Purification and Properties of α-Glucosidase from Bacillus cereus

α-Glucosidase has been isolated from Bacillus cereus in ultracentrifugally and electrophoretically homogeneous form, and its properties have been investigated. The enzyme has a sedimentation constant of 1.4 S and a molecular weight of 12,000. The highly purified enzyme splits α-d-(1→4)-glucosidic linkages in maltose, maltotriose, and phenyl α-maltoside, but shows little or no activity toward polysaccharides, such as amylose, amylopectin, glycogen and soluble starch. The enzyme has α-glucosyltransferase activity, the main transfer product from maltose being maltotriose. The enzyme can also catalyze the transfer of α-glucosyl residue from maltose to riboflavin. On the basis of inhibition studies with diazonium-1-H-tetrazole, rose bengal and p-chloromercuribenzoate, it is assumed that the enzyme contains both histidine and cysteine residues in the active center.     

Purification and Properties of α-Glucosidase and Glucoamylase from Lentinus edodes (Berk.) Sing.

An α-glucosidase and a glucoamylase have been isolated from fruit bodies of Lentinus edodes (Berk.) Sing., by a procedure including fractionation with ammonium sulfate, DEAE-cellulose column chromatography, and preparative gel electrofocusing. Both of them were homogeneous on gel electrofocusing and ultracentrifugation. The molecular weight of α-glucosidase and glucoamylase was 51,000 and 55,000, respectively. The α-glucosidase hydrolyzed maltose, maltotriose, phenyl α-maltoside, amylose, and soluble starch, but did not act on sucrose. The glucoamylase hydrolyzed maltose, maltotriose, phenyl α-maltoside, soluble starch, amylose, amylopectin, and glycogen, glucose being the sole product formed in the digests of these substrates. Both enzymes hydrolyzed phenyl a-maltoside into glucose and phenyl α-glucoside. The glucoamylase hydrolyzed soluble starch, amylose, amylopectin, and glycogen, converting them almost completely into glucose. It was found that β-glucose was liberated from amylose by the action of glucoamylase, while α-glucose was produced by the α-glucosidase.

Maltotriose was the main α-glucosyltransfer product formed from maltose by the α-glucosidase.     

Three Forms of α-Glucosidase from Suspension-cultured Rice Cells

Three forms of α-glucosidase (EC 3.2.1.20), designated as I, II, and III, have been isolated from suspension-cultured rice cells by a procedure including fractionation with ammonium sulfate, CM-cellulose column chromatography, and preparative disc gel electrophoresis. The three enzymes were homogeneous by Polyacrylamide disc gel electrophoresis. α-Glucosidase I was secreted in the culture medium during growth, α-glucosidase II was readily extracted from rice cells with the buffer alone, and α-glucosidase III required NaCl to be solubilized. The molecular weights of the three enzymes were 96,000 (I), 84,000 (II), and 58,000 (III). The three enzymes readily hydrolyzed maltose, maltotriose, maltotetraose, amylose, and soluble starch. α-Glucosidase I possessed strong isomaltose-hydrolyzing activity and hydrolyzed isomaltose about three times as rapidly as α-glucosidase III. The three enzymes produced panose as the main α-glucosyltransfer product from maltose. Half the maltose-hydrolyzing activities of the three enzymes were inhibited by 11.25 ng of castanospermine. The inhibition was competitive.     

Purification and Properties of Wall-bound α-Glucosidase from Suspension-cultured Rice Cells

Wall-bound α-glucosidase (EC 3.2.1.20) has been solubilized from suspension-cultured rice cells with Sumyzyme C and Pectolyase Y-23 and isolated by a procedure including fractionation with ammonium sulfate, Sephadex G-100 column chromatography, CM-cellulose column chroma-tography, Sephadex G-200 column chromatography, and preparative disc gel electrophoresis. The molecular weight of the enzyme was 64,000. The enzyme readily hydrolyzed maltose, maltotriose, and amylose, but hydrolyzed isomaltose and soluble starch more slowly. The Michaelis constant for maltose of the enzyme was estimated to be 0.272 mm. The enzyme produced panose as the main α- glucosyltransferred product from maltose.     

