Cloning,Expression And Characterization Of Lipase Gene From Rhizopus Delemar

Lipases (triacylglycerol ester hydrolases EC 3.1.1.3) are well known hydrolases, which can hydrolyze the ester bonds of water in soluble substrates at the interface between substrate and water. Furthermore, contrary to many other enzymes, they show remarkable levels of activity and stability in non-aqueous solvents, which facilitates the catalysis of several unnatural reactions such as esterification and transesterification. Because of these unique properties, lipases are the most attractive enzymes for use in organic chemical processes.Lipase from fungus Rhizopus sp. has a strong potential in a wide range of reactions relevant to industry. In aqueous systems, it has a high specificity as it hydrolyzes the ester bonds at the sn-1 and sn-3, but not the sn-2 position of triacylglycerides and is therefore, referred to as 1,3-specific lipase. This property makes this lipase useful for producing structured lipids. For large-scale applications, sufficient amounts of active enzymes are required. However, the production of this lipase by the fungus is low, and the isolation of the enzyme is difficult. To overcome these problems, the lipase gene has been cloned and transferred into E. coli and Pichia pastoris. The recombinant lipase shows a high degree of positional selectivity for the primary ester bonds of triglycerides in both hydrolytic and synthetic reaction. It shows activity in aqueous and organic solvents and can be used for both the hydrolysis and restructuring of fats and oils and the generation of flavor and essence compounds.In this paper, the lipase gene from Rhizopus delemar 5564 is cloned and expressed Escherichia coli and Pichia pastoris respectively. The major work is listed as follows:(1) By search and alignment of the homologous Rhizopus sp. lipases, the primers were designed based on the high homologous nucleotide sequences. Then we extracted the total RNA of Rhizopus delemar 5564. With the oligo (dT) 15 primer, we synthesised the first strand of cDNA by reverse transcription. After that we succeed cloning the lipase gene preproRDL.(2) BY using preproRDL lipase gene as a template, mature RDL was amplified by PCR method. The amplified fragments were inserted into pET-28b vector according to the manufacturer's instruction. After investigated the transformed strains of inducible expressing conditions, we found the target protein at expected position by SDS PAGE. Although the majority of the target protein was expressed in the form of inclusion bodies, but there also had the soluble protein in the supernatant. Followed by the optimization of soluble expression conditions, included the choice of host bacteria, IPTG concentration in the determination of the optimal temperature-induced, cell concentration of OD600 and so on., the final conditions of inducible expression were determined. The recombinant plasmid was transformed into Rosetta (DE3) for protein expression. The transformed strains were grown in LB medium at 37℃until the OD600 reached 1.2. Protein expression was induced by adding IPTG to a final concentration of 0.2 mM, and the culture was shaken for 4 h at 30℃, then the cells were harvested by centrifugation.After ultrasonic disruption, we used the Ni-NTA affinity chromatography to purify the supernatant, the purification of fold was 68.58, and the recovery rate was 5.98%. The purpose of protein purification is up to electrophoresis grade.The results of the characterization of recombinant RDL showed that the optimal temperature of the recombinant lipase was 35℃,so this lipase had a better low temperature hydrolysis capacity; And the optimum pH value was 8.8; in different acyl chain length of p-nitrophenol ester hydrolysis study, the highest hydrolytic activity of RDL was obtained to the substrate of pNPC12. The activity detection by using olive oil emulsion as substrate demonstrated that hydrolysis activity of RDL is 90.98 U/mg. The result indicated that RDL was a true lipase. Metal ions Na+ and K+ can increase the activity of RDL; Mn2+, Cu2+ had little impact on the enzyme activity; Zn2+,Al3+,Ca2+ had shown varying degrees of inhibition of the enzyme activity; while non-ionic suifactants Triton X-100, Tween 20 and Tween 80 inhibited the hydrolytic activity.(3) The expression vector pPIC9K-RDL(His6) was constructed and linearized by Bgl II, and then the linearized vector was transformed into methylotrophic yeast Pichia pastoris(GS115) by electroporation. We obtained a large number of recombinants by screening; the lipase activity was detected by using tributyrin plates. We optimized the expression conditions of the recombinant yeast: the induce temperature is 30℃,induction time is 12 hours, adding 0.5% methanol, as well as the BMMY's pH is 7.0 and the dissolved oxygen is more than 80%. After the induction, the enzyme activity is up to 305.6 mU/mg.