Production and properties of ∝-mannosidase from aspergillus sp.

Summary Aspergillus sp NCIM 508 produced 22 U/L of extracellular -mannosidase activity in a medium containing 8 % brewer's yeast cells. The optimum period and pH range for maximum production of the enzyme were 7 days and 4.0–6.0, respectively. The optimum pH and temperature for enzyme activity were 6.0 and 50°C, respectively. The enzyme was stable for 24 h at 28°C, in the pH range 6.0–7.0. The enzyme retained 100 and 65 % of its original activity after heating for 15 min at 45 and 55°C, respectively. The Km and V_max for p-nitrophenyl--D- mannoside (PNPM) were 71M and 7.5 × 10^–2 moles/min/mg, respectively. The enzyme was strongly inhibited by 1 mM Hg⁺⁺ and Cu⁺⁺ and partially by Co.⁺⁺ (NCL Communication No.; 5780)     

Purification and Characterization of a Novel α-Agarase from a Thalassomonas sp.

An agar-degrading Thalassomonas bacterium, strain JAMB-A33, was isolated from the sediment off Noma Point, Japan, at a depth of 230 m. A novel -agarase from the isolate was purified to homogeneity from cultures containing agar as a carbon source. The molecular mass of the purified enzyme, designated as agaraseA33, was 85 kDa on both SDS-PAGE and gel-filtration chromatography, suggesting that it is a monomer. The optimal pH and temperature for activity were about 8.5 and 45°C, respectively. The enzyme had a specific activity of 40.7 U/mg protein. The pattern of agarose hydrolysis showed that the enzyme is an endo-type -agarase, and the final main product was agarotetraose. The enzyme degraded not only agarose but also agarohexaose, neoagarohexaose, and porphyran.     

Purification and some properties of β-mannanase from Bacillus sp.

-Mannanase produced by Bacillus sp. W-2, isolated from decayed commercial konjak cake, was purified from the culture supernatant by (NH₄)₂ SO₄ precipitation, adsorption to konjak gel, and column chromatography with DEAE-cellulose, Sephadex G-100 and Sephacryl S-200. Its molecular size was estimated by SDS-PAGE as 40 kDa, and by gel filtration as 36 kDa. The enzyme was most active at pH 7 and 70°C and was stable for at least 1 h between pH 5 and 10 and below 60°C. Its activity was completely inhibited by Hg²⁺. The enzyme hydrolysed galactomannan better than glucomannan and mainly produced mannose and mannobiose.The authors are with the Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University. Utsunomiya, Tochigi 321, Japan     

Purification and Characterization of α-Hydroxyglutarate Dehydrogenase from Alcaligenes sp.

NADH-dependent soluble l-α-hydroxyglutarate dehydrogenase (l-2-hydroxyglutarate: NAD⁺ 2-oxidoreductase) was found in a bacterium belonging to the genus Alcaligenes obtained from soil by citrate enrichment culture. A mutant with about 2.5-fold higher activity of the enzyme was derived from the bacterium and used as the enzyme source. High level of the enzyme was produced at the late stage of cultivation in the presence of citrate and with limited aeration. The enzyme was purified from the cells to homogeneity to give crystals, and its enzymatic properties were studied. The enzyme strongly reduced α-ketoglutarate to stereochemically pure l-α-hydroxyglutarate with NADH as a coenzyme, but it oxidized d-α-hydroxyglutarate with about 1/10 of the rate for l-form oxidation.     

Purification and properties of α-d-mannosidase from jack-bean meal

1. α-Mannosidase from jack-bean meal was purified 150-fold. β-N-Acetyl-glucosaminidase and β-galactosidase were removed from the preparation by treatment with pyridine. Zn²⁺ was added during the purification to stabilize the α-mannosidase. 2. At pH values below neutrality, α-mannosidase undergoes reversible spontaneous inactivation at a rate dependent on the temperature, the degree of dilution and the extent of purification. The enzyme is also subject to irreversible inactivation, which is prevented by the addition of albumin. 3. Reversible inactivation of α-mannosidase is accelerated by EDTA and reversed or prevented by Zn²⁺. Other cations, such as Co²⁺, Cd²⁺ and Cu²⁺, accelerate inactivation; an excess of Zn²⁺ again exerts a protective action, and so does EDTA in suitable concentration. 4. Neither Zn²⁺ nor EDTA has any marked effect in the assay of untreated enzyme. In an EDTA-treated preparation, however, Zn²⁺ reactivates the enzyme during assay. 5. It is postulated that α-mannosidase is a dissociable Zn²⁺–protein complex in which Zn²⁺ is essential for enzyme activity.     

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 some properties of cellulose 1,4-β-cellobiosidase from a strain of Penicillium sp.

