Purification and characterization of the amylase of B. subtilis NRRL B3411

The amylase of Bacillus subtilis NRRL B3411 has been purified and partially characterized. The specific activity can be increased from 300,000 units/g to 6,000,000 units/g with a 60% recovery of total units. The purified material consists of one major and one trace anodic component as determined by disc gel electrophoresis. The molecular weight was 48,000 as determined by bio-gel filtration; the molecular weight was 44,900 ± 2400 as determined by sedimentation equilibrium methods. This purified enzyme is stable at, 70°C in the presence of 0.01 M Ca⁺⁺ and 0.1 M NaCl over a broad pH range from 5.5–9.5. The pH activity profile indicates optimum activity at pH 6.0. This amylase exhibits maximum activity at 60°C. The enzyme is a liquefying α-amylase as determined by analysis of hydrolysis products and immunological studies.     

Limited Hydrolysis of Ricin D with Trypsin in the Presence of Sodium Dodecyl Sulfate

An α-amylase which produces both maltotetraose and maltopentaose from starch as the main products was found in the culture filtrate of a strain of Bacillus circulans which was newly isolated from soil. The enzyme was purified to be almost homogeneous on disc electrophoresis in polyacrylamide gel by means of ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephadex G-200 gel filtration.

The optimum pH and temperature of the enzyme were around pH 7.0 and around 50°C, respectively. Metal ions such as Hg²⁺, Cu²⁺, Ni²⁺, Zn²⁺, Fe²⁺ and Co²⁺ strongly inhibited the enzyme activity. The molecular weight was about 45,000. The yields of maltotetraose and maltopentaose from potato starch were 30 ~ 40% and 20 ~ 30%, respectively.     

A thermophilic extracellular -amylase from Bacillus licheniformis

A strain of Bacillus licheniformis isolated from soil produced an extracellular α-amylase(s) with unusual characteristics. The enzyme was purified 126-fold by starch adsorption, DEAE-cellulose treatment, and CM-cellulose column chromatography. Four active protein bands were detected by disc electrophoresis in poly-acrylamide gel although the enzyme behaved as a single peak during both ultracen-trifugation and chromatography using CM-cellulose and Sephadex G-100. The enzyme showed a very broad pH-activity curve and had substantial activity in the alkaline range. The optimal temperature was 76 °C at pH 9.O. The enzyme was stable between pH 6 and 11 at 25 °C, and below 60 °C at pH 8.0. Using Sephadex G-100 gel filtration, a molecular weight of 22,500 was estimated for the enzyme. The action pattern on amylose and amylopectin is unique in that the predominant product during all stages of hydrolysis is maltopentaose.     

Purification and characterization of an extracellular glucoamylase from the yeastCandida tsukubaensis CBS 6389

The starch-degrading yeastCandida tsukubaensis CBS 6389 secreted amylase at high activity when grown in a medium containing soluble starch. The extracellular α-amylase activity was very low. The major amylase component was purified by DEAE-Sephadex A-50 chromatography and Ultrogel AcA 44 gel filtration and characterized as a glucoamylase. The enzyme proved to be a glycoprotein with a molecular weight of 56000. The glucoamylase had a temperature optimum at 55°C and displayed highest activity in a pH range of 2.4–4.8. Acarbose strongly inhibited the purified glucoamylase. Debranching activity was present as demonstrated by the release of glucose from pullulan.     

Optimization, production, and partial characterization of an alkalophilic amylase produced by sponge associated marine bacterium Halobacterium salinarum MMD047

An endosymbiont Halobacterium salinarum MMD047, which could produce high yields of amylase, was isolated from marine sponge Fasciospongia cavernosa, collected from the peninsular coast of India. Maximum production of enzyme was obtained in minimal medium supplemented with 1% sucrose. The enzyme was found to be produced constitutively even in the absence of starch. The optimum temperature and pH for the enzyme production was 40°C and 8.0, respectively. The enzyme exhibited maximum activity in pH range of 6∼10 with an optimum pH of 9.0. The enzyme was stable at 40°C and the enzyme activity decreased dramatically above 50°C. Based on the present findings, the enzyme was characterized as relatively heat sensitive and alkalophilic amylase which can be developed for extensive industrial applications.     