Purification and Some Properties of Saccharomyces logos α-Glucosidase

α-Glucosidase was purified from Saccharomyces logos by precipitation with ethanol, and chromatographies on Sephadex G–200, DEAE-Sephadex, DEAE-ceiluiose and Duolite A–2. The purified α-glucosidase was homogeneous on ultracentrifugation and zone electrophoresis using cellulose acetate membrane. The sedimentation coefficient was calculated to be 9.6 S. The molecular weight was estimated to be approximately 2.7 × 10⁵ by gel-filtration technique.

The optimum pH was found to be in the range of 4.6~5.0, and the optimum temperature was 40°C. The enzyme exhibited higher hydrolytic activity toward maltose rather than toward phenyl-α-glucoside and turanose, and no activity toward sucrose.

The enzyme was a glycoprotein containing carbohydrate of about 50%.     

Purification and Some Properties of a Neutral α-Glucosidase from Pig Serum

A neutral α-glucosidase was purified from pig serum by precipitation with ammonium sulfate, chromatographies on DEAE-cellulose and -Sephadex A–50, and gel filtration on Bio-Gel P–300 and Sephadex G–200. The purified enzyme was homogeneous in ultracentrifugal and disc electrophoretic analysis. The sedimentation coefficient (s_20,w) was calculated to be 10.7 S, and the isoelectric point, 4.0. The molecular weight was estimated to be approximately 2.7 × 10⁵ by thin-layer gel filtration and SDS-disc electrophoresis.

The enzyme exhibited also glucoamylase activity. The optimal pH was found to be in the pH range of 6.0 to 7.0 for maltose and soluble starch. The ratio of velocity of hydrolysis for maltose (Km, 0.72 mg/ml), soluble starch (Km, 9.8 mg/ml) and shellfish glycogen (Km, 55.6 mg/ml) was calculated to be 100: 110: 5.15 in this order.     

Purification and Some Properties of Flint Corn α-Glucosidase

An α-glucosidase was purified from flint corn by precipitation with ammonium sulfate, chromatographies on CM-cellulose and Hydroxylapatite and gel-filtrations on Sephadex G-100. The purified enzyme was homogeneous in ultracentrifugal and disc electrophoretic analysis. The sedimentation coefficient was calculated to be 6.5 S. The molecular weight was estimated to be approximately 6.5×10⁴ by gel-filtration technique.

The optimal pH was found to be 3.6 for both maltose and soluble starch. The enzyme lost about 80% of the activity by incubation at 60°C for 10 min.

The ratio of velocity of hydrolysis for maltose, phenyl-α-glucoside and soluble starch was estimated to be 100:14.3:6.1 in this order. The αglucosidase hydrolyzed soluble starch exo-wisely.     

Purification and Some Properties of β-Glucosidase from Streptomyces sp

β-Glucosidases I, II, and III were isolated from the culture filtrate of a Streptomyces sp. by ammonium sulfate fractionation, hydroxylapatite column chromatography, filtration on Bio-Gel P-100, and DE-52 column chromatography. β-Glucosidase III had a single active band on disc-gel electrophoresis. Its optimum pH and temperature for activity were 6.0 and 60°C, respectively. The isoelectric point and molecular weight of the enzyme were pH 4.5 and 45,000, respectively. From an experiment using ¹⁴C-labeled glucose, gentiobiose seemed to be formed from laminaribiose as isomaltose is formed from maltose by fungal α-glucosidase. The enzyme showed transglucosylation and produced gentiobiose from β-gluco-disaccharides and 4-O-β-d-glucopyranosyl-d-manno-pyranose (epicellobiose). The enzyme acted on phenolic β-d-glucosides to produce unknown transfer products.     

