Substrate Specificity of Saccharomyces logos α-Glucosidase

The substrate specificity of Saccharomyces logos α-glucosidase has been investigated.

The enzyme was active especially on maltose and phenyl-α-maltoside. The ratio of hydrolysis for maltose : phenyl-α-maltoside : phenyl-α-glucoside was estimated to be 100:110: 5.5. Therefore, the substrate specificity of the enzyme was quite different from those of other Saccharomyces species, though similar to those of mold α-glucosidases.

Km values for maltose, phenyl-α-maltoside and phenyl-α-glucoside were calculated to be 7.7 mм, 3.6 mм and 8.7 mм, respectively. Of the substrates tested, the enzyme showed a preference for phenyl-α-maltoside.     

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%.     

Amino Acid Sequence of αkSubstance,a Mating Pheromone of Saccharomyces kluyveri

A fraction containing IgA (IgA-rich fraction) was prepared from bovine colostrum by anion exchange chromatography using DEAE-Sephadex A-50 and gel filtration on Sephadex G-200. A large amount of IgG1-dimer was found in this fraction, which could not be separated from IgA by repeated gel filtration.

The Fc fragment of bovine colostral IgG (IgG-Fc) was prepared from papain digestion mixtures. IgG-Fc was found to be heterogeneous on DEAE-cellulose column chromatography. Two IgG-Fc fractions were obtained, but no antigenic difference was found between them. Anti-IgG-Fc antibodies raised in rabbits by injection of these Fc preparations reacted only with IgG1 and IgG2. An immunoadsorbent (anti-IgG-Fc-Sepharose) was prepared by coupling these anti-IgG-Fc antibodies to CNBr-activated Sepharose 4B.

IgA was purified from the IgA-rich fraction by affinity chromatography on anti-IgG-Fc-Sepharose adsorbent. IgG1-dimer was effectively removed by this treatment. The purified sample gave only one precipitin arc characteristic of IgA on immunoelectrophoresis with multiple anti-bovine colostral whey antiserum. A small amount of IgA was found to be adsorbed to the affinity column nonspecifically.

When a rabbit was immunized with the purified IgA, besides anti-IgA antibodies, antibodies against the secretory component (SC) were found in the antiserum. This finding leads us to expect that the purified IgA is secretory IgA containing SC.     

Formation of N-Carboxymethyl Amino Acid from the Reaction of α-Amino Acid with Glyoxal

Enrichment cultures in a medium containing 0.1% methanol and 0.1% bicarbonate at pH 7.0 under anaerobic conditions in the light became mainly green in color. Forty-four enrichment cultures, which showed abundant growth, were obtained from 46 different sources and found to contain cells of methanol-utilizing bacteria and green algae as predominant members. From these enrichment cultures, two strains of bacteria and two strains of algae were isolated. The microorganisms isolated were designated as bacterium No. 7, bacterium No. 8, Chlorella sp. A-1 and Chlorella sp. B-1, respectively. Stable mixed cultures were easily formed by mixing the isolated cultures of bacteria and algae. Both methanol and bicarbonate were necessary for the growth of the mixed cultures under anaerobic-light conditions. Growth behavior of the mixed cultures was examined on a medium containing 0.1% methanol and 0.1 % bicarbonate at 30°C in the light (about 6000 lx). The maximum specific growth rate for the cultures, µ_max, was 0.092 hr^?1 (doubling time, 7.5 hr). The maximum cell yield was 0.87 g dry-cell weight per g of methanol used. The protein content of the biomass was 65%.     

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 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.     

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.     

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.     

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.     

Antioxidant Activity and α-Glucosidase Inhibitory Activities of the Polycondensate of Catechin with Glyoxylic Acid

In order to investigate polymeric flavonoids, the polycondensate of catechin with glyoxylic acid (PCG) was prepared and its chemically antioxidant, cellular antioxidant (CAA) and α-glucosidase inhibitory activities were evaluated. The DPPH and ABTS radical scavenging activities and antiproliferative effect of PCG were lower than those of catechin, while PCG had higher CAA activity than catechin. In addition, PCG had very high α-glucosidase inhibitory activities (IC₅₀ value, 2.59 μg/mL) in comparison to catechin (IC₅₀ value, 239.27 μg/mL). Inhibition kinetics suggested that both PCG and catechin demonstrated a mixture of noncompetitive and anticompetitive inhibition. The enhanced CAA and α-glucosidase inhibitor activities of PCG could be due to catechin polymerization enhancing the binding capacity to the cellular membrane and enzymes.     

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.     