Characteristic Decrease of Nuclear Protein Kinase Activity in G1-Arrested Cells of Saccharomyces cerevisiae cdc28

An alkalopsychrotrophic strain, Micrococcus sp. 207, inducibly and extracellularly produced amylase and pullulanase. The main hydrolysis product from amylose, with a crude enzyme preparation, was maltotetraose. The optimum temperature for activity of the amylase was 60°C and that for pullulanase 55°C. The activities at 0 to 30°C exhibited similar activation energy values. In an optimized production medium at pH 9.7, the highest yields of these enzymes were obtained after cell growth at 18°C for 4 days. At pH 8.5, the yields of amylase and pullulanase became maximum after 3 days cultivation. With more prolonged cultivation, the yield of amylase but not that of pullulanase activity decreased. These enzymes were not produced at temperatures above 30°C. Sucrose was not effective as an inducer, but it stimulated cell growth and enhanced the enzyme productivities with soluble starch.     

Multiple Shoot Formation from Almond Seeds and an Excised Single Shoot

An amylase which produces maltotriose from starch as the main product was found in the culture filtrate of a strain of Bacillus subtilis newly isolated from soil. The enzyme was purified to almost complete homogeneity, as judged by disc electrophoresis in polyacrylamide gel, by means of ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephadex gel filtration.

The optimum pH and temperature of the enzyme were around 6~7 and 50°C, respectively. Metal ions such as Hg²⁺, Cu²⁺, Zn²⁺, Ni²⁺ and Fe²⁺ strongly inhibitied the enzyme activity. The molecular weight was found to be about 25,000 by gel filtration. The yields of maltotriose from short-chain amylose (DP 17), amylopectin, soluble starch and glycogen were about 69, 56, 56 and 40%, respectively.     

Studies on Tannin Acyl Hydrolase of Microorganisms

Tannin acyl hydrolase (Tannase) from Asp. oryzae No. 7 was purified. The purified enzyme was homogenous on column chromatography (DEAE-Sephadex A50, Sephadex G100), ultra centrifugation and electrophoresis.

The molecular weight of the enzyme estimated by gel filtration method was about 200,000.

The enzyme was stable in the range of pH 3 to 7.5 for 12 hr at 5°C, and for 25 hr at the same temperature in the range of pH 4.5 to 6. The optimum pH for the reaction was 5.5. It was stable under 30°C (over one day, in 0.05 M-citrate buffer of pH 5.5), and the optimum temperature was 30~40°C (reaction for 20min). The activity was lost completely at 55°C in 20 min at pH 5.5, or at 85°C in 10 min at the same pH.

Any metal salt tested did not activate the enzyme, Zink chloride and cupric chloride inhibited the activity or denatured the enzyme. The activity was lost completely by dialysis against EDTA-solution at pH 7.25, although it was not affected by dialysis against deionized water.     

Purification, partial characterization, and covalent immobilization–stabilization of an extracellular α-amylase from Aspergillus niveus

An extracellular amylase secreted 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 % polyacrylamide gel electrophoresis (PAGE) and 10 % sodium dodecyl sulfate (SDS-PAGE). The enzyme exhibited 4.5 % carbohydrate content, 6.6 isoelectric point, and 60 and 52 kDa molar mass estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The amylase efficiently hydrolyzed glycogen, amylose, and amylopectin. The end-products formed after 24 h of starch hydrolysis, analyzed by thin layer chromatography, were maltose, maltotriose, maltotetraose, and maltopentaose, which classified the studied amylase as an α-amylase. Thermal stability of the α-amylase was improved by covalent immobilization on glyoxyl agarose (half-life of 169 min, at 70 °C). On the other hand, the free α-amylase showed a half-life of 20 min at the same temperature. The optima of pH and temperature were 6.0 and 65 °C for both free and immobilized forms.     

