Immobilization Of SMG1-F278N Lipase And Its Application In Synthesis Of Propylene Glycol Monooleate

Propylene glycol monoesters(PGME)are good water-in-oil emulsifiers.They are widely applied in the field of food,pharmaceutical and cosmetics as addictives.After immobilization,the properties of lipase were improved such as reusability,thermal and operational stability.In the past,in-depth and comprehensive researches on the enzymatic synthesis of unsaturated monoacylglycerols have been carried out,but very few studies on enzymatic synthesis of unsaturated PGME and the purity of PGME was low in a sovent-free system which caused low-quality pruducts by enzymatic synthesis.Till date,there was no report on the synthesis of PGME using mono-and diacylglycerol lipases.Hence,this research focuses on the screening of mutants of mono-and diacylglycerol lipase of SMG1 and study of immobilization of SMG1-F278 N lipase which possessed highest esterification activity,characterization of immobilized lipase and its application in the synthesis of propylene glycol monooleate.It will provide more information for the synthesis of propylene glycol monooleate.The results are as follows:1.Immobilization of SMG1-F278 N lipase.It was found that the mutant lipase SMG1-F278 N possessed the highest esterification activity which was three fold to wild type SMG1.The best conditions for covalent bonding were: optimal support of ECR8285 epoxy resin,buffer initial pH of 6.0,buffer ionic strength of 1.5M,lipase/support ratio of 20mg/g and immobilization time of 2h.At the moment,the protein loading,esterification activity and specific activity of immobilized SMG1-F278 N were 44.62mg/g,466.96U/g and 10.47U/mg,respectively.2.Characterization of immobilized SMG1-F278 N.The results of SEM and FT-IR indicated that SMG1-F278 N was successfully immobilized onto ECR8285 resin.Meanwhile,compared with free lipase,the immobilized SMG1-F278 N preserved the enzyme activity in a wider pH and temperature ranges and the thermal stability was much better.Immobilized SMG1-F278 N had good reusability which the activity retention was 98% after 7 consecutive cycles(the first esterification reaction catalyzed by immobilized SMG1-F278 N was set as fist cycle).3.Application of immobilized SMG1-F278 N in the synthesis of propylene glycol monooleate.It was found that immobilized SMG1-F278 N preferred 1,3-propylene glycol over 1,2-propylene glycol.A molecular docking trial was carried out to help comprehend the selectivity of the substrate.The results showed that 1,3-propylene glycol interact with Ser171 of active center of lipase SMG1-F278 N and had additional hydrogen bond with Asn278.Nevertheless,the distance between 1,2-propylene glycol and Ser171 was far which was not favorable for enzymatic catalysis.The optimum reaction conditions were: 1,3-propylene glycol /oleic acid ratio of 5:1,reaction temperature of 30 oC,enzyme loading of 7.5% and water addition of 7%,and it was found that the content and purity of 1,3-propylene glycol monooleate was 70% and 84.67% respectively after 12 h.