Relationship Between Activity And Conformation Of Lipase From Pseudomonas Cepacia In Nonaqueous Media

Compared to chemical catalysts, lipases have many advantages, such as mild reactionconditions, higher catalytic efficiency, low energy consumption and environmentalfriendly etc., but high cost, deactivation and low stability still block their wideapplications in industry. To solve these problems, many scholars have attempted toimprove the activity and stability of lipase by solvent engineering and protein engineeringetc, thus reducing the cost of catalytic reaction. Pseudomonas cepacia lipase （PCL） waswidely applied to catalyze organic reactions, but the mechanism of PCL activity varianceswas not totally clear in nonaqueous media. The activity and conformation of PCL wasstudied in5organic solvents,19ionic liquids and compressed CO₂to screen out suitablereaction solvent for PCL and explain the internal mechanism of PCL activity variances innonaqueous media; it could provide theory basis and technical support for the applicationof PCL. The main contents and results are as follows:1. Effect of organic solvents on the activity and conformation of PCL. The reactioncatalyzed by PCL was studied in the system of organic solvent at40oC for12h; GC wasused to analyze biodiesel yield. Results demonstrated that biodiesel yield decreased withthe increase of organic solvent logP, higher biodiesel yield of PCL was obtained inhydrophilic organic solvent, such as tert-amyl alcohol and tert-butanol, while lower yieldwas got in hydrophobic organic solvent （petroleum ether, n-hexane and isooctane）. It maybe attributed to the fact that the substrate and product could form a homogeneous systemin hydrophilic organic solvent, thus the contact probability between the substrates andenzyme was increased, and the toxic effect of methanol and glycerol on PCL was reduced.FT-IR spectra analysis showed that the secondary structure of PCL was affected byorganic solvents, and the increase of activity may be due to the decrease of α-helix content,which led to the active site open and easier to contact with the substrates. Fluorescencespectrum analysis indicated that the tertiary structure of PCL has also been changed byorganic solvents; the fluorescence intensity of lipase was increased with the increase ofactivity. 2. Effect of ionic liquids on the activity and conformation of PCL. The activity ofPCL was investigated in ionic liquids with different cation and anion, the substrates ofsoybean oil and methanol were reacted at40oC for12h, and the biodiesel yield wasanalyzed by GC. Results indicated that the effect of anion on PCL activity is much largerthan the cation, ionic liquids with hydrophobic anions [PF6]-and [Tf2N]-are moresuitable as reaction solvents, and the highest biodiesel yield,82.3%was obtained in ionicliquid [Ompy][BF4]; ionic liquids with strong hydrophilicity and nucleophilicityexhibited low biodiesel yields, it may be due to the fact that the hydrophilic ionic liquidcould affect lipase’s essential water in the microenvironment, and the nucleophilic ionicliquids easily adsorbed around the active site of lipase, thus preventing the contact ofcatalytic center and substrates. FT-IR spectra analysis showed that the secondarystructure of PCL has been changed in ionic liquids, the decrease of α-helix content wasrelated to the increase of PCL activity, it may be attributed to the fact that the active siteof lipase was exposed and substrates were easier to access.3. Effect of compressed CO₂on the catalytic properties of liquid PCL. The effect ofhigh-pressure CO₂on the activity, conformation and stability of PCL was investigated; thehydrolysis activity of PCL was measured using olive oil as substrate. Results showed thatpressure and temperature could significantly improve the hydrolysis activity of PCL, itmay be due to the interactions between CO₂and lipase under high pressure conditions, ledto structural changes. The reaction kinetics demonstrated that high pressure treatmentimproved the maximum reaction rate of PCL and reduced the activation energy of thereaction, but reduced the affinity of PCL on substrate and catalytic efficiency, the thermalstability and organic solvent tolerance of PCL also significantly decreased. SDS-PAGEshowed that high pressure treatment did not make protein aggregation or fracture occur，indicating the primary structure of protein was not changed. Circular dichroism andfluorescence spectra analysis indicated that variances of PCL activity were mainly due tothe changes of the secondary structure in6MPa/35oC; while the variances of PCL activitywere related to changes of the secondary and tertiary structure in10MPa/40oC. Theexposure time has little effect on the tertiary structure of PCL during subcritical andsupercritical conditions. 4. Effect of compressed CO₂on the catalytic properties of solid PCL. The centralcomposite design was taken to study the effect of pressure, temperature and exposure timeon the activity, conformation and stability of solid PCL. Experimental results showed thatsub-and supercritical CO₂treatment could improve the activity of PCL, and the highestwas125.9%and130.4%, respectively, the effect of supercritical treatment was better thansubcritical treatment, but the pressure, temperature and exposure time all have nosignificant influence on lipase activity. FT-IR and fluorescence spectra analysis indicatedthat the activity of PCL decreased with the increase of α-helix content under subcriticalcondition, while the variances of solid PCL activity were related to changes of thesecondary and tertiary structure during supercritical CO₂, the fluorescence intensitygradually reduced as the pressure increased. Lipase’s stability analysis showed that thetolerance of PCL towards hydrophilic organic solvent was significantly decreased, thethermal stability of PCL after supercritical treatment was also significantly decreased,which was confirmed by DSC.