Lipase Immobilization Based On Its Interfacial Activation

Lipase, a kind of significant hydrolytic enzymes, most of which have a section of-helix named lid‘over their active site. The lid‘structure endows them with thespecial catalytic activity, interfacial activity, on the oil/water interface. With theproperty of interfacial activity, it has become an important method to tailor thechemical groups on the support surface in designing lipase immobilization technology.In this work, functionalized polystyrene were used as supports. And the catalyticactivity of immobilized lipase was studied. Then, the biosurfactants were mixed withtwo sources of lipases（from fungi lipase, CRL, Candida rugosa and from mammallipase, PPL, Porcine pancreatic）. To understand the effect of bile salts concentrationon lipase adsorption, the catalytic activity, surface tension and C-H vibration atair/water interface were investigated. The chiral resolution ability of free lipase andimmobilized lipase were studied by changing bile salts concentration and modifyingsupports with bile salts, respectively. Lastly, LB film technology was used asimmobilization method. At the same time, horseradish peroxidase （HRP） wasimmobilized with the LB film technology to control its orientation. In our work, amethod to design lipase immobilization technology was established by controlling thelipase interfacial activation property. The conclusions are as follows:1. A series polystyrene supports with different functional groups were obtained andthe contact angles of the supports were:-COOH（68.4°）＜-OH（76.3°）＜NH₂（80.1°）＜PS（87.7°）＜-CH₃（98.9°）＜-CF₃（99.6°）. The activity order of these immobilizedlipase activity retention were:-OH（92.78%）＞-NH₂（76.22%）＞-COOH（20.03%）＞-CH₃（2.64%）＞PS（1.91%）＞-CF₃（1.23%）. The immobilized lipase activity wascodetermined by the amount of adsorbed lipase, substrate or product affinity andlipase structure. The amount of adsorbed protein on these supports were:-CF₃（42.45mg/m²）>-CH₃（28.67mg/m²）> PS（20.32mg/m²）>-COOH（11.86mg/m²）>-NH₂（3.96mg/m²）>-OH（3.11mg/m²）。The hydrophobic supports hadhigher lipase affinity than the hydrophilic supports. Further, lipase structure was changed greatly on the hydrophobic surface shown by SFG data.2. The effect of bile salt on CRL and PPL were very similar. At the low NaDCconcentrations, lipase competed with NaDC adsorbing on the interface. The SFGspectra showed that at this concentration the strongest resonant signals of thelipase methylene and methyl as modes could be identified, indicating that parts oflipase adsorbed on the interface. At high NaDC concentration the SFG spectra wasquite similar with pure NaDC spectra （at2870and2945cm-1belongs to themethyl symmetric stretching and its Fermi resonance, respectively）, showing thatNaDC completely replaced lipase molecules from the air/solution interface. At thelow NaDC（＜2mM） concentrations, NaDC acted as an promoter of lipase activity,inducing the lipase lid to open and exposing lipase hydrophobic surface. Theminimum of surface tension were41.26mN/m for CRL,40.22mN/m for PPL.3. For the chiral resolution of1-phenylethanol, the hydrolysis activity was improvedat low bile salt（NaDC and NaC） concentration. The maximum activities could beobtained at between2mM and4mM for NaDC, and8mM for NaC, respectively.The activity was inhibited at higher bile salt concentration. The enantiomerexcess（eep） was also improved at low bile salt concentration. Especially for PPL,eepwas improved from60%to93%at4mM for NaDC and73%at2mM for NaC.4. A series of polyacrylonitrile（PAN） supports were used to lipase immobilization,the contact angles of the supports: PAN-COOH（37.9°）> PAN-NH₂（58.0°）>PAN-CA（79.8°）> PAN-DCA（82.5°）> PAN（88.7°）. Lipase had higher affinity onhydrophobic supports. For PPL, the order of the amount of adsorbed protein were:PAN（8.29mg/g）>PAN-NH₂（6.17mg/g）>PAN-DCA（4.83mg/g）>PAN-CA（4.60mg/g）>PAN-COOH（2.97mg/g）. For CRL, the order of the amount of adsorbedprotein were: PAN（3.12mg/g）> PAN-NH₂（2.54mg/g）> PAN-CA（2.19mg/g）>PAN-DCA（2.03mg/g）> PAN-COOH（1.94mg/g）. On hydrophilic PAN supports,the lid of lipase was closed with low specific activity and high selectivity, whileon hydrophobic PAN supports, the lid was open with high specific activity andlow selectivity.5. Lipase was ordered compactly and well organized through LB immobilization method. The activity of lipase was improved by nearly one order of magnitudecompared with adsorption immobilization. The active site of lipase was towards toair side when transferred on hydrophilic glass, remaining higher catalysis activitythan hydrophobic PS. With the method of LB film, HRP could be immobilized inorder and its activity was more than two times higher than that of adsorbed HRP.Furthermore, compared with adsorption immobilization, the advantages of LBmethod were that could keep high activity, high storage stability and structurestability. On the support with higher hydrophility, HRP showed lower activity.