amino Acid Sequence of Crystalline (α–Glucosidase from Aspergillus niger. Bioscience, Biotechnology, and Biochemistry: Vol. 56, No. 8, pp. 1368-1370.

Transglucosylation of an α-glucosidase (I) from honeybee was investigated. The honeybee α- glucosidase catalyzed the predominant formation of α-1,4-glucosidic linkage from sucrose, phenyl α-glucoside, or maltose. The major product from sucrose was 4G-a-glucosyl-sucrose (erlose), which differs from the report by Huber and Thompson that 3F-a-glucosyl-sucrose (melezitose) is mainly synthesized from sucrose by the transglucosylation of the same α-glucosidase: Biochemistry, 12, m 4011 (1973). Melezitose was not detected as the transglucosylation product of this enzyme.

Enzymatic synthesis of α-glucosyl-sucrose through the transglucosidase action of immobilized buckwheat α-glucosidase was attempted in a reaction system containing maltose and sucrose. Three kinds of nonreducing trisaccharides were produced, and a large portion of the trisaccharide was 4G-α-d-glucosyl-sucrose (erlose). Another was a new trisaccharide, O-α-d-glucopyranosyl-(l → 3)-O-α-d-glucopyranosyl-(l → 2)-O-d-fructofuranoside (3G-α-d-glucosyl-sucrose; [α]d+120°, in water), and the other was 6G-α-d-glucosyl-sucrose (theanderose; [α]d+107°, in water). The new trisaccharide was named “esculose.”

A gas-liquid chromatographic method was applied to the determination of anomeric forms of sugar produced by carbohydrases. Anomeric forms of glucose released from maltotriose, phenyl amaltoside and phenyl a-glucoside were determined quantitatively. Thirteen a-glucosidases tested, including a-glucosidase from honey bee and acid α-glucosidase from pig's liver, were found to produce a-glucose exclusively, and two kinds of glucoamylases, only β-glucose. This method proved very useful for the determination of the anomeric forms of sugar produced. It was confirmed that mammalian acid a-glucosidase does not belong to the category of exo-1, 4-α-glucosidase (EC 3.2.1.3).

A gas-liquid chromatographic method was applied to the determination of anomeric forms of sugar produced by carbohydrases. Anomeric forms of glucose released from maltotriose, phenyl α-maltoside and phenyl α-glucoside were determined quantitatively. Thirteen α-glucosidases tested, including α-glucosidase from honey bee and acid α-glucosidase from pig′s liver, were found to produce α-glucose exclusively, and two kinds of glucoamylases, only β-glucose. This method proved very useful for the determination of the anomeric forms of sugar produced. It was confirmed that mammalian acid α-glucosidase does not belong to the category of exo-1,4-α-glucosidase (EC 3.2.1.3).

Anomeric forms of glucose and maltose produced from phenyl, p-nitrophenyl, p-tert-butylphenyl, p-ethylphenyl and p-chlorophenyl α-maltosides and maltopentaose by α- and β-amylases were determined quantitatively by a gas-liquid chromatographic method. All of the three kinds of α-amylases tested, B. subtilis saccharifying α-amylase, Taka-amylase A, and porcine pancreas α-amylase, were found to produce only α-maltose from the maltosides. Sweet potato and barley β-amylases produced β-maltose from maltopentaose.Saccharifying α-amylase from B. subtilis also released α-maltose from all the maltosides mentioned above, contrary to the report by Shibaoka et al. that the enzyme released β-maltose from maltosides other than phenyl α-maltoside: FEBS Lett., 16, 33 (1971); J. Biochem., 77, 1215 (1975). It appears unlikely that the α-amylase releases β-maltose, depending on the kind of substrate.

Two kinds of α-glucosidase (I and II) were isolated from honey bees by salting-out chromatography with ammonium sulfate. α-Glucosidase I was purified by chromatography on CM-cellulose and gel filtration on Sephadex G-100. α-Glucosidase II was purified by chromatographies on DEAE- and CM-cellulose and by gel filtration on Bio-Gel P-150. Both enzyme preparations were homogeneous in tests by disc electrophoresis. The molecular weight of α-glucosidases I and II was estimated to be approximately 9.8 × 104 and 7.6 × 104, respectively, by SDS disc electrophoresis.α-Glucosidases I and II were glycoproteins whose carbohydrate moieties were about 25% and 15%, respectively. Their pH optima were 5.0. Both α-glucosidases readily hydrolyzed phenyl-α-glucoside, sucrose and maltose. α-Glucosidase I showed no activity toward isomaltose and soluble starch, but α-glucosidase II showed relatively high activity toward isomaltose and slight activity toward soluble starch.