Amylolytic enzymes are among the most important enzymes in industry and biotechnology. Alpha amylases (endo-1, 4- D-glucan glucanohydrolase, E.C. 3.2.1.1) are extracellular endo enzymes that randomly cleave the 1, 4-a linkage between adjacent glucose units in the linear amylose chain and ultimately generate glucose, maltose and maltotriose units. Amylases stand out as a class of enzymes, which are of useful applications in the food, textile, detergent and pharmaceutical industries. In the present study, a strain of amylolitic ANOXYBACILLUS FLAVITHERMUS was identified and 16S rRNA analysis was carried out on this thermophilic strain. Bacterial growth optimal production was investigated. Then, protein was partially purified by 85% ammonium sulfate and dialyzed. Besides, biochemical properties such as effect of temperature, pH and effect of denaturant such as SDS on the stability of enzyme were investigated. On the other hand, encode gene of enzyme was amplified and sequenced. Results indicate that the optimal conditions for bacterial growth are after 20 h, at 60 °C and pH 7.0. Also, maximum enzyme activity was obtained at 70 oC at pH 6.0 after 72 h of incubation and optimal temperature is more than other species and stability of the enzyme in the presence of SDS is the same as other species. Moreover, sequencing result confirms existence of the gene with the length of about 1500 bp.

The thermostable and alkaliphile xylanases are beneficial industrial enzymes in food industry (bakery and alcohol production or fermentation industries) pulp-kraft, and pharmaceutic industry. Xylanases hydrolyse xylan (the hemicellulose of plant cell wall) and produced by different kinds of microorganisms like bacteria, fungi and some algae. In this study, xylanase gene from thermoalkaline ANOXYBACILLUS FLAVITHERMUS was isolated from hot spring of Meshkinshahr (Iran) and was cloned in E. coli (BL21). At first, total DNA was extracted from bacteria. PCR was then accomplished and xylanase gene was isolated and amplified. Amplification of xylenase coding gene was confirmed by electrophoresis. Cloning was performed by digestion of xylanase gene and pET21a+ by restriction enzymes (NheI and XhoI), following by ligation and transformation to E. coli (BL21). Finally, the results were confirmed by colony-PCR and sequencing of selected colonies. PCR and sequencing results, demonstrated a high similarity between xylanases of ANOXYBACILLUS species with xylanase of this study.

Biomacromolecules of thermophilic organisms are often thermostable. Apart from high temperature they are also known to withstand denaturants of extremely acidic and alkaline conditions. Thermostable enzymes have capable potentioal in the food and paper industries, detergents, drugs, toxic wastes removal and oil drilling. The enzymes can be produced from the thermophiles through either optimized fermentation of the microorganisms or cloning of fast growing mesophiles by recombinant DNA technology. Metalloproteases are metal ion dependent protease. Metalloproteases that used in this study was a zinc-dependent endopeptidase. Metalloprotease gene was cloned from ANOXYBACILLUS FLAVITHERMUS. For the amplification of the gene, PCR was performed. A pair of primers designed with a cleavage site for enzymes XhoI and NheI and vector pET21a (+) clone was transformed into BL21 host. The results of the cloned gene sequence analysis showed similarity to the protease gene different variants of the species ANOXYBACILLUS FLAVITHERMUS.

Background & Objectives: Thermophilic alpha-amylase can be used in different industries such as starch processing and detergents. This study was performed to isolate alpha-amylase-producing bacteria and characterization of the enzyme.Materials & Methods: After sample collection from Gorooh hot spring in Kerman province, Iran, thermophilic alpha-amylase- producing bacteria were isolated using the starch-agar medium.16S rDNA sequencing was used to identify the bacterial strain. Characterization of the thermophilic alpha-amylase was performed in the presence of various factors such as pH, temperature, metal ions, chemical compounds, and organic solvents. Also, kinetic parameters of the enzyme were determined in different concentrations of starch.Results: ANOXYBACILLUS gonensisAT23 was identified as the best thermophilic alpha-amylase- producing strain. The alpha-amylase enzyme showed the optimal activity at pH 5 to 6. Sevenfold increase in the enzyme activity was observed in the presence of NaCl (3M). Mn2+ and Zn2+ increased the enzyme activity about 95% and 31%, respectively. Kinetic parameters including K m and Vmax were estimated about 1.657 mg/ml and 0.0059 mg/ml/min, respectively. Also, enzyme activity was also improved about 2 folds in the presence of organic solvents including n-butanol and 10% cyclohexane.Conclusion: Our results indicated that AT23 alpha-amylase is a halophile and organic solvent-tolerant enzyme. Therefore, it can be used in different industries.

Thermophilic bacteria are valuable sources of thermostable hydrolytic enzymes. Heat-stable enzymes have immense importance in industry and are generally utilized for a variety of commercial Purposes. Among the starch hydrolyzing enzymes, a-amylases have considerable commercial value, especially in sweetener industry. For the production process of sweeteners from starch, the temperature should be 50ºc or higher and the pH close to 7 in order to prevent browning effects and reduce viscosity. It thus requires enzymes able to withstand the elevated process temperatures.Bacteria were isolated from two hot spring waters of Kharaghan (in Gazvin province) and Mahallat (in Arak). Water samples were collected from 50cm below the spring water surface.The water temperatures at the time of sampling at the Kharaghan and Mahallat sites were 56oc and 48oc, respectively. Following centrifugation of the water samples, any remaining pellets were cultured in trace element enriched nutrient broth and incubated at proper elevated temperatures.The four recovered bacteria were identified molecularly by PCR and 16S rRNA gene ribotyping. They were identified as Aneurinibacillus aneurinilyticus, Aneurinibacillus thermoaerophilus, ANOXYBACILLUS pushinoensis and Bacillus licheniformis.Amylase production was assayed on starch agar plates by dinitrosalicylic assay. The optimal pH and temperature for enzyme activity were characterized. The maximum activity for the B.licheniformis enzyme was observed at 70ºcand at pH 7.0. The A. aneurinilyticus enzyme maximum activity was at 70º c and at pH 8.0; whereas, these for the A.pushinoensisenzyme was at 80oc and at pH 8.0.