Characterization Of Thermostable Α-amylase Research Articles

Purification and Characterization of Thermostable α-Amylase from Soil Bacterium Bacillus sp.

Amylases are used in several industrial and biotechnological sectors, including those producing textiles, detergents, paper and bakery products. This study aimed to purify an industrially important α-amylase from Bacillus sp. For this purpose, a single and rapid α-amylase purification was performed using the starch affinity method. Characterization of the purified enzyme was determined by investigating temperature, pH stability, detergents, and metal ions. The purification coefficient of 29.8-fold with a yield of 9.2% was found. The molecular weight of the purified α-amylase was determined to be 53 kDa by SDS-PAGE, and thermostability was confirmed with 100% activity at 30ºC and 40ºC after 1 h. The purified enzyme was stable over a wide range of pH values, with optimum activity at pH 6.0, 7.0 and 8.0 after 2 h. The study also investigated the effects of the metal ions and detergents on the purified amylase and found that Mg2+ and Ca2+ ions were the activators of the enzyme, while Zn2+, Co2+ and Na+ ions decreased the activity. Furthermore, Hg2+; indicated complete inhibition of amylase activity. The detergents Triton X-100 and Tween 20 increased the α-amylase activity, while sodium dodecyl sulfate inhibited the activity. The purified α-amylase obtained from Bacillus sp. is considered to be environmentally friendly, can be processed in a short time, and has a low cost.

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The purpose of the study is isolation and application of novel Hot spring bacterial enzymes. It also reports on purification and characterization of thermostable α-amylase from a newly hot spring isolate, Exiguobacterium sp. This thermostable amylase is Ca2+-independent an added improvement in starch saccharification process at a higher temperature because it removes the addition of Ca2+ for improving the stability of amylases. Maximum enzyme activity was obtained at 45 °C at pH 8.5 and stability at concentration of 3.0% NaCl. Thermostable α-amylase from Exiguobacterium sp was purified by 3.9 fold with 54.6% recovery and specific activity was 1083 U/ml. The molecular weight of α-amylase was 54 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Apparent Km and Vmax value was 5.88 mg/ml and 250µmol/min/ml, respectively for the hydrolysis of soluble starch. An initial analysis of the circular dichroism (CD) spectrum in the ultraviolet range revealed that the amylase is predominantly turn structure and a detailed structural composition showed alpha helix 10.8%, Beta sheet 27.1%, Turn 29.8% and Randomness 32.3% respectively. The amylase combined with soap-nut extract was able to de stain blood stained cloth within 30 min

Purification and characterization of thermostable α-amylase from germinating Sword bean (Canavalia gladiata (Jacq.) DC.) seeds.

The thermostable α-amylase from germinating sword bean (Canavalia gladiata (Jacq.) DC.) seeds (CgAmy) was successfully purified by a combination of ammonium sulphate fractionation and Epoxy-activated Sepharose 6B affinity chromatography. The purified α-amylase showed 507.8 fold with a specific activity of 750.0 U/mg. SDS-PAGE of the purified enzyme revealed a single protein band of 50.0 kDa. Purified enzyme was confirmed as α-amylase type by LC-MS/MS analysis and activity on specific substrate of ethylidene-pNP-G7. The CgAmy revealed extreme activity at a high temperature of 50.0-70.0°C with optimum activity at 70.0°C. The optimal pH of enzyme activity was observed at 6.0. The CgAmy exhibited stability in pH range of 5.0-8.0 and highly thermostable with a temperature of 40.0-60.0°C. The kinetic parameters K m for hydrolysis of starch were found to be 3.12 mg/ml. The α-amylase activity was enhanced in the presence of Co2+ and β-mercaptoethanol. While, Na2+, K2+, Ca2+, Mg2+, Zn2+, Ba2+, Fe2+ and Cd2+ slightly inhibited α-amylase activity. Interestingly, the CgAmy displayed stability towards some organic solvents and detergents. Stability at high temperature and some metal ions, organic solvents and detergents indicated that this enzyme has potential for various applications.