Multiple Forms of α-Glucosidase from Pig Duodenum

Multiple forms of neutral α-glucosidase (pH optima, 6.0~6.5) were purified from pig duodenal mucosa by a procedure including Triton X-100 treatment, fractionation with ammonium sulfate, fractionation with ethyl alcohol, DEAE-cellulose column chromatography and preparative polyacrylamide disc gel electrophoresis. All of the α-glucosidases, I_a, II_a, I_b and II_b, were found to be homogeneous on polyacrylamide disc gel electrophoresis. The molecular weights, isoelectric points and optimum temperatures of α-glueosidases I_a and II_a were 145,000~150,000, pH 3.5~3.7 and 55°C, respectively, and both enzymes were stable up to 55°C on treatment at pH 6.0 for 15 min; whereas those of the other two α-glucosidases, I_b and II_b, were 80,000, pH 4.0~4.1 and 65°C, respectively, and both enzymes were stable up to 70°C on the same treatment. The Km values of enzyme II_a for maltose, maltotriose and amylose were 1.72mm, 0.37 mm and 1.67mg/ml, while those of enzyme II_b were 3.33 mm, 2.61 mm and 11.8 mg/ml, respectively. All enzyme hydrolyzed α-1,4-, α-1,3- and α-1,2-glucosidic linkages in substrates, but showed no activity on sucrose or isomaltose. Enzymes II_a and II_b hydrolyzed phenyl α-maltoside to glucose and phenyl α-glucoside, and maltotriose was formed as the main α-glucosyltransfer product from maltose. It was revealed that two types of neutral α-glucosidases having no activity toward sucrose or isomaltose existed in pig duodenal mucosa, and that one type comprised α-glucosidase having both maltose- and amylaceous α-glucan-hydrolyzing activities and the other type heat-stable maltooligosaccharidases which hydrolyzed amylaceous α-glucan weakly.     

Purification and Properties of β-Glucosidase from Humicola insolens YH-8

The thermophilic fungus, Humicola insolens YH-8 exhibited high β-glucosidase activity when grown in solid wheat bran medium. The β-glucosidase was purified from the culture extract by consecutive column chromatographies and found to be homogeneous on polyacrylamide gel disc electrophoresis. The molecular weight was estimated to be 250,000 by SDS-gel electrophoresis, and the isoelectric point was at pH 4.23. The enzyme had an optimum pH of 5.0, an optimum temperature of 50°C, and showed significant resistance to urea, dimethyl sulfoxide and ethyl alcohol.     

Transglucosylation Activities of Multiple Forms of α-Glucosidase from Spinach

Transglucosylation activities of spinach α-glucosidase I and IV, which have different substrate specificity for hydrolyzing activity, were investigated. In a maltose mixture, α-glucosidase I, which has high activity toward not only maltooligosaccharides but also soluble starch and can hydrolyze isomaltose, produced maltotriose, isomaltose, and panose, and α-glucosidase IV, which has high activity toward maltooligosaccharides but faint activity toward soluble starch and isomaltose, produced maltotriose, kojibiose, and 2,4-di-α-D-glucosyl-glucose. Transglucosylation to sucrose by α-glucosidase I and IV resulted in the production of theanderose and erlose, respectively, showing that spinach α-glucosidase I and IV are useful to synthesize the α-1,6-glucosylated and α-1,2- and 1,4-glucosylated products, respectively.     

Certain Properties of α-Glucosidase from Mucor racemosus

The substrate and inhibitor specificities, and α-glucosyltransfer products of the purified α-glucosidase from the mycelia of Mucor racemosus were investigated. The enzyme hydrolyzed maltose, maltotriose, phenyl α-maltoside, isomaltose, soluble starch, and amylose liberating glucose, but did not act on sucrose. The enzyme hydrolyzed phenyl a-maltoside into glucose and phenyl α-glucoside. Maltotriose was the main a-glucosyltransfer product formed from maltose, and isomaltose was that from soluble starch. Tris and turanose inhibited the enzyme activity, but PCMB and EDTA did not. The enzyme hydrolyzed amylose liberating a-glucose. The enzyme was a glycoprotein containing 4.1% of neutral sugar. The neutral sugar was identified as mannose in the acid hydrolyzate of the enzyme.     

