Production Of Highly Pure Diacylglycerol By Lipase SMG1

Lipase SMG1from Malassezia globosa is a novel mono-and di-acylglycerol lipase which hasbeen studied recently. On the basis of the specific substrate selectivity of mono-and di-acylglycerol lipases, lipase SMG1shows potential in oil modification, production of partialglycerides, etc. In this project, we aim to evaluate the catalytic properties of lipase SMG1and itsapplication in fats and oils. The specific research activities include the production of lipaseSMG1in a10-L fermentor and the evaluation of its catalytic properties, lipase SMG1catalyzedesterification reaction of oleic acid and glycerol and its reaction kinetics, the immobilization oflipase SMG1and its application in catalyzing the esterification reaction and the purification ofDAG in the esterification products by molecular distillation and the characteristics of highly pureDAG products. The results are as follows:1. Lipase SMG1was expressed in recombinant Pichia pastoris and its large-scale productionwas conducted in a10-L fermentor. The hydrolytic activity of the prepared crude lipase SMG1was6100U/g. Lipase SMG1shows positive activity towards DAGs and MAGs but negativeactivity to TAGs. The hydrolysis of plant oil, which catalyzed by the combination of lipaseSMG1and Palatase20000L (a Sn-1,3specific lipase) was performed and the hydrolysis wasenhanced by combining with lipase SMG1.2. Lipase SMG1was employed to catalyze the esterification of oleic acid and glycerol toproduce DAG. The optimized results were with an enzyme loading of120U/g (U/w, with respectto total reaction mixture mass), a oleic acid/glycerol molar ratio of1:4, a reaction temperature of30oC, a water content of1%(w/w, with respect to total reaction mixture mass) and a stirring rateof200rpm. After24h of reaction under the above conditions, the DAG content reached to51.25%and no TAG was detected. A kinetic model based on esterification and isomerization wasestablished to explain reactions in lipase SMG1-catalyzed esterification. The kinetic model is, tosome extent, effective for describing the esterification by lipase SMG1catalysis.3. Immobilization of Lipase SMG1and its application in catalyzing the esterification of oleicacid and glycerol were investigated. The immobilized lipase SMG1, with an esterification activityof22.91U/g was used to catalyze the esterification, and high esterification degree of68.37%was obtained under the conditions of enzyme at a concentration of7.5%(w/w, with respect to totalreactants), a oleic acid/glycerol molar ratio of1:7, and40oC. The immobilized lipase retained49.7%of its initial activity after use for six reaction times. A two-stage molecular distillation was adoptedto separate and purify the DAG from esterification products, and the DAG content was up to96.20%(1,3-DAG,63.07%;1,2-DAG,33.13%) in the final products.In this study, we explored the large production and immobilization of lipase SMG1, andevaluated the kinetics study of lipase SMG1-catalyzed esterification reaction. On this basis, theenzymatic process for the production of highly pure diacylglycerol was established. With thedevelopment of the research and the improvement of the process and instrument, this processcould be performed eventually.