Enzymatic Properties of a Novel Liquefying α-Amylase from an Alkaliphilic Bacillus Isolate and Entire Nucleotide and Amino Acid Sequences

A novel liquefying α-amylase (LAMY) was found in cultures of an alkaliphilic Bacillus isolate, KSM-1378. The specific activity of purified LAMY was approximately 5,000 U mg of protein⁻¹, a value two- to fivefold greater between pH 5 and 10 than that of an industrial, thermostable Bacillus licheniformis enzyme. The enzyme had a pH optimum of 8.0 to 8.5 and displayed maximum activity at 55°C. The molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa, and the apparent isoelectric point was around pH 9. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction. Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzyme. The structural gene for LAMY contained a single open reading frame 1,548 bp in length, corresponding to 516 amino acids that included a signal peptide of 31 amino acids. The calculated molecular mass of the extracellular mature enzyme was 55,391 Da. LAMY exhibited relatively low amino acid identity to other liquefying amylases, such as the enzymes from B. licheniformis (68.9%), Bacillus amyloliquefaciens (66.7%), and Bacillus stearothermophilus (68.6%). The four conserved regions, designated I, II, III, and IV, and the putative catalytic triad were found in the deduced amino acid sequence of LAMY. Essentially, the sequence of LAMY was consistent with the tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C and which include the well-known (α/β)₈ barrel motif in domain A.α-Amylase (1,4-α-d-glucan glucanohydrolase [EC 3.2.1.1]) and pullulanase (pullulan 6-glucanohydrolase [EC 3.2.1.41]) are amylolytic enzymes of industrial importance, particularly in the food and detergent industries. We have found and characterized some unique debranching enzymes, such as a high-alkaline pullulanase (2), an alkali-resistant neopullulanase (16), and an alkaline isoamylase (3), from cultures of alkaliphilic Bacillus strains, and these enzymes can be used as effective additives in dishwashing and laundry detergents under alkaline conditions, especially when used in combination with α-amylase. We have also found the first known alkaline amylopullulanase from alkaliphilic Bacillus sp. strain KSM-1378 (4), which is very unique in that it efficiently hydrolyzes the α-1,6 linkages of pullulan, as well as the α-1,4 linkages of various carbohydrates at different active sites (1, 13).Liquefying α-amylases, particularly the Bacillus licheniformis enzyme (BLA) (35), are used widely in technical application fields, such as in bread making, production of glucose and fructose syrup and fuel ethanol from starch materials, and textile treatment. The demand for α-amylase for use in laundry and automatic dishwashing detergents has also been growing for several years (42). However, most of the Bacillus liquefying amylases, such as the enzymes from Bacillus amyloliquefaciens (BAA) and Bacillus stearothermophilus (BSA) (28), including BLA (35), have pH optima of between 5 and 7.5 (44). These neutrophilic enzymes are essentially not good for use in detergents, because the working pH range between 8 and 11 is relevant to washing in detergents (17). Since Horikoshi (15) first reported an alkaline amylase from alkaliphilic Bacillus sp. strain A-40-2, many alkaline amylases have been found in cultures of, for example, Bacillus sp. strain NRRL B-3881 (31), Bacillus sp. strain H-167 (14), Bacillus alcalothermophilus A3-8 (7), and Bacillus sp. strain GM8901 (21). The alkaline amylases from these alkaliphilic Bacillus strains reported to date are all of the saccharifying type, except for the enzymes from Bacillus sp. strain 707 (22, 41) and B. licheniformis TCRDC-B13 (5). However, very limited or no information about enzymatic properties of these two liquefying amylases is available. In this paper, we report the isolation of a novel liquefying α-amylase (LAMY) from cultures of the alkaline amylopullulanase producer Bacillus sp. strain KSM-1378 (13). This enzyme is highly active at alkaline pH compared with those of other liquefying α-amylases reported to date. Furthermore, analysis of the gene for this α-amylase (amyK) indicates that LAMY exhibits low amino acid identity to the reported liquefying α-amylases.     

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.     

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.     

Chemical Modification of Amino Groups of Acid-stable α-Amylase and Acid-unstable α-Amylase from Aspergillus niger

Monochlorotrifluoro-p-benzoquinone (CFQ) was used for investigating the state of the amino groups of acid-stable α-amylase and acid-unstable α-amylase. About half of the total amino groups in both enzyme molecules were reacted with the reagent. The unreactive amino groups seemed to exist in a different state from the reactive ones. Both enzymes whose amino groups were modified by CFQ still maintained the α-phenylmaltosidase activity in spite of losing or decreasing the amylase activity. These facts suggest that the amino groups of both enzymes were not in the active site but the modification of them caused steric hindrance.

The pH-stability of the acid-unstable α-amylase whose one or two amino groups were modified with succinic anhydride or 2,4,6-trinitrobenzene-l-sulfonate (TNBS) increased on the acidic side and decreased on the alkaline side, but further modification of them led to decrease the stability on both sides.     

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.     

Chemodiversity and α-Glucosidase Activity of Eucalyptus Species from Northwestern Himalaya,India

The aim of current work was to determine essential oils (EOs) composition from three Eucalyptus species, including E. citriodora, E. camaldulensis and E. globulus and assess their α-glucosidase inhibitory activity. The EOs were collected using the hydrodistillation technique and characterized by GC/MS, GC-FID and NMR. The isolated EOs from leaves parts of Eucalyptus species varied from 0.56 to 1.0 % on fresh weight basis. The content of the EOs was distinct according to the species. The most abundant metabolites were identified as citronellal (0–83.0 %), 1,8-cineole (0.2–44.8 %), spathulenol (0.4–16.1 %) α-pinene (0.4–15.9 %), p-cymene (3.7–11.9 %), citronellol (0–8.6 %), β-eudesmol (5.3–8.6 %) and β-pinene (0–7.1 %). The EOs obtained from targeted samples exhibited strong α-glucosidase inhibitory activity. These results are encouraging and underline that the EOs of Eucalyptus species may be a promising alternative source of natural antidiabetic.