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OBTAINING OF RECOMBINANT LIPASE AND ITS PRACTICAL APPLICATION

A. A. Tolkacheva

Abstract

Industry is constantly forced to look for new ways to make production processes environmentally safe and products more competitive. In this regard, microbiological enzyme preparations increasingly replace conventional chemical catalysts in a number of industrial processes. Such preparations, in addition to environmental friendliness and high activity, have a number of advantages over enzyme preparations of vegetable and animal origin. With the aim of creating new recombinant lipase, we analyzed data on the activity, specificity and structure of this enzyme, as well as on its coding genes, and searched for suitable producer in microorganism collections. The target gene of lipase from microorganisms was isolated and cloned by integration into the plasmid vectors of the PET system (Novagen). Expression of cloned gene was carried out in E. coli cells (Bl-21, C-41). The optimal time for induction of biosynthesis of recombinant enzyme was determined, high levels of target enzyme in cell culture was achieved - up to 40% of the total protein volume. Isolation and purification of recombinant enzyme was performed by Ni-affinity chromatography, using Ni-NTA agarose (Qiagen, USA). The activity of the obtained enzymes was determined by standard biochemical methods. The activity indices of the obtained recombinant lipase are 12 times higher than the activity of the native enzyme. A method for purifying wastewater using the obtained recombinant lipase was developed by the authors. The maximum yield of fatty acids and their salts as a result of hydrolysis by lipase is observed when the enzyme is introduced in an amount of 10 U/g of fat after 4 days of hydrolysis of waste water from the fat and oil enterprise at a temperature of 40В±2 В°C in a stationary mode. After 24 hours of hydrolysis, the concentration of fat in the wastewater is reduced to the maximum permissible concentration.

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DOI resolver: doi.org/10.5593/sgem2018/6.2/s25.053

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