الفهرس 
REVIEW OF LITERATURE
Sources of ?-amylaseOnly 14 pages are availabe for public view
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Abstract
Amylases are among the most important enzymes which are of great significance for biotechnology and have almost completely replaced chemical hydrolysis of starch in the starch processing industry. α-amylases constitute a group of enzymes that yields dextrin and numerous monomer products by endo-hydrolysing α-1, 4-O-glycosidic linkages of starch molecules to give diverse products including dextrins and progressively smaller polymers composed of glucose units. α-amylase (EC 3.2.1.1) find potential applications in pharmaceuticals, baking, brewing, textile, paper, syrup industries and detergent manufacturing processes.
Enzymes obtained from halophilic or halotolerant microorganisms could be considered as a recent alternative for use in the harsh industrial processes because of their capacity to be thermostable, tolerate a wide range of pH, withstand denaturation and tolerate high salt concentrations. Moreover, bacterial amylases have abroad application in industries due to their stability, high enzyme activity at various parameters and cost-effective production.
So, our work focused on the isolation and purification of halophilic bacteria from habitats with high salt concentrations such as sea water and slatterns to obtain α-amylase with distinctive properties.
The obtained results could be summarized as the following points: 1. The present study has been performed on the collection of samples from water and sediment of marine and salterns at different locations for isolation of bacteria. The samples were taken from Rashid, Sidi Bisher beach at Alexandria, Tiba rose village (El Sāḥel Al-shamali), Hurghada, mangrove tree around the rizosphere area (Marsa Alam), Safaga, El-Ain Elsokhna beach, and Wadi El-natron. The serial dilution method was used for isolation of bacteria. Starch agar medium was used for bacterial isolation. 100 μl from the diluted samples of 10-410-6 concentrations were placed on each starch agar plate and spreads with sterile L-shaped glass rod by spread plate technique for isolation of bacteria. The inoculated plates were incubated at 50°C for 24 hrs after that single colonies showed different morphological characteristics such as size, shape, colour, elevation and margin were isolated from different plates streaked with diluted samples. The appeared colonies per plate of each sample were subjected to purification to obtain single pure colonies on the plates.
2. The bacterial isolates were screened for their ability to produce α-amylase using starch agar plates and iodine solution as an indicator.
3. The promising isolates were characterized according to their morphological features, biochemical and physiological properties. A molecular technique was used to prove and further confirm the identification of the isolate to the species level. The partial 16S rRNA sequence was determined and was compared to the GenBank databases in the National Center for Biotechnology Information (NCBI) using the BLASTN 2.2.6 program. The two promising isolates were identified as Bacillus sp. NRC12017 and Bacillus sp. NRC22017. Bacillus sp. NRC22017 followed by Bacillus sp. NRC12017 showed the most active species for α-amylase production.
4. Bacterial growth and α-amylase production from Bacillus sp. NRC12017 and Bacillus sp. NRC22017 in shake-flask cultures were carried out using five different media at pH 7.0 and incubated for 72 hrs at 50°C. The highest production of α-amylase was obtained when medium 5 was used (10.5 and 11.0 U/ml), and it was chosen for completed other factors.
5. Bacillus sp. NRC12017 and Bacillus sp. NRC22017 were selected to study the effect of different physical and culture conditions on microbial growth and α-amylase production. It was found that the incubation period of 72 hrs, inoculum level of 200 μl, volume of the fermentation medium of 15 ml in 100 ml Erlenmeyer flask, incubation temperature 45°C, initial pH 6.5 and supplementation of starch at 2.5% concentration and peptone plus yeast extract at 0.7% concentration resulted in a maximum production of α- amylase (18.41 U/ml) for Bacillus sp. NRC12017. For Bacillus sp. NRC22017, the highest yield of α-amylase (15.14 U/ml) occurred at 72 hrs of incubation, inoculum size of 500 μl, volume of fermentation medium of 20 ml, incubation temperature 45°C and initial pH 6.0. Maltose as a sole carbon source and yeast extract plus meat extract as a nitrogen source were found to be the best medium components for maximum α-amylase production. But due to the availability of starch and low price comparing with maltose it was used for α-amylase production. The suitable starch concentration as a sole carbon source and yeast extract plus meat extract concentration as a nitrogen source that provided the highest enzyme production were found to be 2 and 1.05% (w/v), respectively.
6. The obtained α-amylases from both strains were applied to partial purification by different concentrations of ammonium sulfate. Only 60 and 80% ammonium sulfate fractions showed α-amylase activity and designated as FIII and FIV for fractions from Bacillus sp. NRC12017 and FIIIa, FIVa for fraction from Bacillus sp. NRC22017. It was found that the ammonium sulfate precipitation resulted in remarkable increase in specific activities of each fraction than the crude extract. Crude extracts from both strains and partially purified proteins obtained by 60 and 80% ammonium sulfate precipitation were subjected to SDS-PAGE. All fractions showed the main band at 30 KDa.
7. The properties of the partially purified α-amylases from different sources, Bacillus sp. NRC12017 and Bacillus sp. NRC22017 were analyzed. All fractions from both strains had maximal activity at 250 μl starch concentration in a reaction mixture. The best incubation time for FIII, FIIIa, FIVa was 20 min. and for FIV was 30 min. Activities of (FIII, FIVa) and (FIV, FIIIa) increased with increase in temperature and reached to the maximum at 50 and 55°C, respectively. Optimum pH of both FIII and FIV (from Bacillus sp. NRC12017) was 6.0-7.0, while the ideal pH for α-amylase activities (FIIIa, FIVa) from Bacillus sp. NRC22017 was 7.0.
8. Concerning thermal and pH stability, all fractions from both strains were stable up to 65°C. pH stability study revealed that both fractions of the enzyme (FIII and FIV) from Bacillus sp. NRC12017 were stable over a wide range of pH (5.0-11.0) when pre-incubated for different times (1, 3 and 24 hrs) as both fractions exhibited a slight decrease in their original activity. After 24 hrs of pre-incubation, both FIII and FIV fractions showed a residual activity of 94.86 and 92.67%, respectively at pH 11.0. Also, FIIIa from Bacillus sp. NRC22017 showed high stability in a wide range of pH from 5.0 to 11.0 after 1 and 3 hrs and showed a residual activity of 92.67 and 90.68 % after pre-incubation in pH 10.0 and 11.0 for 24 hrs. FIVa was less stable than FIIIa when pre-incubated in different buffer systems for 1, 3, and 24 hrs and presented a residual activity of 75.42 and 70.17 %, respectively at pH 10.0, 11.0 after 24 hrs.