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Vol.2 , No. 6, Publication Date: Feb. 3, 2016, Page: 82-91
, triacylglycerol hydrolases, are a significant group of biotechnologically applicable enzymes and they find enormous applications in food, detergent and pharmaceutical industries. Lipases are largely produced from microbes, and they play a fundamental role in commercial ventures. A lipase from thermophilic <i>Burkholderia pseudomallei</i> bacterial strain was isolated from Saudi Arabian environment. Based on this strain, a lipase gene encoding 399 amino acids was cloned, and expressed in <i>E. coli</i> BL21 (DE3). The lipase protein fused with glutathione S-transferase was purified to homogeneity 128.2 fold. SDS- PAGE of the purified enzyme revealed it has <i>Mr</i> of 32 kDa. The recombinant lipase was efficiently immobilized in calcium alginate gelatin composites. The optimum temperature for free enzyme highest activity was recorded at 65°C however the immobilized enzyme exhibits the highest activity at 70°C. The immobilized enzyme retains most of its activity and shows high stability for 120 min at 70°C compared to 45 minutes for the native enzyme when incubated at 70°C. The free enzyme has an optimum pH at 7.5 but this optimum pH is shifted to 8.5 for the immobilized enzyme. The free and immobilized lipase catalytic function were enhanced in the presence of 1 mM of Ba<sup>++</sup>, Ca<sup>++</sup> and Na<sup>+</sup>, but inhibited by 1mM of Ni<sup>++</sup>, Hg<sup>++</sup>, Cu<sup>++</sup> and Co<sup>++</sup>. The free and the immobilized enzyme activities increased in the presence of 5 mM Fe<sup>++</sup>, Co<sup>++</sup> or Li<sup>+</sup>.&picture=http://www.aascit.org/aascit/decorators/img/journal/919.jpg)
| [1] | Magdy M. Youssef, Department of Chemistry, College of Science, King Faisal University, Hofuf, Saudi Arabia; Department of Chemistry, Faculty of Science, Mansoura University, Mansoura, Egypt. |
Lipases (EC 3.1.1.3), triacylglycerol hydrolases, are a significant group of biotechnologically applicable enzymes and they find enormous applications in food, detergent and pharmaceutical industries. Lipases are largely produced from microbes, and they play a fundamental role in commercial ventures. A lipase from thermophilic Burkholderia pseudomallei bacterial strain was isolated from Saudi Arabian environment. Based on this strain, a lipase gene encoding 399 amino acids was cloned, and expressed in E. coli BL21 (DE3). The lipase protein fused with glutathione S-transferase was purified to homogeneity 128.2 fold. SDS- PAGE of the purified enzyme revealed it has Mr of 32 kDa. The recombinant lipase was efficiently immobilized in calcium alginate gelatin composites. The optimum temperature for free enzyme highest activity was recorded at 65°C however the immobilized enzyme exhibits the highest activity at 70°C. The immobilized enzyme retains most of its activity and shows high stability for 120 min at 70°C compared to 45 minutes for the native enzyme when incubated at 70°C. The free enzyme has an optimum pH at 7.5 but this optimum pH is shifted to 8.5 for the immobilized enzyme. The free and immobilized lipase catalytic function were enhanced in the presence of 1 mM of Ba++, Ca++ and Na+, but inhibited by 1mM of Ni++, Hg++, Cu++ and Co++. The free and the immobilized enzyme activities increased in the presence of 5 mM Fe++, Co++ or Li+.
Keywords
Cloning, Expression, Purification, Immobilization, Characterization, Glutathione S Transferase
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