Purification and properties of α-d-mannosidase from rat epididymis

1. alpha-d-Mannosidase from rat epididymis was purified 300-fold. beta-N-Acetyl-glucosaminidase and beta-galactosidase were removed from the preparation by treatment with pyridine. Zn(2+) was added during the purification to stabilize the alpha-mannosidase. 2. Mammalian alpha-mannosidase is most stable at pH6. At lower pH values it undergoes reversible spontaneous inactivation. The enzyme is also subject to irreversible inactivation, which is delayed by the addition of albumin. 3. Reversible inactivation of alpha-mannosidase is accelerated by EDTA and reversed or prevented by Zn(2+). Other cations, such as Co(2+), Cd(2+) and Cu(2+), accelerate inactivation and the action of a toxic cation can be prevented by Zn(2+) or by EDTA in suitable concentration. 4. The enzyme is stabilized by substrate and neither Zn(2+), EDTA nor a toxic cation has more than a small effect in the assay of an untreated preparation. The addition of Zn(2+) is necessary, however, for a constant rate of hydrolysis during prolonged incubation of the enzyme with substrate. In an EDTA-treated preparation, Zn(2+) reactivates the enzyme during the assay. 5. Evidence is presented that alpha-mannosidase is a dissociable Zn(2+)-protein complex, in which Zn(2+) is essential for enzyme activity.     

Purification and properties of α-d-mannosidase from the limpet, Patella vulgata

1. alpha-Mannosidase from the limpet, Patella vulgata, was purified nearly 150-fold, with 40% recovery. beta-N-Acetylglucosaminidase was removed from the preparation by treatment with ethanol. The final product was virtually free from beta-galactosidase. 2. Limpet alpha-mannosidase was assayed at pH3.5 and at this pH it was necessary to add Zn(2+) for full activity. At pH5, the enzyme had the same activity in the presence or absence of added Zn(2+). 3. On incubation at acid pH, the enzyme underwent reversible inactivation, which was prevented by adding Zn(2+). 4. EDTA accelerated inactivation and the addition of Zn(2+) at once restored activity. No other cation was found to reactivate the enzyme. 5. Cl(-) had an unspecific effect on hydrolysis by limpet alpha-mannosidase. It increased the rate of reaction with substrate. The anion did not prevent or reverse inactivation by EDTA. 6. It is concluded that alpha-mannosidase is a metalloenzyme or enzyme-metal ion complex, dissociable at the pH of activity, and that it requires Zn(2+) specifically.     

Purification and properties of an α-amylase inhibitor from wheat

Four inhibitors of α-amylase (EC 3.2.1.1) were separated from an alcohol extract of wheat by ion-change chromatography on DE52-cellulose. One inhibitor, which showed the greatest specificity for human salivary amylase relative to human pancreatic amylase, has been purified by the following steps: (a) alcohol fractionation (60–90%) of water extract (b) ion-exchange chromatography on QAE-Sephadex A-50; (c) re-chromatography on DE52-cellulose and (d) gel filtration on Sephadex G-50. The purified inhibitor is 100 times more specific for human salivary amylase than for human pancreatic amylase. It shows an electrophoretic mobility of 0.2 on disc gel electrophoresis and a molecular weight of about 21 000. This inhibitor contributes about 16% to the total salivary amylase inhibiting power of the wheat extract.     

Purification and properties of α-galactosidases from Vicia faba seeds

Two forms of alpha-galactosidase, I and II, exist in Vicia faba seeds and these have been purified 3660- and 337-fold respectively. They behaved as homogeneous preparations when examined by ultracentrifugation, disc electrophoresis and gel filtration. The apparent molecular weights of enzymes I and II, as determined by gel filtration, were 209000 and 38000 respectively. The carbohydrate contents of enzymes I and II were 25% and 2.8% respectively, and the enzymes differed in their aromatic amino acid compositions. Enzyme I was split into six inactive subunits in the presence of 6m-urea. alpha-Galactosidases I and II showed different pH optima and K(m) and V(max.) values with p-nitrophenyl alpha-d-galactoside and raffinose as substrates, and also differed in their thermal stabilities.     

Purification and properties of heat stable α-amylase from Bacillus brevis

Summary An extracellular -amylase has been isolated from a continuous culture of a thermophilic strain of Bacillus brevis. This enzyme was purified eightfold and obtained in electrophoretically homogenous form. The enzyme had a molecular weight of about 58000, a pH optimum from 5.0 to 9.0 and a temperature optimum at 80°C. The half-life of the purified enzyme in the presence of 5 mM CaCl₂ at 90° C and pH 8.0 was 20 min. The K _m value for soluble starch was calculated to be 0.8 mg/ml.     

Purification and properties of α-l-arabinofuranosidase from plant Scopolia japonica calluses

α-l-Arabinofuranosidase (α-l-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) from the culture medium of Scopolia japonica calluses was partially purified.Various properties of the enzyme were studied and the effects of lactones on the activity were determined.     