Properties of Crystalline α-Glucosidase from Mucor javanicus

Substrate and inhibitor specificities, and transglucosylation action of crystalline α-glucosidase from the mycelia of Mucor javanicus have been investigated. The enzyme hydrolyzed maltose, methyl-α-maltoside, and soluble starch liberating glucose, but little or not phenyl-α-glucoside, methyl-α-glucoside, sucrose, isomaltose, panose and dextran. The enzyme hydrolyzed phenyl-α-maltoside to glucose and phenyl-α-glucoside. The enzyme acted also as a glucosyltransferase when it was incubated with glucosyl donor such as maltose. Maltotriose was the principal transglucosylation product formed from maltose. The enzyme also catalyzed transglucosylation from maltose to riboflavin, pyridoxine, esculin and rutin. Tris and turanose inhibited the enzyme activity, but PCMB and EDTA did not. It is suggested that the enzyme activity is closely related to the histidine residue in the active center, from the inhibition experiments using diazonium-1-H-tetrazole and rose bengal.     

High Recovery Purification and Some Properties of a β -Glucosidase from Aspergillus niger

A β-glucosidase was intensively purified with high recovery from a commercial preparation of Aspergillus niger by consecutive column chromatography. The enzyme was an acidic protein with a pI of 3.8, and split cellotriose to produce specifically β-D-glucose. Substrate specificity studies demonstrated that the purified enzyme required absolutely the C-4 configuration of the terminal, nonreducing β-D-glucose residues in the substrate molecules.     

Purification and Some Properties of α-Galactosidase from Tubers of Stachys affinis

An α-galactosidase from tubers of S. affinis was purified about 130 fold by ammonium sulfate fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The purified enzyme showed a single protein band on disc gel electrophoresis. The molecular weight of the enzyme was determined to be approximately 42,000 by gel filtration and 44,000 by SDS disc gel electrophoresis. The optimum reaction pH was 5.2. The enzyme hydrolyzed raffinose more rapidly than planteose. The activation energy of raffinose and planteose by the enzyme was estimated to be 7.89 and 11.4 kcal/mol, respectively. The enzyme activity was inhibited by various galactosides and structural analogs of d-galactose. Besides hydrolytic activity, the enzyme also catalyzed the transfer reaction of d-galactosyl residue from raffinose to methanol.     

Certain Properties of Crystalline α-Glucosidase from Mucor javanicus

The crystalline α-glucosidase from Mucor javanicus has a sedimentation constant () of 6.1 S, a diffusion constant (D_{20, w}) of 4.8 × 10^?7 cm² · sec^?1, and an average molecular weight, as determined by two different methods, of 124,600. The α-glucosidase is a glycoprotein containing the following constituents; tryptophan₂₃, lysine₈₁, histidine₃₉, arginine₃₄, aspartic acid₁₀₂, threonine₆₉, serine₄₆, glutamic acid₇₈, proline₅₅, glycine₇₈, alanine₅₅, half cystine₈, valine₅₃, methionine₁₇, isoleucine₅₈, leucine₈₁, tyrosine₅₁, phenylalanine₄₁, glucosamine₁₂, and mannose₃₈.

The low content of half cystine, the high contents of aspartic acid, lysine, and histidine, and the presence of mannose as the sole constituent of neutral sugar are the characteristics of this enzyme.     

Purification and Some Properties of α-Galactosidase from Candida guilliermondii H-404

Candida guilliermondii H-404, isolated from soil, produced thermostable α-galactosidase, but small amounts of other glycosidases (such as β-galactosidase, α-glucosidase, and β-glucosidase). The enzyme was separated into two fractions by DEAE-Toyopearl 650M chromatography, and the two enzymes were designated galactosidase I and II. These two enzymes had the same molecular weight (270,000 by gel filtration, 64,000 by SDS-PAGE). The isoelectric points of α-galactosidase I and II were 6.16 and 6.21, respectively. These two enzymes were different from each other in pH stability, temperature stability, and effects of Fe^{2 +} and Cu^{2 +} ion on α-galactosidase activity. The enzyme had stronger transfer activity and wider acceptor specificity than α-galactosidases which have been reported.     

