Research Of Immobilized Lipase Chromatographic Column

Thanks to its unique advantages of efficiency,mild condition and environment friendly as a natural catalyst,enzyme plays an important role in biological,chemical and pharmaceutical industries.While having a wide range of sources,lipase is capable of catalyzing esterification,hydrolysis and trans-esterification with chemo-selectivity,region-selectivity,enantiomer-selectivity both in aqueous and non-aqueous phase.However,the sensitivity and high cost of the free enzyme limit the use of enzymes.The development of immobilized enzyme technology has overcome the majority of free enzyme's drawbacks.After being immobilized,enzymes gain improvements on stability and reusability.As a result,immobilized enzyme could be used conveniently at lower cost.Reactive chromatography combines reaction and separation onto one unit,which could simplify the chemical process and reduce the cost of production.Moreover,reactive chromatography could improve the conversion of reversible reaction by breaking the chemical equilibrium limits.Reactive chromatography and enzyme immobilization technologies could be combined through using immobilized enzyme column as immobilized enzyme reactor.The high-performance liquid chromatography with automated online determination can make the data more easily to get,which reduce the complexity of the process.In this paper,hybrid carriers composed of silica and cellulose were consumed for lipase immobilization through physical adsorption and cross-linking respectively.The resulted immobilized lipases were packed into a chromatographic column which could be used for the online hydrolysis reaction through HPLC system.Firstly,we choose hydrolysis of nitrophenyl palmitate(nPP)in solvent which consist of hexane,isopropanol and water.When the water content in the reaction solvent is more than 17%,lipase showed higher catalytic activity Immobilized lipase possessed good thermal stability within a range from 15 ? to 45 ?.Ethyl cellulose coated silica was the best carrier for immobilized lipase chromatographic column(ILCC).The resulted ILCC was a kind of reactive chromatography.The ILCC was stable at a temperature range from 18 ? to 50 ?with a long half-life time of 160 h.Amino silica and amino cellulose was prepared for cross linking immobilized lipase.The immobilized lipase possessed good operation stability and good tolerance on nitrophenol(nP).The immobilized lipase could recover from inhibition or activation.The packed immobilized lipase column was hydrophilic,which adsorbed nP stronger than nPP.The immobilized lipase column could hydrolysis nPP at a conversion of 50% while the water content is 10%,which is better than free lipase.To get a high conversion,the water content of mobile phase should be 20.7%.Increase of hexane content could inhibit the catalytic activity of the immobilized lipase column.Moreover,the immobilized lipase chromatographic column showed little attenuation of catalytic activity after being used for 160 h.