Co-expression of lipase isozymes for enhanced expression in Pichia pastoris.

To enhance the overall expression level of lipase isozymes which catalyse the same reaction in Pichia pastoris through co-expression of isozymes from different sources; several types of co-expression ways were constructed to determine the co-expression efficiencies of lipase isozymes in P. pastoris. The results showed that the Kex2-mediated co-expression of lipase isozymes could express Rhizomucor miehei lipase (RML) and Thermomyces lanuginosus lipase (TLL) simultaneously, and GS-RMk-kTL displayed an average lipase activity of 306·91 U ml-1 , higher than GS-RML and GS-kTL (2·89 and 300·59 U ml-1 ) expressed independently in P. pastoris, and the sum of both (303·48 U ml-1 ), implying the potential of isozyme co-expression mediated by Kex2 in increasing the overall recombinant expression, but the low recombinant expression of RML in P. pastoris weakened the overall increasing effect on lipase expression in the isozyme co-expression strains. In addition, the fusion isozymes were successfully expressed, but with low lipase activities. Furthermore, 2A peptide could successfully mediate the co-expression and secretion of lipase isozymes, but it seriously affected the expression of TLL downstream of 2A peptide. SIGNIFICANCE AND IMPACT OF THE STUDY: The low production level is one of the limitation factors for decreasing the prices of enzymes and expanding their application in industry as the biocatalysts. This research focuses on developing lipase isozyme co-expression strategies in Pichia pastoris to enhance the expression level of overall lipase isozymes which catalyse the same reaction. The Kex2-mediated co-expression strategy of lipase isozymes could potentially enhance the overall isozyme expression, and isozyme co-expression might provide a new direction for improving the recombinant isozyme expression, and decreasing the production and application prices of these mixed enzymes as biocatalysts.

Economic Method for Extraction/Purification of a Burkholderia cepacia Lipase with Potential Biotechnology Application.

In order to recover biomolecules, a single downstream processing step is carried out. In this sense, an aqueous two-phase system (ATPS) containing polyethylene glycol (PEG) and potassium phosphate salts is used. Intending the purification of Burkholderia cepacia (Bc) lipase, the effects of the molecular masses of 1500 (PEG 1500), 4000 (PEG 4000), and 6000 (PEG 6000), pH (6, 7, and 8) and distinct tie line lengths are perfomed. Although this is reasonable reported in literature, a study covering an economical production aspect considering the Bc is scarce. This characterizes a novelty proposed in this investigation. Lipase is recovered in a polymer phase at lower pH value. PEG 1500/phosphate salt ATPS at pH 6 is considered a good method with ~ 98% of the extraction efficiency. Another contribution of this proposed investigation concerns to a biotechnological material synthesis, which is applied in several advanced and revolutionize engineering practices. Additionally, an economic analysis of the proposed method indicates a minimal sale price (~ US$410/L) inducing to a future and potential commercial application.

Studies on the kinetics of Isopropyl Palmitate synthesis in packed bed bioreactor using immobilized lipase.

The objective of this research was to study the kinetics of synthesis of a commercially important ester - Isopropyl Palmitate (IPP) using immobilized lipase (Lipozyme IM). It was studied in a packed bed differential reactor. In order to establish the kinetics of the reaction, parameters such as linear velocity of the fluid through the reactor, particle size, substrate concentration, substrate molar ratio, temperature and water activity were studied. Operational and storage stability of the enzyme were also assessed. The reaction followed Michaelis-Menton kinetics as observed from the relationship of initial rate of the reaction as a function of substrate concentration. It was found that the optimum substrate concentration was 0.15M palmitic acid and isopropyl alcohol in 1:1 stoichiometric ratio. Inhibition by excess of isopropyl alcohol has been identified. The optimum temperature for the esterification reaction was found to be around 50 degrees C. The activation energy of this process was determined to be 43.67 kJ/mol. The optimum water content was 0.50%. The reaction rates were measured in the absence of any significant external diffusional limitations. Since internal diffusional limitations could not be eliminated, the kinetics observed is only apparent.