A New High-throughput Screening Method For Lipase And The Immobilization And Application Of Rhizomucor Miehei Lipase

Lipases (triacylglycerol ester hydrolases) form a widespread family of enzymes digesting fats and oils. Lipases are widely applied in the chemical synthesis, food, medical science, cleaning industry and dynamophore exploitation. Rhizomucor miehei Lipase (RML) is a1,3-position specific lipase, which is of special interest in the efficient and economical production of oils and additives based on triglycerides.First of all, we compared different methods to determine lipase activity and studied the activity properties of RML. The p-NPP assay is only sensitive to measure low levels of lipase activity. The cupric acetate assay and the alkali titration assay are sensitive to measure the lipase activity from low to level. The activity of RML is highest at the working conditions35-38℃and pH8-10. The activity of RML is activated by Ca2+, Mg2+, but not by Cu2+.A new and efficient method was developed for the first time using the targeted substrate, triacylglycerol, for the rapid screening of a large library of mutants for lipase directed evolution. The high-throughput method applies calcium chloride to convert the fatty acid produced from hydrolysis reaction to hydrochloric acid which is easy to release H+detected by pH indicator. This new method exhibits significant advantages compared with the conventional method of applying a substituent of the actual targeted substrate,p-nitrophenyl palmitate as a high-throughput screening substrate. The results determined by the new method correlate well with those obtained from alkali titration and HPLC analysis. By applying the new method, a significantly enhanced mutant enzyme was obtained with lipase activity increased more than five-fold compared to wild type after only two cycles of directed evolution.Magnetic nanoparticles (MNPs) have attracted increasing attention in biorelated research due to its biocompatibility, stability, large surface area, and super-paramagnetic properties. A method for covalently immobilized RML on polymer-modified magnetic Fe3O4nanoparticles by means of graft copolymerization of polyethylacrylate was established. This method includes an acyl azide activation. The procedure is particularly mild since the enzymes never come into contact with chemical reagents, thus avoiding all denaturing processes. A specific loading of250mg of RML/g of MNPs was achieved and the activity was100%of its free lipase activity. Even after10cycles, the nanobiocatalyst was still fully active. 1,3-Diacylglycerol (1,3-DAG), because of its multifunctional and nutritional properties, attracts considerable attention recently. In this article, the methods for enzymatic synthesis of DAG were studied. By esterification of fatty acid and glycerol,1,3-DAG was produced in an86%yield and in91%purity. By glycerolysis of olive oil,1,3-DAG was produced in82%yield and in41%purity.