A Study On Catalytic Mechanism Of Lipase In Perhydrolysis And Its Application

Lipases are the most widely used class of enzymes in biotechnology.This work studied the catalytic propoties and applications of lipase in perhydrolysis reaction.Peracetic acids can introduce oxygen into olefins,cyclic ketones,and amines compounds,making them essential oxidants for organic synthesis.Lipase can catalyze perhydrolysis,in which the peroxycarboxylic acids were produced from carboxylic acids and hydrogen peroxide,then the peracid used as oxidant to epoxidation of alkenes or to Baeyer-Villiger(BV)oxidation of ketones.However,the application of lipases in industry is still limited due to their catalytic characteristic,e.g.lipase can catalyze the synthesis of ester from ketones in BV oxidation reaction,howerer,the hydrolysis of ester happened in the same time.To resolve such problems,a mono-and diacylglycerol lipase from Malassezia globosa(SMG1)and a lipase B from the yeast Candida antarctica(CalB)were selected to investigate the mechanism of perhydrolysisof lipase.Engeering lipase derivated from SMG1 and CalB with desired catalysis capability was also studied.Firstly,the wild type of SMG1 and CalB were prepared,then cloned,expressed and purified.The mutants of lipase SMG1 and CalB were based on the plasmids of pGAPZ?A-SMG1 and pGAPZ?A-CalB.The hydrolysis and esterification activities of SMG1 and CalB were determined,and these resuts further confirmed that SMG1 was a monoand diacylglycerol lipase which can‘t synthesize triacylglycerol,but mono-and diacylglycerol,while CalB can hydrolyze and synthesize triacylglycerol.To understand the substrate selectivity of SMG1 better,the hydrolysis ability of SMG1 on phosphatidylcholine and lysophosphatidylcholine was investigated.The results indicated that SMG1 lipase was preferred to DAGs like subsrates.Secondly,the epoxidation reaction of SMG1 and its mutants were studied.The results indicated that the Ser171 Ala mutant can catalyze epoxidation reaction,but His281 Ala can‘t.This indicated that histidineis essential for catalytic activity while Ser171 was not important in epoxidation.Therefore,we considered that His281 took part in the stabilization of hydrogen peroxide.Interestingly,the formation of acylenzyme intermediate is not probable as the Ser171 is not present in the active mutant.It is also unlikely that thetetrahedral intermediate is formed because the distance between carbonof the carboxyl group and oxygens of peroxide are too big for bond formation.The essential His281 side chain can form hydrogen bonds both to peroxide and carboxyl oxygen,therefore it mostlikely intermediates in the formation of the peroxide.Therefore,the epoxidation mechanism of SMG1 is His281 mediated and not relying on theacyleenzyme complex and tetrahedral intermediate formation.Besides,SMG1 catalyzes epoxidation reaction is a two-step process: SMG1 catalyzes formation of peroxidesfrom carboxylic acids that subsequently react with double bonds of alkenes to produce epoxides.Finally,to remove the side reaction of hydrolysis in BV reaction,lipase CalB-wt and Ser105 Ala mutant were used as research objects,and their effects on BV oxidation of ketones were evaluated.It was found that hydrolysis side reaction in BV oxidationwas effiectively inhibited when Ser105 Ala mutant was used as catalyst.In addition,the converstion of ketone was enhanced when deep eutectic solvent was used as reaction medium.Subsequently,it was found that deep eutectic solvent facilitated to increase the interface area and decrease the interface tension of oil/water.This could be the reason of the enhancement of converstion.In conclusion,the work studiedthe substates selectivity of SMG1 lipase and its catalytic mechanism in epoxidation reaction.Those informations not only pave a road to modify enzyme suitable for lipids modification,but also can improve the usage of lipase in epoxidation reaction.Besides,Ser105 Ala mutant of CalB can catalyze perhydrolysis reaction in BV oxidation simultaneously avoid the happenning of hydrolysis side reaction.This result expands the usage of lipase in BV oxidation.At last,this study firstly explains the reason of deep eutectic solven in enhancing the efficiency of reaction.