Purification, immobilization and characterization of thermostable α-amylase from a thermophilic bacterium Geobacillus sp. TF14

Abstract Objective In this study, α-amylase from a thermophilic bacterium Geobacillus sp. TF14 was purified and immobilized on two different supports. Methods Ion exchange and hydrophobic interaction chromatography techniques were employed for the purification. Results The enzyme was purified as 17.11 fold and determined as a single band of 54 kDa on SDS-PAGE. Purified enzyme showed two pH optimums of pH 5.00 and pH 9.00 and the enzyme is quite stable at these pHs over a period of 48 h. Purified enzyme showed maximal activity at 75°C and stability at this temperature over a period of 72 h. It was observed that Ca2+ activated the enzyme at about 70% at 5 mM final concentration. SDS, Triton X100, Triton X114 and Tween 20 caused around 50% loss of initial activity at a final concentration of 1% (w/v). Purified enzyme was immobilized on the surface of Dowex and chitin. Immobilization highly enhanced temperature optima and thermal stability. Dowex immobilized enzyme maintained most of its initial activity in the presence of SDS, Triton X100, Triton X114 and Tween 20 at a concentration of 1%. Conclusion It can be concluded that the purified enzyme may find application in many fields of starch based industries.

Thermostable amylase production from hot spring isolate Exiguobacterium sp: A promising agent for natural detergents

The purpose of the study is isolation and application of novel Hot spring bacterial enzymes. It also reports on purification and characterization of thermostable α-amylase from a newly hot spring isolate, Exiguobacterium sp. This thermostable amylase is Ca2+-independent an added improvement in starch saccharification process at a higher temperature because it removes the addition of Ca2+ for improving the stability of amylases. Maximum enzyme activity was obtained at 45°C at pH 8.5 and stability at concentration of 3.0% NaCl. Thermostable α-amylase from Exiguobacterium sp was purified by 3.9 fold with 54.6% recovery and specific activity was 1083U/ml. The molecular weight of α-amylase was 54kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Apparent Km and Vmax value was 5.88mg/ml and 250µmol/min/ml, respectively for the hydrolysis of soluble starch. An initial analysis of the circular dichroism (CD) spectrum in the ultraviolet range revealed that the amylase is predominantly turn structure and a detailed structural composition showed alpha helix 10.8%, Beta sheet 27.1%, Turn 29.8% and Randomness 32.3% respectively. The amylase combined with soap-nut extract was able to de stain blood stained cloth within 30min

Purification and characterization of thermostable α-amylase from thermophilic Anoxybacillus flavithermus

This study reports on the purification and characterization of thermostable α-amylase (α-1-4 d-glucan glucanohydrolase EC 3.2.1.1) from a newly isolated Anoxybacillus flavithermus. A. flavithermus was used, which was isolated from hot water springs of Ömer, Afyonkarahisar, Turkey. The gram-positive, spore-forming, motile, moderately thermophilic bacteria were found to be a strain of A. flavithermus analysed by 16S rRNA comparison. The optimal conditions for bacterial growth were determined to be at 20thh, 55°C and pH 6.0. Maximum α-amylase activity was obtained at 55°C at pH 7.0 after 24h of incubation. Thermostable α-amylase from A. flavithermus was purified by 70% (NH4)2SO4 and ion-exchange chromatography (5.2-fold; 65.8% yield). The molecular weight of α-amylase was 60kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The α-amylase hydrolyzed soluble starch at 55°C with Km: 0.005mM and Vmax: 3.5μmolmin−1.

Bioprocess development for the production, purification and characterization of thermostable α-amylase by Bacillus licheniformis local isolate using response surface methodology

Bioprocess development for the production, purification and characterization of thermostable α-amylase by Bacillus licheniformis local isolate using response surface methodology

Characterization of Thermostable α-amylase from Thermophilic andAlkaliphilic Bacillus sp. Isolate DM-15

Bacillus sp. DM-15 was isolated from Ciftehan Thermal Spring, Turkey produced thermostable α-amylase at 55°C at pH 9. Analysis of the enzyme for molecular mass and amylolytic activity carried out by SDS-Starch-PAGE electrophoresis revealed a single band with high molecular mass 126 kDa. After extraction, the enzyme remained stable in a range of temperature and pH between 40-100°C and 4.5-10, respectively. The optimum enzyme activity was displayed at 60°C and pH 5.5-6.0. α-amylase production by thermophilic Bacillus sp. strain cultivated in liquid media reached a maximum at 12 h with levels of 27.5 μmol mg-1 protein min-1. The enzyme was stable for 15 min at 60°C while 24% of the original activity was lost at 100°C. Enzyme activity was increased in the presence of 5 mM CaCl2, Na2SO3 and KCl (105%) and inhibited in the presence of 5 mM EDTA, CuSO4, FeSO4, SDS (1%), Urea (8 M), Co up to 98, 79, 57, 36, 33 and 10%, respectively. The DM-15 α-amylase may be suitable for in liquefaction of starch in detergent and textile industries and in other industrial applications.

Production and characterization of thermostable α-amylase from a newly isolated strain of Bacillus subtilis KIBGE-HAR

Production and characterization of thermostable α-amylase from a newly isolated strain of Bacillus subtilis KIBGE-HAR