Isolation and characterization of an extracellular lipase producing Bacillus Corynebacterium sp. from petroleum contaminated soils

Author Name: *Sushma Poddar, Preetha Singha
Author Email: sushmapoddar696@gmail.com

Abstract

The use of microorganisms as bioreactors has been the trend of the year. Lipase which is one of the most commonly used enzyme in food and textile industry is of high demand. The current work aims to use microbes as bioreactors to produce the required enzyme lipase in laboratory conditions. The organisms used for lipase production were isolated from soil and screened for their efficiency. The positive organisms were further identified as Corynebacterium species as per the Bergey’s manual. The substrate used for the enzyme production was olive oil and tween 80. The enzyme thus produced was purified by Salt precipitation and Dialysis. The enzyme produced was estimated quantitatively and the activity and efficiency of the enzyme was calculated qualitatively. The standard conditions of temperature and pH were also estimated for maximum enzyme production. Thus maximum enzyme production with economic sources has been achieved.

Keywords

Lipase, Oil, Extracellular, Purification, Corynebacterium, Bioreactors

Introduction

Lipase (Triacylglycerol lipases,EC 3.1.1.3) are water soluble enzymes which have the ability to hydrolyse triacylglycerols to release free fatty acids and glycerol. Lipase constitutes a major group of biocatalysts that have immense biotechnological applications. Lipase have been isolated and purified from fungi, yeast, bacteria, plant and animal sources.[1] Lipoprotein lipase (LPL) is a member of the lipase gene family, which includes pancreatic lipase, hepatic lipase andendothelial lipase. LPL is water soluble enzyme that hydrolyzes triglycerides in lipoproteins, which is found in cylomicrons and very low density lipoproteins. Lipoprotein lipase which is a hydrolytic enzyme produced by many tissues and is rate-limiting for the removal of lipoprotein triglycerides from the circulation. It also has other important roles in many normal tissues, as well as in certain metabolic diseases including obesity. [2] Lipases are ubiquitously produced in plants, animals and microorganisms. Microbes can easily cultivate and microbial lipases are mostly extracellular and hence have received particular attention with respect to their industrial production and applications. [3] They catalyze both the hydrolysis of triglycerides and the synthesis of esters from glycerol and long chain fatty acids. In additions they also serve as biocatalyst for alcoholysis, acidolysis, esterification and amionlysis. The common mode of lipases as a biocatalyst is shown below:
Fats or oils +Water →fatty acid + Glycerol
Lipases of microbial origin have greater industrial attraction because they are available in large quantities and can be produced with high yields. Numerous lipases have been characterized and efforts have been made to improve their stability in organic solvents for varied applications. Cost effectiveness, the main concern of industries is also found suitable in lipase catalyzed process as compared to traditional downstream processing where major bottleneck are energy consumption and toxic byproducts. [4] The aim of the study was therefore, to purify and study the kinetics of lipase enzyme produced by Bacillus sp. A local strain isolated from a petroleum contaminated soil sample.

Conclusion

The current work was aimed to produce maximum concentration of efficient lipase from cheaper sources like soil bacteria. The bacterium that was positive towards lipase production was identified to be Corynebacterium species. The organism was prepared as pure culture and used for further analysis. Production media was prepared by incorporating tween 80 and olive oil in the basic media. The  production media was incubated for 3 days and the enzyme thus produced was extracted and purified. The pure enzyme was thus assayed qualitatively and the efficiency was found to be 0.123U/ml. The concentration of the enzyme produced was calculated and found to be 2μl/ml in sample. The study was further focussed to study the optimal conditions for the maximum enzyme production. The optimum temperature and Ph was calculated to be 370C and 7 respectively. The enzyme thus produced was maximum under these conditions.

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