Yoshiki Yamasaki

β-Amylase in germinating millet seeds

Abstract β-Amylase (EC 3.2.1.2) was isolated from germinating millet (Panicum miliaceum L.) seeds by a procedure that included ammonium sulfate fractionation, chromatography on DEAE-cellulofine and CM-cellulofine, and preparative isoelectric focusing. The enzyme was homogeneous by SDS–PAGE. The Mr of the enzyme was estimated to be 58,000 based on its mobility on SDS–PAGE and gel filtration with TSKgel G4000SWXL, which showed that it is composed of a single unit. The isoelectric point of the enzyme was 4.62. The enzyme hydrolyzed malto-oligosaccharides more readily as their degree of polymerization increased, this being strongest for malto-oligosaccharides larger than 13 glucose residues and very weakly for maltotriose. Amylose, amylopectin and soluble starch were the most suitable substrates for the enzyme. While the enzyme showed some activity against native starch by itself, starch digestion was accelerated 2.5-fold using α-amylase, pullulanase and α-glucosidase. This enzyme appears to be very important for the germination of millet seeds.

Enzymatic degradation of pectic substances and cell walls purified from carrot cell cultures

Abstract The purified pectic substances from carrot ( Daucus carota ) cell cultures were depolymerized by purified exo -polygalacturonase and endo -pectate lyase. The degraded pectic fractions were fractionated by gel filtration chromatography, and the degree of polymerization and glycosyl composition determined for each fraction. The results indicate that subfractionated pectic substances contain a homogalacturonan region with a degree of polymerization of ca 70 and neutral glycosyl residues such as side-chains arranged in blocks (‘hairy’ regions) of different M r s. In addition, the endo -pectate lyase released four pectic fragments from isolated cell walls. Based on the analysis of glycosyl composition of each fragment, the pectic substances of carrot cell walls are characterized.

Two forms of α-glucosidase from sugar-beet seeds

Two forms of α-glucosidase (EC 3.2.1.20), designated as I and II, have been isolated from sugarbeet (Beta vulgaris L.) seeds by a procedure including fractionation with ammonium sulfate and ethanol, carboxymethyl-cellulose column chromatography, and preparative disc gel electrophoresis. The two enzymes were homogeneous by polyacrylamide disc gel electrophoresis. Their molecular weights were 98,000 (I) and 60,000 (II). α-Glucosidase I readily hydrolyzed maltose, isomaltose, kojibiose, maltotriose, panose, amylose, soluble starch, amylopectin and glycogen. α-Glucosidase II also hydrolyzed maltose, kojibiose and maltotriose but hydrolyzed the other substrates only very weakly or not at all. α-Glucosidase I hydrolyzed soluble starch at a faster rate than maltose. It produced isomaltose and panose as the main α-glucosyltransfer products from maltose, whereas maltotriose was the main product of α-glucosidase II. α-Glucosidase I hydrolyzed amylose liberating α-glucose. The neutral-sugar content was calculated to be 2.7% for α-glucosidase I and 8.8% for α-glucosidase II. The main neutral sugar was mannose in α-glucosidase I, and glucose in α-glucosidase II.

Purification and properties of α-glucosidase from millet seeds

Abstract Two forms of α-glucosidase (EC 3.2.1.20), designated as I and II, have been isolated from millet ( Panicum miliaceum L.) seeds by a procedure including fractionation with ammonium sulphate, CM-cellulofine column chromatography, Sephadex G-100 column chromatography, preparative isoelectric focusing and preparative disc gel electrophoresis. The two enzymes showed identical M r , calculated to be 85 000 on SDS-PAGE and 93 000 on gel filtration. The two enzymes readily hydrolysed maltose and malto-oligosaccharides, and native starch weakly. The two enzymes hydrolysed amylose liberating α-glucose.

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 gl...

