Optimization of recombinant amylase expression using response surface methodology (RSM)
The Anoxybaccilus DT3-1 is a newly found bacterium that is able to express amylase. The gene that encodes the amylase was recently cloned and expressed in E. coli system. However, the expression level was far too low to be used. The main objective of this study is to enhance the recombinant amylase...
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| 格式: | Thesis |
| 语言: | English |
| 出版: |
2010
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| 主题: | |
| 在线阅读: | http://eprints.utm.my/id/eprint/12710/6/KavithaMuniandyMFBB2010.pdf |
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| 总结: | The Anoxybaccilus DT3-1 is a newly found bacterium that is able to express amylase. The gene that encodes the amylase was recently cloned and expressed in E. coli system. However, the expression level was far too low to be used. The main objective of this study is to enhance the recombinant amylase expression level using pET-22b vector. Another objective of this study is to determine the end product release by the reaction of this amylase. The media optimization was carried out with five different media i.e. LB, TB, SB, CDM 1 and CDM 2. Medium LB was found to be the best medium to support the cell growth and amylase production (72 U/ml). Relevant factors such as the inducer (IPTG) concentration, yeast extract concentration and induction time (OD600nm) were optimized through two Response Surface Methodology (RSM) methods, which were the Two-level factorial and Central Composite Design (CCD). After the final optimization using CCD, 83 U/ml of amylase activity was obtained with the optimal condition of 0.007 mM IPTG, 0.3% of yeast extract and induction should be done when the cells optical density was at 1.52. Upon achieving the optimal conditions, the end products were determined using High Performance Liquid Chromatography (HPLC). The amylase was able to degrade various starches like rice, corn, wheat and soluble starch and produced a wide variety of oligosaccharides such as the glucose, maltose and isomers of maltose. |
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