Construction And Properties Of Rice Lipase Genetic Engineering Bacteria

Lipases (EC.3.1.1.3, triacylylgcerol acylhydrolases) are a group of enzymes having the ability to hydrolyze triacylglycerols at an oil water interface. They widely distributed among microorganisms, plants and animals. They can catalyze the reactions of ester hydrolysis, ester synthesis and trans esterification, which are widely used in the processing of fats and oils, detergents and degreasing formulations, food processing, the synthesis of fine chemicals and pharmaceuticals, paper manufacture, and production of cosmetics. To explore their biotechnological potentials, microbial lipases have attracted much attention.Rice lipase has a high activity and presented in rice paddy context. Due to the cell structure been damaged in rice processing, the lipase and ester oils meet and hydrolysis reaction can easily cause rancidity. Thus, the researches of rice lipase have been mainly focused on destroy the enzyme passivation, while (pays) little attention has been paid to the the development and utilization of it.Chosen Rice Lipase (RL) gene as the main research object, cloned the RL gene and expressed it (expressing) in genetically engineered bacteria, we compared the expression of RL gene in the two expression system of E. coli BL21 and Pichia pastoris with each other, and reached on the enzymatic properties of the recombinant lipase, which including the effects of temperature, pH, metal ions and substrates on lipase activity.The main conclusions are as follows:(1) Pichia pastoris expression system is a more suitable microbial expression system for the expression of RL gene.(2)RL has a good thermal stability and pH stability. The optimum temperature of RL is 35～45℃ and can maintain over 80% relative enzyme activity after incubated 3 hours at 55℃. The optimum pH is 8.0, and can keep more than 80% of the enzyme activity in the range pH of 7.0 to 10.0.(3) By Pichia pastoris high density fermentation, an abundant of yeast cells with RL enzyme activity can be obtained, and they can be reused five times still remain more than 80% of the activity and without any purification and immobilization treatment.(4)Exhibits a better hydrolytic activity to short chain fatty acid (C4-C8).(5)Metal ions and surfactants can improve the RL activity.Through this study provides a theoretical basis for future applications.