A β-glucosidase associated with cell walls from cell suspension cultures of carrot

Abstract The activity of β-glucosidase (EC 3.2.1.21) in the protein fraction solubilized with 3 M LiCl from cell walls of carrot cell cultures was found to be much higher than those of the other glycan-hydrolases. The cell wall-associated β-glucosidase was purified to electrophoretic homogeneity. The M r of the purified enzyme was estimated to be 46 000 by Sephacryl S-200HR gel-permeation, and 48 000–52 000 by SDS-PAGE under denaturing conditions. The enzyme contained carbohydrate and protein in a ratio of 1 : 15 (w/w) and was rich in Ser, Gly, Glx and Ala. The isoelectric point was pH 8.2, the pH optimum 4.6–5.2 and the temperature optimum 50°. The activity was inhibited by Cu 2+ , Ag + , Hg 2+ , p -chloromercuribenzoate, and d -glucono-1,5-lactone. The K m and V max values for p -nitrophenyl (PNP)-β-glucopyranoside were 0.12 mM and 0.13 mmol (mg protein) −1 hr −1 , respectively. The enzyme also acted on PNP-β-cellobioside, lichenan and laminarin, but was not capable of hydrolysing the glucose-containing polymers isolated from cell walls of carrot cell cultures.

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 characterization of β-galactosidase from cell suspension cultures of Marchantia polymorpha

Abstract A β-galactosidase (β-D-galactoside galactohydrolase, EC 3.2.1.23) has been isolated and purified to homogeneity from cell homogenates of cell suspension cultures of a thalloid liverwort, Marchantia polymorpha . The enzyme in 0.1 M phosphate buffer soluble protein fraction was dialyzed at pH 5.2 and further purified by a combination of chromatographic techniques including DEAE-Sephadex A-50, p -aminophenyl β- d -thiogalactopyranoside-linked Sepharose 4B and Sephadex G-200. The molecular weight of β-galactosidase by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by Sephadex G-200 gel filtration is 62 000 and 67 000, respectively. The isoelectric point of the enzyme is found to be pH 4.2. The optimal activity occurs at pH 3.4 with McIlvaine buffer, and at pH 2.6 with glycineHCl buffer. The K m - and V max -values are 0.625 mM and 658 units/mg protein for p -nitrophenyl β- d -galactopyranoside. The enzyme catalyzes the transfer of the d -galactosyl residues from p -nitrophenyl β- d -galactopyranoside to d -glucose. Furthermore, the enzyme acts on the galactan extracted from citrus pectic polysaccharides in an exo-fashion.

Two Forms of Glucoamylase from Mucor rouxianus: II. Properties of the Two Glucoamylases

Both of the two forms of glucoamylase (glucoamylases I and II) from the wheat bran culture of Mucor rouxianus hydrolyzed amylopectin, amylose, glycogen, soluble starch, maltotriose, and maltose, but did not act on isomaltose and isomaltotriose. Phenyl α-maltoside was hydrolyzed into glucose and phenyl α-glucoside by both glucoamylases. Maltose was hydrolyzed about one-fifth as rapidly as amylopectin. Both enzymes produced glucose from amylopectin, amylose, glycogen, soluble starch in the yields of almost complete hydrolysis. They hydrolyzed amylose with the inversion of configuration, producing the β-anomer of glucose. Glucoamylase II hydrolyzed raw starch at 3-fold higher rate than glucoamylase I. The former hydrolyzed rice starch almost completely into glucose, whereas the latter hydrolyzed it incompletely (nearly 50%).

α-Glucosidases of suspension-cultured sugar-beet cells

Abstract Four α-glucosidases have been fractionated from sugar-beet cells. They are divisible into two groups in terms of soluble starch-hydrolysing activity. One group hydrolyses soluble starch at a faster rate than maltose. The enzymes of this group are not detected during the phase of starch accumulation in cells, but undergo a marked increase with the decrease of starch content. A possible role for these enzymes is in the metabolism of starch in the cell. Another group hydrolyses soluble starch only very weakly. The former group are highly active in the release of α-1,3-linked glucose from nigerose.