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.     

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 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 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 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 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 characterization of α-glucosidase from Aspergillus carbonarious

Summary An -glucosidase was purified from Aspergillus carbonarious CCRC 30414 over 20 fold with 37 % recovery. Its molecular mass was estimated to be 328 kDa by gel filtration with an optimum pH from 4.2 to 5.0, and pI=5.0. The optimum temperature is at 60°C over 40 min. The enzyme was partially inhibited by 5 mM Ag⁺, Hg²⁺, Ba²⁺, Pb²⁺, and Aso₄ ⁺.     

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 Some Properties of β-Mannanase from Penicillium purpurogenum

A β-mannanase was purified from the culture filtrate of Penicillium purpurogenum No. 618 by 1st and 2nd DEAE-cellulose column chromatographies, and subsequent Ultro-gel chromatography. The final preparation thus obtained showed a single band on polyacrylamide disc-gel and SDS-polyacrylamide gel electrophoresis. The molecular weight and isoelectric point were determined to be 57,000 and pH 4.1 by SDS-polyacrylamide gel electrophoresis and isoelectric focusing, respectively. The purified mannanase contained the following amino acids: glycine > serine >glutamic acid > alanine > aspartic acid. The mannanase exhibited maximum activity at pH 5 and 70°C, and was stable in the pH range of 4.5 to 8 and at temperatures up to 65°C. The enzyme activity was not affected considerably by either metal compounds or ethyl- enediaminetetraacetic acid. Copra galactomannan (Gal: Man =1 :14) was finally hydrolyzed to galactose, mannose and β-1,4-mannobiose through the sequential actions of the purified mannanase and the α-galactosidase purified from the same strain.     

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.     

Purification and Some Properties of β-Xylosidase from Emericella nidulans

β-Xylosidase was purified 662 fold from a culture filtrate by ammonium sulfate fractionation, gel filtration on Biogel P-100, DEAE-Sephadex chromatography, and gel filtration on Sephadex G-200. With isoelectric focusing, the purified β-xylosidase found to be homogeneous on SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis. The molecular weight was estimated by gel filtration to be 240,000, and 116,000 by SDS polyacrylamide gel electrophoresis. The purified β-xylosidase had an isoelectric point at pH 3.25, and contained 4% carbohydrate residue. The optimum pH was found to be in the range of 4.5 ~ 5, and the optimum temperature was 55°C. The enzyme activity was inhibited by Hg^{2 +}, SDS, and N-bromosuccinimide at a concentration of 1 × 10^?3 m, and also p-chloromercuribenzoate at a concentration of 1 × 10^?4m. The purified enzyme hydrolyzed phenyl β-d-xyloside (k_o = 302.6 sec^?1),β-nitrophenyl β-d-xyloside (k_o = 438.9 sec^?1), o-nitrophenyl β-d-xyloside (k_o = 431.0 sec^?1), p-chlorophenyl β-d-xyloside (k_o = 207.9 sec^?1), o-chlorophenyl β-d-xyloside (k_o = 211.8 sec^?1), β-methylphenyl β-d-xyloside k_o = 96.5 sec^?1), o-methylphenyl β-d-xyloside (k_o = 83.1 sec^?1), p-methoxyphenyl β-d-xyloside (k_o = 99.3 sec^?1), o-methoxyphenyl β-d-xyloside (k_o= 100.0 sec^?1), xylobiose (k_o = 992A sec^?1), xylotriose (k_o = 1321.9 sec^?1), xylotetraose (k_o = 7S9.1 sec^?1) and xylopentaose (k_o = 508.0 sec^?1). On enzymic hydrolysis of phenyl β-d-xyloside, the reaction product was found to be β-d-xylose with retention of the configuration. The purified β-xylosidase was practically free of a-xylosidase and β-glucosidase activities.     

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.     

Purification and Properties of Leucine Aminopeptidase I–III from Aspergillus oryzae

An aminopeptidase from Aspergillus oryzae 460 was purified from the rivanol precipitable fraction. The partially purified enzyme was not homogeneous in disc electrophoresis, although symmetric profiles were obtained for enzyme protein and activity in Sephadex gel filtration. Its optimum pH is at pH 8.5 for l-leucyl-β-naphthylamide. The enzyme activity was inhibited by metal chelating agents and S-S dissociating agents, but not inhibited by SH reagents. The molecular weight of the enzyme was estimated to be about 26,500 by gel filtration. The enzyme was named leucine aminopeptidase I of Asp. oryzae 460, since it preferentially hydrolyzed oligopeptides that possess leucine as the amino terminal amino acid.     

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 biochemical characterization of a novel α-glucosidase from Aspergillus niveus

An extracellular α-glucosidase produced by Aspergillus niveus was purified using DEAE-Fractogel ion-exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5% PAGE and 10% SDS–PAGE. The enzyme presented 29% of glycosylation, an isoelectric point of 6.8 and a molecular weight of 56 and 52 kDa as estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The enzyme showed typical α-glucosidase activity, hydrolyzing p-nitrophenyl α-d-glucopyranoside and presented an optimum temperature and pH of 65°C and 6.0, respectively. In the absence of substrate the purified α-glucosidase was stable for 60 min at 60°C, presenting t ₅₀ of 90 min at 65°C. Hydrolysis of polysaccharide substrates by α-glucosidase decreased in the order of glycogen, amylose, starch and amylopectin. Among malto-oligosaccharides the enzyme preferentially hydrolyzed malto-oligosaccharide (G10), maltopentaose, maltotetraose, maltotriose and maltose. Isomaltose, trehalose and β-ciclodextrin were poor substrates, and sucrose and α-ciclodextrin were not hydrolyzed. After 2 h incubation, the products of starch hydrolysis measured by HPLC and thin layer chromatography showed only glucose. Mass spectrometry of tryptic peptides revealed peptide sequences similar to glucan 1,4-alpha-glucosidases from Aspergillus fumigatus, and Hypocrea jecorina. Analysis of the circular dichroism spectrum predicted an α-helical content of 31% and a β-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.     

