Lipases Oriented-immobilized On Magnetic Nanoparticles And Its Application

Recently, magnetic nanoparticles exployed for enzyme immobilization has attracted more and more attention. The advantages of magnetic nanoparticles include higher specific surface, lower mass transfer resistance, facility of reusability related to the selective separation under a magnetic field and modification on the surface with various active groups, etc. However, conventional covalent immobilization of enzymes was usually associated with a significant activity decrease because the active site may be blocked from substrate accessibility, multiple point-binding, or the enzyme may be denatured due to the random cross-linking between proteins and supports, especially the covalent-bondes formed between the residues closed to the catalytic sites of enzyme and supports. In this article, we proposed to develop a suitable oriented immobilization technology based on the structural information of enzyme molecules obtained by bioinformatics analysis.Lipases from Yarrowia lipolytica LIP2(YLLIP2) and Burkholderia cepacia (BCL) were oriented immobilized on Fe3O4magnetic nanoparticles (MNPs) by different strategies. The derivatives were employed to enrich DHA from fish oil and to prepare biodiesel from soybean oil and ethanol. The main work and innovations were summarized as follows:1. MNPs were synthesized by co-precipitation in water phase, then, covered with (3-aminopropl) triethoxysilane (APTES). The APTES-MNPs (A-MNPs) were characterized by X-ray diflfractometer (XRD) and Fourier transform infrared spectroscopy (FT-IR). The XRD pattern suggests that MNPs is Fe3O4with high purity, and FT-IR spectrum indicates that the preparation of Fe3O4MNPs, functionlization were successful. The diameters of MNPs and A-MNPs were17.2nm and18.4nm, respectively.2. The contents of different active groups in YLLIP2and BCL molecules were estimated. Then, the three dimensional structrures and the dittibutions of free ε-NH2,-COOH,-OH and-SH in YLLIP2and BCL molecules were studied using Rasmol software. It shows that the free ε-NH2groups were regional distribution which tends to locate away from the catalytic sites, while the other active groups distributed evenly in the molecules of both lipases. Based on this result, two immobilized strategies were proposed respectively using free amino groups and carboxyl groups as covalent-bonded sites. Consequently, the relative specific activities of YLLIP2covalently bound to amino groups and carboxyl groups were respectively36.9%and17.3%,as well as128.2%and94.6%for BCL, These results verified the bioinformatic analysis.3. Consequently, YLLIP2and BCL were immobilized on glutaraldehyde-activated-MNPs (G-MNPs) covalent-bound to the free amino groups in aqueous phase. The immobilization efficiencies were over85%. The hydrolysis acivity recovery of the immobilized YLLIP2was42.4%under the optimized conditions. The esterification activity recovery of the oriented-immobilized BCL using oilive oil as activative and protective agent attained1081.9%.4. For the first time, a novel strategy to orientated-immobilized YLLIP2and BCL in reverse micelles system was proposed. The results showed that both YLLIP2and BCL were successfully oriented-immobilized on G-MNPs. The hydrolysis activity recovery of YLLIP2immobilized in Span20microemulsions with the molar ratio of H2O to surfactant Wq=1.12and Z=2.26ran up to1466.1%. The esterification activity recovery of BCL immobilized in AOT microemulsions with W0=10reached989.5%.5. The enzymatic properties of the oriented-immobilized lipases were further examined. For YLLIP2, after oriented immobilization, the optimum temperature rised from25℃to30℃, and the optimum pH value was8.0, similar to free YLLIP2. The thermal and pH stability were improved significantly. When incubated at40℃for1h, the the immobilized YLLIP2kept61.8%original activity, compared with34.3%of free YLLIP2. Accordingly, when incubated at pH7.5for1h, the immobilized YLLIP2kept81.5%original activity, compared with36.4%of free YLLIP2.For BCL, the optimum temperature of the immobilized rised from40℃to45℃, and the optimum pH value was7.0which was equal to the free BCL. The thermal stability improved significantly. The residual activity of the immobilized BCL was kept82.4%while28.9%of free BCL when incubated at65℃. The residual activity of the immobilized BCL kept81.5%while36.4%of free BCL at pH7.5. The pH stability of the immobilized BCL was still slightly improved though free BCL possessed strong pH stability. 6. The oriented-immobilized YLLIP2was employed to enrich DHA in fish oil. The hydrolysis degree of fish oil reached40%while that of the free lipase was only27.3%after reacting12h. Correspondingly, DHA contents of the modified glycerides attained36.8%and31.6%, which were1.9-fold and1.6-fold of the original DHA content. After20cycles of successive usage, it still remained over80%of relative hydrolysis degree, which shows a good recyclability.The immobilized BCL was used to catalyze transesterification reaction of soybean oil and ethanol to produce biodiesel. Biodiesel yield was84.9%in30%tert-butanol system with ethanol/oil molar ratio4:1at45℃. There was no loss in its activity after being recycled10times and the biodiesel yield always kept over80%, demonstrating a high operational stability.