Production, Cloning And Characterization Of Thermostable Lipase From Geobacillus Thermodenitrificans Ibrl-Nra
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Date
2015-04
Authors
Balan, Anuradha
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Abstract
Lipases from thermophiles have gained interest in recent years as they have various applications in industries. They play significant roles in industries as they have high stability at elevated temperatures and resistant to chemical denaturation. Geobacillus thermodenitrificans IBRL-nra exploited in this study was originally isolated from a hot spring in Labok, Kelantan, Malaysia with growth temperatures ranging from 45ºC to 70ºC. The production of thermostable lipase by G. thermodenitrificans IBRL-nra at 65˚C was checked qualitatively by streaking the bacteria on lipase screening agar plates. The production of extracellular thermostable lipase by G. thermodenitrificans IBRL-nra was carried out in a shake flask system and 5L stirred-tank bioreactor. The production of thermostable lipase in stirred-tank bioreactor was improved five fold compared to the production in shake flask system and an increment of three fold was observed in the cell growth. The enzyme activity was improved by 30% while the cell growth was also increased approximately 20% after the enhancement of physical parameters in the bioreactor. Lipases isolated from different sources exhibit diverse and unique characteristics. Therefore, thermostable lipase from G.thermodenitrificans IBRL-nra was purified and characterized to determine its properties. The extracellular crude thermostable lipase was purified to homogeneity by using ultrafiltration, Heparin affinity chromatography and Sephadex G-100 gel-filtration chromatography by 34 fold with a final yield of 9% and specific activity of 73.4 U/mg. The molecular weight of the purified enzyme was estimated to be 27.3 kDa on SDS-PAGE. Thermostable lipase gene, LipGt from G.thermodenitrificans IBRl-nra was cloned and over-expressed in Escherichia coli system for bulk enzyme production. Gene coding for LipGt from G. thermodenitrificans IBRL-nra was amplified from the genomic DNA, cloned into pGEM-T Easy and then expressed in expression vector pET-15b. The plasmid harbouring the thermostable lipase gene was verified for the presence of insert by DNA sequencing and restriction enzyme digestion and transformed in E. coli BL21 (DE3) and OverExpress C43 (DE3) pLysS. The recombinant protein was purified by employing heat treatment, ultrafiltration and gel-filtration chromatography. The purified recombinant thermostable lipase, LipGt was screened for crystal formation using Hampton Research Crystal Screen Cryo, HR2-121 and HR2-122 using hanging drop vapour diffusion and microbatch methods. The findings of the study reveal that the recombinant thermostable lipase gene from G. thermodenitrificans IBRL-nra was cloned and overexpressed in E. coli system and the recombinant thermostable lipase exhibits similar characteristics with the wild-type extracellular thermostable lipase. The properties of the enzyme are unique and therefore it holds a promising role in the biotechnological and industrial applications.thermodenitrificans IBRl-nra was cloned and over-expressed in Escherichia coli system for bulk enzyme production. Gene coding for LipGt from G. thermodenitrificans IBRL-nra was amplified from the genomic DNA, cloned into pGEM-T Easy and then expressed in expression vector pET-15b. The plasmid harbouring the thermostable lipase gene was verified for the presence of insert by DNA sequencing and restriction enzyme digestion and transformed in E. coli BL21 (DE3) and OverExpress C43 (DE3) pLysS. The recombinant protein was purified by employing heat treatment, ultrafiltration and gel-filtration chromatography. The purified recombinant thermostable lipase, LipGt was screened for crystal formation using Hampton Research Crystal Screen Cryo, HR2-121 and HR2-122 using hanging drop vapour diffusion and microbatch methods. The findings of the study reveal that the recombinant thermostable lipase gene from G. thermodenitrificans IBRL-nra was cloned and overexpressed in E. coli system and the recombinant thermostable lipase exhibits similar characteristics with the wild-type extracellular thermostable lipase. The properties of the enzyme are unique and therefore it holds a promising role in the biotechnological and industrial applications.
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Keywords
Lipolysis , Lipase