Purification and Some Properties of Acid-stable α-Amylases from Shochu koji (Aspergillus kawachii)

Aspergillus kawachii α-amylase [EC 3.2.1.1] I and II were purified from shochu koji extract by DEAE Bio-Gel A ion exchange chromatography, Sephacryl S-300 gel chromatography (pH 3.6), coamino dodecyl agarose column chromatography and Sephacryl S-200 gel chromatography. By gel chromatography on a Sephacryl S-300 column, the molecular weights of the purified α-amylase I and II were estimated to be 104,000 and 66,000, respectively. The isoelectric points of α-amylase I and II were 4.25 and 4.20, respectively. The optimal pH range of α-amylase I was 4.0 to 5.0, and the optimum pH of α-amylase II was 5.0. The optimum temperatures of both α-amylases were around 70°C at pH 5.0. Both α-amylases were stable from pH 2.5 to 6.0 and up to 55°C, retaining more than 90% of the original activities. Heavy metal ions such as Hg^{2 +} and Pb^{2 +} were potent inhibitors for both α-amylases.     

Primary Structures of α- and β-Subunits of α-Amylase Inhibitors from Seeds of Three Cultivars of Phaseolus Beans

The primary structures of three α-amylase inhibitors (TAI, DAI, and MAI-2) consisting of glycoprotein subunits α and β from the respective seeds of three cultivars of Phaseolus beans, Toramame (Phaseolus vulgaris L.), Daifukumame (Phaseolus vulgaris L.), and Murasakihanamame (Phaseolus coccineus L.) were determined by sequencing the peptide fragments derived from their enzymatic digestions. Major sugar chains of the inhibitors were also assessed by analyzing glycopeptides in the enzymatic digests. The subunits, α and β, were shown to be composed of 76 and 139 amino acid residues, respectively, in each inhibitor. The overall amino acid sequences of the inhibitors were slightly different from one another. Furthermore, the sequence of TAI was the same as that deduced from a cDNA clone encording α-amylase inhibitor-1 from the common bean (Phaseolus vulgaris L.). It was also revealed that there were two N-glycosylation sites in each α-subunit: PA-derivatives of the major N-glycans were estimated to be M6B at Asn(12) and M9A at Asn(65). Each β-subunit of TAI and MAI-2 had two N-glycosylation sites, while the β-subunit of DAI had only one site. The major N-glycans pyridylaminated were estimated to be M3X at Asn(63) in each β-subunit and M3FX at Asn(83) in β-subunits of TAI and MAI-2.     

Purification and Properties of β-Galactosidases from Bacillus circulans

Six compounds, Z- and E-fadyenolide (3, 4), 1-ally1-2,3-(methylenedioxy)-4,5-dimethoxy-benzene (5), 4-methoxy-3,5-bis (3′-methyl-2′-butenyl)-benzoic acid (6), 2,6-dihydroxy-4-methoxy-dihydrochalcone (7), and 5-hydroxy-7-methoxyflavanone (8) were isolated from three species of Jamaican Piper, Piper fadyenii, C.D.C., Piper aduncum L. and Piper hispidum Sw. Three amides (9 ~ 11) of 3,5-dimethoxy-4-oxo-5-phenylpent-2-enoic acid using piperidine, pyrrolidine and morpholine, respectively, were synthesized from compounds 3 and 4, and tested for insecticidal activity against the tick Boophilus microplus (Canestrini) and the flour feetle, Tribolium confusum Duval. In our experiment, compounds 9 ~ 11 inhibited ovogenesis of B. microplus and were toxic to T. confusum. Compounds 3 ~ 8 were found to have no activity.