Purification and characterization of maltooligosaccharide-forming amylase from Bacillus circulans GRS 313

A maltooligosaccharide-forming amylase that hydrolyzes starch into maltotriose and maltopentaose was found in the culture filtrate of a strain of Bacillus circulans GRS 313 isolated from local soil. The enzyme was purified by organic solvent fractionation, Sephadex G-100 gel filtration and CM-Sephadex column chromatography. Optimum pH and temperature of amylase were evaluated using response surface methodology (RSM) and were found to be 48°C and 4.9, respectively. The enzyme was stable up to 60°C and its pH stability was in the range of 5.0–8.0. The K _m and V _max of the amylase with starch were 11.66 mg/ml and 68.97 U, respectively, and the energy of activation, E _a, was 7.52 kcal/mol. Dextrin inhibited the enzyme competitively, with a K _i of 6.1 mg/ml, and glucose caused noncompetitive inhibition with a K _i of 9.5 mg/ml. The enzyme was inhibited by Hg²⁺, Mn²⁺, Fe³⁺ and Cu²⁺ and enhanced by Co²⁺ and Mg²⁺. EDTA reversed the inhibitory effect of the metals. Paper chromatographic and high-performance liquid chromatography analysis of the products of the amylolytic reaction showed the presence of maltotriose, maltotetraose, maltopentaose, maltose and glucose in the starch hydrolysate. Journal of Industrial Microbiology & Biotechnology (2002) 28, 193–200 DOI: 10.1038/sj/jim/7000220 Received 11 December 2000/ Accepted in revised form 22 October 2001     

Purification and partial characterization of β-1,3-glucanase from <Emphasis Type="Italic">Chaetomium thermophilum</Emphasis>

A thermostable extracellular β-1,3-glucanase from Chaetomium thermophilum was purified to homogeneity by fractional ammonium sulfate precipitation, Pheny1-Sepharose hydrophobic interaction chromatography, ion exchange chromatography on DEAE-Sepharose and gel filtration on Sephacryl S-100. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 76.3 kDa. The enzyme exhibited optimum catalytic activity at pH 6.0 and 60 °C. It was thermostable at 50 °C, and retained 90% activity after 60 min at 60 °C. The half-life at 65 °C, 70 °C and 80 °C was 55 min, 21.5 min, and 5 min, respectively. The N-terminal amino acid sequence (8 residues) of the enzyme was HWLGDIPH. The HPLC analysis showed that the only enzymatic product formed from laminarin by the purified β-1,3-glucanase was glucose, indicating that the enzyme is an exo-β-1,3-glucanase (EC 3.2.1.58).     

Purification and Properties of Alcohol Oxidase from Candida methanosorbosa M-2003

Alcohol oxidase from Candida methanosorbosa was purified about sixfold with a yield of 37.6% from the culture broth of Candida methanosorbosa M-2003. The enzyme preparation was homogeneous on slab gel electrophoresis. The purified enzyme had an optimal pH from 6.0 to 9.0 and was stable in the range 6.0–8.5. Its optimal temperature of reaction was 50°C, and it was stable below 50°C. In the presence of NaN₃, the enzyme retained its initial activity at 30°C for 35 days, indicating stability for a long term, so far. The isoelectric point was pH 4.3. Its molecular weight was 620,000 by gel filtration chromatography and 80,000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. These results indicate that the enzyme consists of 8 subunits. Received: 1 October 1996 / Accepted: 12 December 1996     

Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose.     

Purification and properties of an acidic β-glucosidase from Acidobacterium capsulatum

Acidobacterium capsulatum, an acidophilic, mesophilic and chemoorganotrophic bacterium, produced an inducible, acidic β-glucosidase in the cellobiose medium. The enzyme was successively purified 109 times by CM-Sepharose, Sephacryl S-200 chromatography and preparative discontinuous polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis of the purified enzyme gave a single band at pH 4.3. The enzyme had an optimum pH of 3.0 and optimum reaction temperature of 55°C, being stable from pH 1.5 to 6.0 and at temperatures from 20 to 45°C. No activity was detected above pH 6.5 or above 65°C. The molecular weight of 90,000 was estimated by gel filtration and the enzyme had an isoelectric point of 7.0. The enzyme hydrolyzed aryl-β-glycosides and β-linked disaccharides.     

Purification and Characterization of a Thermostable Alkaline Protease from Alkalophilic Thermoactinomyces sp. H8682

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25, 000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0.

Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu²⁺ and Hg²⁺. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5–13.0 at 37°C and the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl₂, it showed maximum proteolytic activity at 80°C and stability from pH 4–12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca²⁺ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of Microbiol serine protease, although alanine of the NH₂-terminal amino acid was deleted.     