A cellulase was purified from the culture supernatant of a strain of Penicillium sp. The purified enzyme was homogenous on polyacrylamide disc gel electrophoresis. It was a glycoprotein with a molecular weight of 52,000 estimated by gel filtration. The optimum pH was about 4.0 and the optimum temperature was 60°C. The enzyme was stable in the pH range of 3.0–10.0 at 6°C for 48 h and on heating at 60°C for 10 min. The activity of the enzyme toward Avicel was about 3 times higher than toward carboxymethyl cellulose. The enzyme showed a low activity for cotton, newspaper, filter paper and cellulose powder. The main product from Avicel was cellobiose, with a trace of glucose.     

Purification and characterization of a liquefying α-amylase from alkalophilic thermophilic Bacillus sp. AAH-31

α-Amylase (EC 3.2.1.1) hydrolyzes an internal α-1,4-glucosidic linkage of starch and related glucans. Alkalophilic liquefying enzymes from Bacillus species are utilized as additives in dishwashing and laundry detergents. In this study, we found that Bacillus sp. AAH-31, isolated from soil, produced an alkalophilic liquefying α-amylase with high thermostability. Extracellular α-amylase from Bacillus sp. AAH-31 (AmyL) was purified in seven steps. The purified enzyme showed a single band of 91 kDa on SDS-PAGE. Its specific activity of hydrolysis of 0.5% soluble starch was 16.7 U/mg. Its optimum pH and temperature were 8.5 and 70 °C respectively. It was stable in a pH range of 6.4-10.3 and below 60 °C. The calcium ion did not affect its thermostability, unlike typical α-amylases. It showed 84.9% of residual activity after incubation in the presence of 0.1% w/v of EDTA at 60 °C for 1 h. Other chelating reagents (nitrilotriacetic acid and tripolyphosphate) did not affect the activity at all. AmyL was fully stable in 1% w/v of Tween 20, Tween 80, and Triton X-100, and 0.1% w/v of SDS and commercial detergents. It showed higher activity towards amylose than towards amylopectin or glycogen. Its hydrolytic activity towards γ-cyclodextin was as high as towards short-chain amylose. Maltotriose was its minimum substrate, and maltose and maltotriose accumulated in the hydrolysis of maltooligosaccharides longer than maltotriose and soluble starch.     

Purification and Characterization of a Liquefying α-Amylase from Alkalophilic Thermophilic Bacillus sp. AAH-31

α-Amylase (EC 3.2.1.1) hydrolyzes an internal α-1,4-glucosidic linkage of starch and related glucans. Alkalophilic liquefying enzymes from Bacillus species are utilized as additives in dishwashing and laundry detergents. In this study, we found that Bacillus sp. AAH-31, isolated from soil, produced an alkalophilic liquefying α-amylase with high thermostability. Extracellular α-amylase from Bacillus sp. AAH-31 (AmyL) was purified in seven steps. The purified enzyme showed a single band of 91 kDa on SDS–PAGE. Its specific activity of hydrolysis of 0.5% soluble starch was 16.7 U/mg. Its optimum pH and temperature were 8.5 and 70 °C respectively. It was stable in a pH range of 6.4–10.3 and below 60 °C. The calcium ion did not affect its thermostability, unlike typical α-amylases. It showed 84.9% of residual activity after incubation in the presence of 0.1% w/v of EDTA at 60 °C for 1 h. Other chelating reagents (nitrilotriacetic acid and tripolyphosphate) did not affect the activity at all. AmyL was fully stable in 1% w/v of Tween 20, Tween 80, and Triton X-100, and 0.1% w/v of SDS and commercial detergents. It showed higher activity towards amylose than towards amylopectin or glycogen. Its hydrolytic activity towards γ-cyclodextin was as high as towards short-chain amylose. Maltotriose was its minimum substrate, and maltose and maltotriose accumulated in the hydrolysis of maltooligosaccharides longer than maltotriose and soluble starch.     

Separation and properties of α-mannosidase and mannanase from the basidiomycetePhellinus abietis

Proteins of a crude enzyme preparation obtained from the cultivation medium of the basidiomycetePhellinus abietis were separated by gel filtration and ion-exchange chromatography. The preparation contained a minimum of three enzymes capable of splitting α-d-mannosidic bonds: α-mannosidase, exomannanase, and endomannanase, which were separated. Some properties of the mannanase complex of the crude enzyme preparation, and of a partially purified α-mannosidase were examined. The mannanase complex exhibited two pH optima, its temperature optimum being at 46 °C The pH optimum of purified α-mannosidase was at pH 5.0, the temperature optimum was at 60 °C; the enzyme had a relatively high heat stability. The K_m of α-mannosidase forp-nitrophenyl α-d-mannopyranoside was 1.5 x 10⁻⁵ M. Pure α-mannosidase did not split mannan.     

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 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 Some properties of Endo-β-1,6-Glucanase from Acinetobacter sp.

