Lipase Configuration Changes At The Oil-water Interface And Application Of Its Catalytic Mechanism

Lipase can catalyze the hydrolysis of fats and oils at the oil-water interface,and more complex catalytic reactions such as esterification,transesterification,lactide,and acid hydrolysis can occur in the micro-aqueous and non-aqueous phases.Therefore,lipase is considered to be one of the most important catalysts in the biological field at present,and has great commercial value in food,washing,medicine,feed and biodiesel.As an important industrial hydrolase,it has a wide range of substrate selectivity,but also has high regioselectivity and enantioselectivity.Unlike other hydrolases,lipases can only be activated at the oil-water interface.This is because the active center of a lipase is usually covered by an alpha-helix "cap" structure.This structure makes the catalytic reaction conditions more stringent.The opening of the "cap" structure is also affected by the environment in which the lipase is located,thus limiting the application of the lipase in many fields.Based on this,olive oil was selected as the oil substrate and hydrolyzed by Candida.sp.99-125 lipase.Taking the hydrolysis rate of olive oil as an index,the Box-Benhnken center combination and response surface method were used to analyze the influence factors of Candida.sp.99-125 lipase hydrolysis of olive oil.The results showed that at the oil-water interface,there were significant differences in the temperature of lipase hydrolysis of olive oil,the amount of lipase,the water-oil ratio,the water-oil contact rate at the interface and the p H value.The impact is as follows: D(p H)> B(Water oil ratio)> C(Water oil ratio)> A(Reaction temperature).Candida.sp.99-125 lipase hydrolyzed olive oil is most affected by p H and least affected by temperature.The effect of water-oil ratio on lipase hydrolysis is second only to p H,which indicates that water-oil ratio is an important factor affecting lipase hydrolysis at the oil-water interface.A suitable water-oil ratio provides the best activity for the lipase,which is beneficial for the hydrolysis reaction.Then,the conformational changes of Candida.sp.99-125 lipase in aqueous solution,n-hexane water and n-hexane solvent were studied by molecular dynamics simulation.The three-dimensional structure of Candida.sp.99-125 lipase at the n-hexane-water interface shows that one end of the "lid" structure of the lipase is in a solvent of n-hexane and one end is in an aqueous solution,and the entire "lid" structure is in a stretched state.It is possible that the opening of the lipase "lid" structure is related to the ratio of the hydrophobic amino acids of the "lid" structure.Molecular dynamics simulation of lipase in n-hexane-water solution shows that the secondary structure of Candida.sp.99-125 lipase in n-hexane-water solution requires the GLY87-SER90 ring to undergo a large conformational rearrangement to promote the placement of THR88 amide in the appropriate position to stabilize the fat Conformation of the enzyme tetrahedral intermediate.In the ?4 helix,the fluctuation of THR159-LEU175 is more obvious,especially the active center of SER162 lipase is more obvious,indicating that the ?2 helix and the ?4 helix interact.The activation mechanism of lipase at the oil-water interface may involve the conformational rearrangement of ?2 helix and ?4 helix,and is also accompanied by the interaction of GLY87-SER90 and MET101-HIS126.Finally,Candida.sp.99-125 lipase was applied to the detergent on the hard surface of the kitchen oil to get the best detergent formula: triethanolamine 1%,Propylene glycol butyl ether 2.5%,Candida.sp.99-125 lipase 2%,6501 2.5% and AOS 3%.