Study On The Immobilization Of Lipase And Its Catalytic Performances

Lipase is one of the important biocatalysts and widely used in the synthesis and resolution of high optically pure chrial compounds, which is mainly based on their high efficiency, stereoselectivity, mild reaction condition and non-polluting, etc. However, free enzyme is difficult to be widely used in industry because of its insufficient stability, easy deactivation and hard recovery and re-use. Under these conditions, the immobilization of lipase can availably overcome these applied limits, and it has been a hotspot in the field of enzyme engineering. Preparation of perfect immobilization enzyme demand both effective immobilized technology and ideal supports,therefore, the development and application of immobilized lipase possess the great theoretical significance and applied value.The paper investigated the immobilization of Pseudomonas sp. lipase (PSL) with the carrier-bound and carrier-free methods, and several immobilized lipases were obtained and used for the catalytic resolution of two important chiral intermediates. The catalytic and operated performances of immobilized lipases were further studied in the two different reaction systems. 1. Mesoporous molecular sieves immobilized lipasePseudomonas sp. lipase (PSL) was immobilized on the mesoporous molecular sieves with ordered pores and SBA-15 was selected as the optimal support through screening. In the investigation of surface modification of SBA-15, psR-SBA-15(with different functional groups) and SBA-15(X) (with the different amounts of template) were synthesized. Compared with SBA-15, the activity retention of PSL immobilized on PsEtNH2PrNH2-SBA-15 was improved 4 times and the activity retention of PSL was improved 6 times when SBA-15 contained 48% template as carrier. So, the SBA-15(48) was selected as the optimum support for PSL immobilization. The immobilized efficiency and activity retention of SBA-15(48)-PSL were respectively 69.2% and 59.9% under the optimized operating conditions including the ratio of lipase and support (1:15), the phosphate buffer (pH 8.0, 25mmol/L), temperature (4℃) and immobilized time (2h). In the characterization of immobilized PSL and analysis of the course of immobilization, it was also founded that PSL molecules entered into the pore channels of SBA-15 by a monolayer adsorption.2. Lipase immobilized on marcoporous resinsIn order to further improve the protein loading and activity of immobilized PSL, the organic resins with higher pore size and surface area were selected as the supports. HP20 was first selected as the immobilized material by the determining the activity and enzyme loading of various resin beads immobilized lipase. Under the optimized conditions, a fine immobilized lipase—HP20-PSL was prepared. The immobilized efficiency and activity retention of HP20-PSL were respectively 91.8% and 80.5%, and the optimum preparing conditions included the methods (physical adsorption), the ratio of PSL and HP20 (1:5), temperature (25℃) and immobilized time (3h). It was found that PSL adsorbed on HP-20 corresponded to a monolayer immobilization and the immobilized course could be described by the pseudo-second order kinetic model.In addition, PSL was immobilized on the EC-EP with different modified degrees. The results showed that the quick immobilization and higher enzyme activity could be performed when 20% of epoxy groups were modified to amido (–NH2) on the surface of EC-EP. It was also found that the process of PSL immobilized on EC-EP contained the physical adsorption and covalent attachment, and the rapid adsorption and covalent attachment could be realized when EC-EP with 20% modified degree as the support for PSL immobilization.3. Preparation of cross-linked enzyme aggregatesLipase was immobilized by the method of cross-linked enzyme aggregates (CLEAs). During the screening three types of precipitants (salts, polymers and organic solvents), acetone and ammonium sulfate were selected as the optimal precipitators, and Ac-CLEA (acetone as precipitator) and SA-CLEA ((NH4)2SO4 as precipitator) were obtained. For Ac-CLEA, the optimized preparing conditions were PSL (30mg/ml), acetone (5ml) and glutaraldehyde (31.3mM). For SA-CLEA, the optimized preparing conditions included PSL (50mg/ml), (NH4)2SO4 (66.7%), glutaraldehyde (31.3mM), and the activity of SA-CLEA could be improved 1.3 times when 20% SDS was added in the course of immobilization. The activity, immobilized efficiency and recovered activity of resultant Ac-CLEA and SA- CLEA were 534U/g, 70.6%, 45.1% and 475U/g, 70.4%, 48.3%, respectively. In the analyzing the FT-IR spectra of free and immobilized PSL, it was found the second structure of Ac-CLEA had been obviously changed compared with free PSL, but