Purification and physical properties of sweet-almond α-galactosidase

1. α-Galactosidase from sweet almonds was purified about 2000-fold through eight steps. 2. The enzyme preparation was free from other related enzymes known to occur in sweet almonds, and behaved as a homogeneous protein on filtration through Sephadex G-75. 3. A molecular weight of about 33000 was determined from the gel-filtration data. 4. The ultraviolet-absorption spectrum and thermal inactivation of the enzyme are described. 5. The purified enzyme hydrolysed p-nitrophenyl α-d-galactoside at a much faster rate than melibiose. 6. The pH optimum was at 5·5–5·7. 7. Besides hydrolysis, it also catalysed transfer of galactosyl residues, chain elongation of melibiose and the synthesis of oligosaccharides from galactose.     

Purification and properties of a low-molecular-weight α-mannosidase from Cellulomonas sp.

Cellulomonas sp. isolated from soil produces a high level of α-mannosidase (α-mannanase) inductively in culture fluid. The enzyme had two different molecular weight forms, and the properties of the high-molecular-weight form were reported previously (Takegawa, K. et al.: Biochim. Biophys. Acta, 991, 431–437, 1989). The low-molecular-weight α-mannosidase was purified to homogeneity by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was over 150,000 by gel filtration. Unlike the high-molecular-weight form, the low-molecular-weight enzyme readily hydrolyzed α-1,2- and α-1,3-linked mannose chains.     

Purification and properties of a hyperthermoactive α-amylase from the archaeobacterium Pyrococcus woesei

The cultivation of the hyperthermophilic archaeobacterium Pyrococcus woesei on starch under continuous gassing (80% H₂:20% CO₂) caused the formation of 250 U/l of an extremely thermoactive and thermostable -amylase. In a complex medium without elemental sulphur under 80% N₂ and 20% CO₂ atmosphere enzyme production could be elevated up to 1000 U/l. Pyrococcus woesei grew preferentially on poly-and oligosaccharides. The amylolytic enzyme formation was constitutive. Enzyme production was also observed in continuous culture at dilution rates from 0.1 to 0.4 h^-1. A 20-fold enrichment of -amylase was achieved after adsorption of the enzyme onto starch and its desorption by preparative gel electrophoresis. The -amylase consisted of a single subunit with a molecular mass of 70 000 and was catalytically active at a temperature range between 40°C and 130°C. Enzymatic activity was detected even after autoclaving at a pressure of 2 bars at 120°C for 5 h. The purified enzyme hydrolyzed exclusively -1,4-glycosidic linkages present in glucose polymers of various sizes. Unlike many -amylases from anaerobes the enzyme from P. woesei was unable to attack short chain oligosaccharides with a chain length between 2 and 6 glucose units.     

Purification and properties of two different α1-protease inhibitors from mouse plasma

Two similar but distinct forms of α1-protease inhibitor (α1-PI) have been isolated and purified 120-fold to homogeneity from the plasma of female, white Swiss (Ha/ICR) mice. The two inhibitors can be separated by chromatography on DEAE-cellulose using a shallow NaCl gradient at pH 8.9 for elution. Because of their differing specificities for elastase and trypsin we have labeled the two inhibitors α1-PI(E) and α1-PI(T), respectively. The apparent M_r for both proteins, as estimated by gel exclusion chromatography, is approximately 53,000 daltons. However by polyacrylamide gel electrophoresis in the presence of SDS, α1-PI(T) has an apparent m_r of 65,000 while the apparent m_r of α1-PI(E) is 55,000. These results suggest differences in charge and carbohydrate composition. The two mouse inhibitors also have different AT-terminal amino acids. Like human α1-PI the mouse inhibitors form stable complexes with proteases. However they differed from human α1-PI in that they were not found to neutralize either human thrombin or plasmin. While α1-PI(E) inhibits bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase, α1-PI(T) is an effective inhibitor only of trypsin. Plasma levels of α1-PI(E) increase significantly 24 h after stimulation of the acute phase reaction while those of α1-PI(T) do not. Our data suggest that α1-PI(E) and α1-PI(T) are products of different genes.     

Purification and properties of three kinds of α-hydroxysteriod dehydrogenases from Brevibacterium fuscum DC33

The cell-free extract of Brevibacterium fuscum DC33 contained three kinds of hydroxysteriod dehydrogenase (3a-, 7a-, and 12a-hydroxysteriod dehydrogenases). 7a-Hydroxysteroid dehydrogenase (EC 1.1.1.59) was purified to electrophoretical homogeneity by ion exchange chromatography, affinity chromatography, and preparative electrophoresis. Its molecular weight was 104, 000 and the enzyme was composed of four identical subunits. The enzyme had an optimum pH of 5.3 for dehydrocholic acid reduction, and around 10 for cholic acid oxidation. It was stable in a pH range of 5.7 to 10.5 at 5°C overnight. The enzyme was most active at 25° to 30°C. The activity was not affected by incubation at 30°C for 30 min, but it was lost at 40°C for 30 min. Withe the assumption of two-substrate kinetics, we calculated various kinetic constants for dehydrocholic acid, 7, 12-diketolithocholic acid, 12-ketochenodeoxycholic acid, and 3, 12-diketolithocholic acid (for the structure of bile acids, see Table 2) together with NAD⁺ or NADH. The enzyme was active only toward hydroxysteroids with a 7a-hydroxyl group. The production of 7-ketochenodeoxycholic acid from cholic acid and of 3, 12-diketolithocholic acid from dehydrocholic acid by the purified 7a-hydroxysteroid dehydrogenase was confirmed by thin-layer chromatography.12a-Hydroxysteroid dehydrogenase was purified by a similar method. It was active toward hydroxysteroids with a 12a-hydroxyl group.3a-Hydroxysteroid dehydrogenase was purified by preparative electrophoresis. It was active toward hydroxysteroids with a 3a-hydroxyl group.     