A kind of endo-β-1, 6-glucanase has been purified from the culture filtrate of Acinetobacter sp. grown in the medium containing baker’s yeast cells as a carbon source. A 100-fold purified preparation was obtained by DEAE-Sephadex A–50 column chromatography. The enzyme hydrolyzed pustulan giving a series of gentio-oligosaccharides and glucose. Gentiotriose and gentiotetraose were hydrolyzed by this enzyme yielding glucose and gentiobiose, and glucose, gentiobiose and gentiotriose, respectively. Gentiobiose was not hydrolyzed. Baker’s yeast glucans obtained from the isolated cell walls were also hydrolyzed by this enzyme giving a series of oligosaccharides and glucose. From the action patterns on these carbohydrates, we concluded the present enzyme being endo-β-1, 6-glucanase.     

Purification and properties of the raw starch-degrading α- amylase of Bacillus sp. IMD 434

The raw starch-degrading a-amylase of Bacillus sp. IMD 434 was purified to homogeneity by acetone precipitation, ion- exchange chromatography and hydrophobic interaction chromatography. The enzyme had a relative molecular mass of 69,200, displayed maximum activity at pH 6.0 and 65°C and released large amounts of glucose and maltose on hydrolysis of starch.     

Purification and characterization of a novel thermostable α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp.

The purification and characterization of an extracellular α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp. was investigated in this report. The α-l-AFase was purified to homogeneity with a purification fold of 1030. The purified α-l-AFase had a specific activity of 20.6 U mg^?1. The molecular mass of the enzyme was estimated to be 52.9 kDa and 51.6 kDa by SDS–PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.0 and 70 °C, respectively. The enzyme was stable over a broad pH range of 4.0–10.0 and also exhibited excellent thermostability, i.e., the residual activities reached 75% after treatment at 60 °C for 1 h. The enzyme showed strict substrate specificity for the α-l-arabinofuranosyl linkage. The K_m and V_max values for p-nitrophenyl (pNP)-α-l-arabinofuranoside were calculated to be 1.43 mM and 68.3 μmol min^?1 mg^?1 protein, respectively. Furthermore, the gene encoding α-l-AFase was cloned and sequenced and found to contain a catalytic domain belonging to the glycoside hydrolase (GH) family 43 α-l-AFase. The deduced amino acid sequence of the gene showed the highest identity (67%) to the putative α-l-AFase from Neurospora crassa. This is the first report on the purification, characterization and gene sequence of an α-l-AFase from Chaetomium sp.     

Cloning and characterization of a new β-mannosidase from Streptomyces sp. S27

A new β-mannosidase gene, designated as man2S27, was cloned from Streptomyces sp. S27 using the colony PCR method and expressed in Escherichia coli BL21 (DE3). The full-length gene consists of 2499 bp and encodes 832 amino acids with a calculated molecular mass of 92.6 kDa. The amino acid sequence shares highest identity of 62.6% with the mannosidase Man2A from Cellulomonas fimi which belongs to the glycoside hydrolase family 2. Purified recombinant Man2S27 showed optimal activity at pH 7.0 and 50 °C. The specific activity, K_m, and k_cat values for p-nitrophenyl-β-d-mannopyranoside (p-NP-β-MP) were 35.3 U mg^-1, 0.23 mM, and 305 s^-1, respectively. Low transglycosylation activity was observed when Man2S27 was incubated with p-NP-β-MP (glycosyl donor) and methyl-α-d-mannopyranoside (p-NP-α-MP) (acceptor) at 50 °C and pH 7.0, and a small amount of methylmannobioside was synthesized. Using locust bean gum as the substrate, more reducing sugars were liberated by the synergistic action of Man2S27 and β-mannanase (Man5S27), and the synergy degree in sequential reactions with Man5S27 firstly and Man2S27 secondly was higher than that in the simultaneous reactions.     

Purification and properties ofβ-fructofuranosidase fromAureobasidium sp. ATCC 20524

Summary Purification and properties of two -fructofuranosidases, which produce 1-kestose (1^F--fructofuranosyl-sucrose) from sucrose, fromAureobasidium sp. ATCC 20524 are reported. The enzymes were purified to homogeneity by fractionations involving ethanol, calcium acetate and ammonium sulfate and DEAE-Cellulofine and Sephadex G-200 chromatography. Molecular weights of the enzymes were estimated to be about 318000 (P-1) and 346000 (P-2) daltons by gel filtration. The enzymes were glycoproteins that contained about 30% (w/v) (P-1) and 53% (w/v) (P-2) carbohydrate. The optimum pH for the enzymatic reactions were 4.5–5.5 (P-1) and 4.5–6 (P-2). The enzymes were stable over a wide pH range (4–9). The optimum reaction temperatures for both enzymes were 50–55°C and they retained more than 94% (P-1) and 98% (P-2) activities at 50°C after 15 min. TheK _m values for sucrose were 0.47 M (P-1) and 0.65 M (P-2). The enzymes were inhibited by mercury, copper and lead ions as well asp-chloromercuribenzoate.     

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.