| Lipase was widely used in industries such as food, medicine, oil processing and dailychemistry, while its large-scale application was limited by low activity and stability. In thispaper, immobilization and additive were chosen as the means to improve the activity andstability of lipase from porcine pancreas (PPL). Also, the stability and enzymatic reactiondifferences of immobilized and free PPL under ultra high pressure were compared. The mainresults including:Silica gel and sepharose functionalized with different chemical groups and two kinds ofion exchange resin were used as support materials, and PPL was immobilized on them viaphysical adsorption, covalent attachment and ion exchange. The best results were obtained bycovalently immobilizing PPL onto silica gel functionalized with amino-group using EDC. Atthe optimum conditions (molar ratio of EDC to protein was50:1, weight ratio of support toPPL was10:1, pH was6.0and immobilization time was8h), the maximum activity (86.67U·g-1) of immobilized PPL was obtained.The immobilization of PPL could be well assisted by polyvinylalcohol (PVA). Thisfunction was greatly depended on PVA concentration, PVA adding strategy and types ofimmobilization support. The highest activity140.29U·g-1support of immobilized PPL wasobtained by4%PVA treatment during immobilization, which was61.87%higher thanimmobilized PPL without treatment. At this condition, the specific activity of immobilizedPPL10.15U·mg-1protein, which was, respectively,1.27-fold of untreated free PPL and2.36-fold of the untreated immobilized PPL. Moreover, circular dichroism spectra and Fouriertransform infrared spectroscopy were used to inspect the structure changes of free andimmobilized PPL. Higher α-helix content changes were found for immobilized and free PPLafter PVA treatment. The mechanisms of PPL activated by PVA were deduced that on the onehand, PVA increased the hydrophobic interaction between PPL and environment, on the otherhand, PVAdisplayed protection for PPL during immobilization.Immobilized PPL was analyzed by scanning electron microscopy and Fourier transforminfrared spectroscopy. Filiform proteins were observed on the surface of enzyme-immobilizedsupport and the absorption peak at1543cm-1(amideⅡ) were found in the spectra ofimmobilized PPL. Also, the properties of immobilized PPL were studied. The optimum pH ofthe immobilized PPL was shifted from7.0to8.0, and the optimum hydrolysis andesterification temperature were determined to be5and10℃higher than that of free PPL,respectively. Meanwhile, the thermal and storage stability of immobilized PPL weresignificantly improved, and65.67%hydrolytic activity was still reserved after beingrepeatedly used for6times.The effect of high pressure on stability and esterification rate of immobilized and freePPL was studied. At40℃, the stability and esterification rate decreased with the increase ofpressure. However, pressure displayed well protection on PPL when temperature increasedfrom50℃to70℃. Especially, PPL appeared the best stability at60℃and250MPa, and theresidual activity of immobilized and free PPL was97.37%and104.58%after15mintreatment, respectively. After being treated for60min, the residual activity of free PPL was 13.26-fold compared with that of under0.1MPa, and the activity of the free and immobilizedPPL could be recovered22.26%and22.41%after48h at4℃, respectively. Furthermore, theesterification rate of free and immobilized PPL under60℃and250MPa was greatlyimproved, which was1.63-and2.46-fold of free PPL at0.1MPa, respectively. |
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