Purification and some properties of an α-amylase glucoamylase fusion protein from Saccharomyces cerevisiae

A fusion gene containing the Bacillus subtilis -amylase gene and Aspergillus awamori glucoamylase cDNA was expressed in Saccharomyces cerevisiae. The resulting bifunctional fusion protein having both -amylase and glucoamylase activities secreted into the culture medium was purified to apparent homogeneity by affinity chromatography and gel filtration on Sephadex G-100. The enzyme had an apparent molecular mass of 150 kDa and showed an optimum pH and temperature of 6.0 and 60 °C, respectively. The main hydrolysis products from soluble starch were glucose and maltose.     

Purification and properties of β-ketothiolase from Zoogloea ramigera

-Ketothiolase from Zoogloea ramigera I-16-M was purified 140-fold to electrophoretic homogeneity. The bacterium appeared to contain a single isoenzyme of -ketothiolase with a molecular weight of 190000, as determined by Sephadex G-200 gel filtration. The monomer molecular weight was 44000, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The native enzyme thus appeared to be a tetramer with identical subunits.The enzyme showed a pH optimum of 7.5 in the condensation reaction, and 8.5 in the thiolysis reaction. The enzyme employed a Bi Bi ping pong mechanism for the forward thiolysis reaction. The apparent K _m value for acetoacetyl coenzyme A in the thiolysis reaction was 10 M, and that for coenzyme A was 8.5 M. The apparent K _m value for acetyl coenzyme A in the condensation reaction was 0.33 mM. The condensation reaction was inhibited by coenzyme A concentrations lower than 0.1 mM.The enzyme was stable in the presence of dithiothreitol and other SH-compounds, but was strongly inhibited by 0.4 mM p-chloromercuribenzoate.Non-Standard Abbreviation PHB poly--hydroxybutyrate     

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 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.     

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 of α-galactosidase from coconut

α-Galactosidase from coconut endosperm was purified to homogeneity with a 490-fold increase in specific activity. The yield was 70%, and the specific activity was 24.5 units/mg protein. The purification procedure included extraction, acidification, ammonium sulphate fractionation and hydrophobic chromatography. The hydrophobic gel (Sepharose-4B-capranilide) had a capacity of 0.63 mg of α-galactosidase per ml of gel. Purified α-galactosidase was a glycoprotein with a carbohydrate content of 12%. The molar extinction coefficient was 8.7 x 10⁴/M/cm.     

Purification and properties of a β-N-acetylaminoglucohydrolase from malted barley

β-N-Acetylaminoglucohydrolase (β-2-acetylamino-2-deoxy-D-glucoside acetylaminodeoxyglucohydrolase, EC 3.2.1.30) was extracted from malted barley and purified. The partially purified preparation was free from α-and β-glucosidase, α- and β-galactosidase, α-mannosidase and β-mannosidase. This preparation was free from α-mannosidase only after affinity chromatography with p-amino-N-acetyl-β-D-glucosaminidine coupled to Sepharose. The enzyme was active between pH 3 and 6.5 and had a pH optimum at pH 5. A MW of 92000 was obtained by sodium dodecyl sulfate-acrylamide gel electrophoresis and a sedimentation coefficient of 4.65 was obtained from sedimentation velocity experiments. β-N-Acetylaminoglucohydrolase had a K_m of 2.5 × 10^?4 M using the p-nitrophenyl N-acetyl β-D-glucosaminidine as the substrate.     

Purification and properties of recombinant β-galactosidase from Bacillus circulans

A gene encoding β-galactosidase from Bacillus circulans which had hydrolysis specificity for the β1-3 linkage was expressed in Escherichia coli. The β-galactosidase was purified from crude cell lysates of E. coli by column chromatographies on Resource Q and Sephacryl S-200 HR. The enzyme released galactose with high selectivity from oligosaccharides which had terminal β1-3 linked galactose residues. However it did not hydrolyse β1-4 linked galactooligosaccharides. Moreover, Galβ1-3GlcNAc, Galβ1-3GalNAc, and their p-nitrophenyl glycosides were regioselectively synthesized in 10–46% yield by the transglycosylation reaction using this enzyme.     

Purification and properties of neutral β-galactosidases from human liver

Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM₁-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar K_m and V values; the more acidic form (designated β-galactosidase N₁) was more heat stable than the other form (designated β-galactosidase N₂). Antibodies evoked against the N₁ and N₂ β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.