that of SA-CLEA was similar to free PSL. It was also indicated the cross-linking between PSL and glutaraldehyde with the forms of semi-acetal and semi-acetal oligomers were mainly existed by the further investigation of the cross-linked mechanism.4. The enzymatic characteristics of immobilized lipasesThe enzymatic characteristics of obtained immobilized lipases using the above methods were investigated. The results showed the optimal temperature of PSL was increased 15-25℃and the optimal pH-value of PSL was retained in 8.0 without obvious changes, and that the steady ranges to temperature and pH became broad after immobilized. The effects of metal ions on the activity of immobilized PSL was similar to those of free PSL, but the enabled and inhibited influences of metal ions on the immobilized PSL were more inconspicuous than those of free PSL. The protein leaching from immobilized lipases in aqueous solution was lower and the durability of immobilized PSL in organic solvents was significantly improved, compared with those of free PSL. It was also found that the immobilized methods and carriers might affect the stability of immobilized enzyme, generally, the stability of PSL bounded on the supports by the covalent attachment was higher than that of PSL adsorbed on the material, and the effects of outside environment on the carrier-bound immobilized enzyme was lower than that of carrier-free enzyme. 5. Immobilized PSL-catalyzed resolution of N-(2-ethyl-6-methylphenyl) alanine (NEMPA)Enantiomerically pure (S)-NEMPA (e.e.p>99%) could be obtained by the lipase-catalyzed hydrolysis of (R,S)-N-(2-ethyl-6-methylphenyl) alanine methyl ester ((R,S)-NEMPA-ME). In the kinetic resolution of (R,S)-NEMPA-ME, immobilized lipases not only kept excellent enantioselectivity (E-value > 100) as the free PSL, but also showed higher catalytic activity. Comparing with other immobilized PSL, Ac-CLEA-PSL and EC-EP-PSL displayed the better advantages and 50% conversion was achieved at the shorter reaction time,so they were selected for the further investigation. The results showed that the changes of reaction microenvironment didn't alter the high enantioselectivity of immobilized PSL and the susceptivity of PSL to the environment was decreased after immobilization. It was found that the catalytic performances of PSL were evidently meliorated during immobilization. The maximal reaction rate (Vmax) of Ac-CLEA-PSL was 2.0 times higher than free PSL, the catalytic kinetic constant (Kcat) of PSL was increased 2.9 times by immobilized on EC-EP and the affinity toward the substrate of PSL was enhanced during the immobilization. Two immobilized PSL exhibited excellent operating stability, and Ac-CLEA-PSL and EC-EP-PSL respectively retained 81% and 76% of initial activity after ten recycles while free PSL was disused after the first batch. Furthermore, the adding of organic solvents and surfactants could also affect the catalytic activity of immobilized PSL. The activity of Ac-CLEA-PSL and EC-EP-PSL were improved 1.1 times after adding the 5% of acetone and 15% diethyl ester to the respective reaction media, and Ac-CLEA-PSL and EC-EP-PSL could maintain the high stability in acetone and diethyl ester. When the adding of CTAB (30mg/ml), the elevated activity of free and immobilized PSL were observed but the stability of immobilized PSL in CTAB was lower than that of free PSL. It was suggested that the activation of CTAB towards immobilized PSL was not sensitive comparing with free lipase.6. Immobilized PSL–catalyzed resolution of racemic 2-octanolThe immobilized lipases were also applied in the kinetic resolution of (R,S)-2-octanol in organic media, and HP20-PSL, EC-EP-PSL and SBA-15-PSL exhibited higher catalytic efficiency in the transesterification. The reaction parameters (the amount of enzyme, the molar ratio of substrate, temperature, the water content and solvent) of three immobilized lipases-catalyzed resolution of (R,S)-2-octanol were further investigated. The reaction rate of PSL was remarkably improved after immobilized, and the HP20-PSL displayed the highest reaction rate and its initiative velocity (V0) is 21-folds of free PSL. Under the optimized conditions, high optically pure (S)-2-octanol (99.0% e.e.s) was recovered at 53%, 53.4% and 54.5% conversions with HP20-PSL, SBA-15-PSL and EC-EP-PSL, and the E-value of three immobilized PSL were orderly 80, 71 and 54. However, homochiral (S)-2-octanol could be obtained when conversion greater than 60% using free PSL and the E-value was only 24. The stability of three immobilized PSL in the reaction system were also superior than free PSL, and the activity and enantioselectivity of immobilized PSL were retained without changes after 5 recycles while free PSL lost entire activity after the 4th batch.