Studies On The Immobilization Of Lipase From Thermomyces Lanuginosus And Its Catalytic Performances

The lipase from Thermomyces laguginosus(TL) is a basophilic and noticeably thermostable enzyme, which is commercially available in both liquid and immobilized form. Free enzyme is unstable, and can’t be repeat used. Immobilized enzyme has received considerable attention due to their advantages on separation, reusability, and stability.In this study, enzyme was immobilized on various carrier materials by different methods. Firstly, lipase from Thermomyces laguginosus was immobilized on magnetic particles by covalence, on white carbon black and resins by adsorption and adsorption-crosslinking, respectively in one phase system. The activity of immobilized enzyme was employed for interesterification of soybean oil with fully hydrogenated soybean oil at70℃. The best carrier and method were found:WR-6resin, adsorption-crosslinking method. The results showed that the highest interesterification activity of fixed TL was abtained at the optimum immobilized:adsorption time1h, the ratio of enzyme and carrier(V/W)1.5:1,35℃, concentration of crosslinking agent(V/V)0.15%, crosslinking time3h, pH6.7. The TL immobilized under optimum conditions (WR-6-TL1) showed high stability towards temperature and storage time.Secondly, lipase from Thermomyces laguginosus was immobilized on WR-6resin by adsorption and adsorption-crosslinking, respectively in two phase system. The results showed that the biphase and surfactant could enhance the activity of immobilized lipase effectively. Reaction factors including the type, amounts of surfactants, and various solvents were investigated in detail. According to the results, Tween20showed the best effect, and the optimum amount of Tween20was100mg per gram carrier. The kind of organic solvents had little effect on the immobilization effects. And the TL immobilized in two phase system (WR-6-TL2) also showed high thermostability and storage stability.Thirdly, the prepared immobilized TL lipases was used to catalyze the synthesis of l,3-dioleoyl-2-palmitoyl glycerol (OPO) compared with the commercial products TL IM and RM IM. OPO product was achieved29.8%and28.4%catalyzed by WR-6-TL1and WR-6-TL2, separately. According to the analysis of FAC, Sn-2, and TAG, both WR-6-TL1and WR-6-TL2showed better stability, activity and Sn-1,3specificity than commercial RM IM and TL IM. Both these two immobilized enzyme can be repeated for more than nine times, which indicates the potential possible application for industrial scale.Finally free enzyme and immobilized enzyme were analyzed by Fourier transform infrared spectroscopy using Peakfit v4.12software. Conformational changes of secondary structure before and after being immbilized on carriers were analyzed and the possible mechanism was discussed.