Purification and characterization of a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents, from dimethylsulfoxide-tolerant Brachybacterium sp. strain LB25

A bacterium that secretes maltooligosaccharide-forming amylase in a medium containing 12.5% (vol/vol) dimethylsulfoxide (DMSO) was isolated and identified as Brachybacterium sp. strain LB25. The amylase of the strain was purified from the culture supernatant, and its molecular mass was 60 kDa. The enzyme was stable at pH 7.0–8.5 and active at pH 6.0–7.5. The optimum temperature at pH 7.0 was 35°C in the presence of 5 mM CaCl₂. The enzyme hydrolyzed starch to produce maltotriose primarily. The enzyme was active in the presence of various organic solvents. Its yield and product selectivity of maltooligosaccharides in the presence of DMSO or ethanol were compared with those of the industrial maltotriose-forming amylase from Microbacterium imperiale. Both enzymes improved the production selectivity of maltotriose by the addition of DMSO or ethanol. However, the total maltooligosaccharide yield in the presence of the solvents was higher for LB25 amylase than for M. imperiale amylase.     

Purification and characterization of an extracellular acid trehalase fromLentinula edodes

Trehalase from the culture filtrate ofLentinula edodes was purified and characterized. Molecular masses were estimated to be 158 kDa and 79–91 kDa by gel filtration and SDS-PAGE under the reduced condition, respectively. The enzyme was composed of two identical subunits and contained carbohydrate molecules. The optimum temperature and pH were obtained at around 40°C and pH 5.0, respectively. The enzyme was stable up to 40°C and in a range pH of 4–10 at 30°C. It cleaved α-1,1 linkages of trehalose, but not α-1,4, α-1,6 or β-1,4 glycosyl linkages, and was defined as an acid trehalase.     

Purification and characterization of α-amylase fromAspergillus flavus

Aspergillus flavus produced approximately 50 U/mL of amylolytic activity when grown in liquid medium with raw low-grade tapioca starch as substrate. Electrophoretic analysis of the culture filtrate showed the presence of only one amylolytic enzyme, identified as an α-amylase as evidenced by (i) rapid loss of color in iodine-stained starch and (ii) production of a mixture of glucose, maltose, maltotriose and maltotetraose as starch digestion products. The enzyme was purified by ammonium sulfate precipitation and ion-exchange chromatography and was found to be homogeneous on sodium dodecyl sulfate— polyacrylamide gel electrophoresis. The purified enzyme had a molar mass of 52.5±2.5 kDa with an isoelectric point at pH 3.5. The enzyme was found to have maximum activity at pH 6.0 and was stable in a pH range from 5.0 to 8.5. The optimum temperature for the enzyme was 55°C and it was stable for 1 h up to 50°C. TheK _m andV for gelatinized tapioca starch were 0.5 g/L and 108.67 μmol reducing sugars per mg protein per min, respectively.     

Isolation and Purification of Ascorbate Oxidase from Acremonium sp. HI-25

A screening test was undertaken to isolate microorganisms that produced ascorbate oxidase. The enzyme activity was found in a culture filtrate of a fungal strain (HI-25), newly isolated from a soil sample. Based on the morphological characteristics, this isolate was identified as Acremonium sp. From the examinations of cultural conditions, optimum conditions for enzyme production were found; strain HI-25 was aerobically cultured by a jar fermenter at 25°C in a medium containing 5% glycerol, 2% defatted soybeans, 0.1% monosodium L-glutamate, 0.1% KH₂PO₄, 0.02% MgSO₄ ·7H₂O, and 0.01% KCl, pH 6.0. After cultivation, an ascorbate oxidase was purified from the culture filtrate by an ammonium sulfate fractionation, column chromatographies on DEAE-cellulose and Butyl-Toyopearl, and gel filtration twice on Sephadex G-100. The purification was 850-fold with an activity yield of 8.8%. The purified enzyme gave a single band on SDS polyacrylamide gel electrophoresis, and had a molecular weight of 80,000 by SDS polyacrylamide gel electrophoresis and 76,000 by native gel filtration. This enzyme was most active at pH 4.0, 45°C, and was most stable between pH 6.0–10.0 and at temperatures below 60°C.     

Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose.