Amylases are among the most important industrial enzymes, accounting for about 30% of the world's total enzyme production. They have numerous industrial applications extending from general products such as detergents, paper, beer and textiles to clinical biology, health science and even the pharmaceutical industry (Maarel et al., 2002). a-Amylases (E.C.184.108.40.206.) are starch-degrading enzymes that catalyze the hydrolysis of internal a-1,4-O-glycosidic bonds in polysaccharides such as starch, to produce fine diverse products including glucose, dextrin and maltose (Bordbar et al., 2005).
a-amylase can be derived from sources such animals, plants and microbes. However, microbial sources such as fungi and bacteria are widely used in industrial production due to their high consistency and production capacity, lower production cost, faster production, non-pathogenic nature and increased product purity and stability (Burhan et al. 2003). Among bacteria, the Bacillus sp. is widely used in thermo-stable a-amylase production to meet industrial needs. B. subtilis, B. stearothermophilus, B. licheniformis and B. amyloliquefaciens are widely used in the commercial production of a-amylase for various applications (Sivaramakrishnan et al., 2006). However, B. subtilis is the most common and ideal host,mainly because of its enhanced capacity for exo-enzyme secretion, which is advantageous for high yield and purity of product (Kakeshita et al., 2011). a-amylase is synthesized early in the stationary phase in B. subtilis (Yamaguchi et al., 1974). Expression of the a-amylase structural gene, amyE, is usually switched on immediately after the onset of stationary phase (Nicholson et al., 1987). The gene is extracted from B. subtilis and cloned and expressed in the host bacterium, E. coli, to produce recombinant a-amylases.
[...] Tu, He, Li, Chen, Chang, Chen, Yao, Liu, Ye, Huan, Jiantao, S & Wu, X 2005, 'An improved system for competent cell preparation and high efficiency plasmid transformation using different Escherichia coli strains', Journal of Biotechnology, vol no pp. 114- 119. Yamabhai, Emrat, Sukasem, Pesatcha, Jaruseranee, N & Buranabanyat, B 2008, ‘Secretion of recombinant Bacillus hydrolytic enzymes using Escherichia coli expression systems', Journal of Biotechnology, vol no pp. 50-57. Yamaguchi, Nagata, Y & Marua, B 1974, ‘Isolation of mutants defective in α-amylase from Bacillus subtilis: genetic analysis', Journal of Bacteriology, vol no pp. 416-424. [...]
[...] Positive clones were cultured for protein expression using IPTG ( as an inducer for pGEM-3Zf induction. Two set of tubes were prepared. The first set was prepared by harvesting 20mL of induced bacterial culture by centrifuging at 4500g for 10 minutes at 4oC. The supernatant was decanted and the pellet was re-suspended in TE buffer (5mL) µL of lysozyme was added to the re-suspended pellet, thoroughly mixed and stored at -20oC to release bacterial cell content. This lysate was used in later enzyme activity assays. [...]
[...] The cloning and expression of Alpha-Amylase gene from Bacillus subtilis using Escherichia Coli as the host In this experiment, cloning and expression of an industrially important enzyme was studied by amplifying the amyE gene that codes for α-amylase enzyme in Bacillus subtilis. Genomic DNA from B. subtilis was inserted into a vector pGEM-3Zf and expressed into Escherichia coli DH5α cells, allowing induction of IPTG and protein secretion. Using NCBI BLAST, the gene sequence obtained displayed a 99% similarity with a B. [...]
[...] Table 3 Colony count after transformation into competent cells for different samples. The two dilutions at 10 and 40 µL was done to make it easier to count the colonies. It was observed that white colonies did not form in any sample. The result was as expected in the intact pGEM-3Zf sample because there was no transformation so blue colonies would form as lacZ is not disrupted. However for the ligation mixture, white colonies were expected to form as ligation mixture is present, but only blue colonies formed. [...]
[...] The electrophoresis was done at 150V for 90 minutes and the gel was stained using Coomassie blue for viewing. Amylase activity assays. The cell lysate obtained from the first set in part was thawed and centrifuged at 10,000g for 10 min at 4oC to remove any cell debris. The supernatant, which contained the crude proteins, was retrieved carefully and stored on ice. To measure amylase activity mL of this crude protein extract mL of starch solution and 1.7 mL of dH2O was added into 5 tubes and incubated at 37oC for a time-course study. [...]
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