Purification,Crystallization and Some Properties of β-Galactosidase from Saccharomyces fragilis

Crystalline β-galactosidase was prepared from the cell extract of Saccharomyces fragilis KY5463, by procedures including protamine sulfate treatment and DEAE-cellulose, hydroxylapatite and DEAE-Sephadex column chromatographies. Crystals were formed when solid ammonium sulfate was added to solutions of the purified enzyme. This procedure resulted in a 55-fold purification with an over-all yield of l5.4%. The crystalline enzyme appeared to be homogeneous on ultracentrifugation and electrophoresis.

The sedimentation coefficient, , was determined to be 10.0 S. The molecular weight was estimated to be approximately 203,000 by the sedimentation equilibrium method of Yphantis. Electrolysis with carrier ampholytes revealed that this enzyme has an isoelectric point at around pH 4.4.

The enzyme was activated by K⁺ in addition to bivalent cations, such as Mn²⁺, Mg^2? and Co²⁺. The Km values for o-NPG and lactose were 4.0×10^?3m and 21.0×10^?3m, respectively. The enzyme is sulfhydryl dependent and was completely inactivated by mercuric ions or p-chloromercuribenzoate.     

Purification and Some Properties of β-Fructofuranosidase from Bifidobacterium adolescentis G1

A unique β-fructofuranosidase was purified from the extract of Bifidobacterium adolescentis G1 by anion-exchange, hydrophobic, and gel filtration chromatographies, and preparative electrophoresis. The molecular mass was 74kDa by SDS–PAGE, and the isoelectric point was pH 4.5. The enzyme was a monomeric protein. The pH optimum was at 6.1. The enzyme was stable at pH from 6.5 to 10.0, and up to 45°C. The neutral sugar content was 1.2%. The enzyme hydrolyzed 1-kestose faster than sucrose or inulin. The hydrolytic activity was strongly inhibited by Cu²⁺, Ag⁺, Hg⁺, and ρ-chloromercuribenzoic acid. The K_m (mM) and k₀ (s^?1) were: 1-kestose, 1.1 and 231; sucrose, 11 and 59.0; inulin, 8.0 and 149, respectively. From the kinetic results, β-fructofuranosidase from B. adolescentis G1 was concluded to have a high affinity for 1-kestose, thus differing from invertases and exo-inulinases in substrate specificity.     

Purification and Some Properties of Chaetomium trilaterale β-Xylosidase

A β-xyloside hydrolytic enzyme of the fungus Chaetomium trilaterale was further purified by a modification of Kawaminami’s procedure (DEAE-Sephadex A-25 and Sephadex G-75 column chromatography), followed by isoelectric focusing. The purified preparation was homogeneous by polyacrylamide disc gel electrophoreses at pH 4.3 and pH 8.3. The purified enzyme hydrolyzed β-d-glucopyranosides as well as β-d-xylopyranosides, and the ratio of β-glucosidase activity against β-xylosidase activity increased about 3 fold during the purification steps. The molecular weight of this preparation was estimated to be about 240,000 by Sephadex G-200 gel filtration and 118,000 by SDS-polyacrylamide slab gel electrophoresis. The isoelectric point was 4.86 and the amino acid composition was also determined.

The optimum pH was at 4.2 for phenyl β-d-glucoside and around 4.5 for phenyl β-d-xyloside. The β-xylosidase activity was relatively stable but β-glucosidase activity was rapidly inactivated, at the alkaline pH range above 11. The heating of the preparation at 60°C didn’t show a parallel inactivation of the two activities. N-Bromosuccinimide strongly inactivated both enzyme activities. Nojirimycin and glucono-l,5-lactone showed a stronger inhibition on β-xylosidase activity than on β-glucosidase activity. The maximal velocities decreased in the order; phenyl β-d-glucoside > cellobiose > phenyl β-d-xyloside > xylobiose; the value with phenyl β-d-glucoside was about 28-fold higher than that with phenyl β-d-xyloside.     

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.     

Purification and properties ofβ-fructofuranosidase from Aspergillus japonicus

-Fructofuranosidase fromAspergillus japonicus, which produces 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose, was purified to homogeneity by fractionation with calcium acetate and ammonium sulphate and chromatography with DEAE-Cellulofine and Sephadex G-200. Its molecular size was estimated to be about 304,000 Da by gel filtration. The enzyme was a glycoprotein which contained about 20% (w/w) carbohydrate. Optimum pH for the enzymatic reaction was 5.5 to 6. The enzyme was stable over a wide pH range, from pH 4 to 9. Optimum reaction temperature for the enzyme was 60 to 65°C and it was stable below 60°C. The K_m value for sucrose was 0.21m. The enzyme was inhibited by metal ions, such as those of silver, lead and iron, and also byp-chloromercuribenzoate.