为满足工业生物催化需求,寻求一种简单、快捷、有效的脂肪酶固定化方法来扩大其实际应用范围。采用声化学方法,在10 min内将脂肪酶固定于由磷酸铜沉淀所构成的无机杂化纳米花中,并进一步通过扫描电子显微镜 (scanning electron microscope,SEM)、X射线衍射(X-ray diffraction,XRD)和傅里叶红外光谱 (Fourier transform infrared spectroscopy,FT-IR)等技术对制备的脂肪酶杂化纳米花进行表征。随后,研究了不同金属离子、金属盐离子类型、合成条件对脂肪酶杂化纳米花活性的影响。结果表明,Cu2+可实现最佳固定化效果,且不同含铜无机盐类型对所形成的杂化纳米花活性存在影响。其中,使用CuSO4合成纳米花可实现100%的包封率,而CuCl2可使最终合成的纳米花相对酶活力达到145.7%。酶动力学研究结果表明,CuSO4和CuCl2纳米花的催化效率(kcat/Km)分别是游离酶的106%和134%。此外,该固定化方法可有效提高固定化酶的储藏稳定性,在室温下储藏30 d其酶活力仍能保持在80%以上。在重复使用性研究中发现,固定化酶在连续使用5次后仍具有40%以上的催化活性。该研究结果有助于提高脂肪酶在工业生产中的适用性,并为其在生物柴油合成、洗涤剂及高温生产环境等多方面领域的应用奠定基础。
Performed by enzyme, biocatalysis is considering as a green and sustainable technology that could eliminate the production of toxic and harmful by-products during the industrial manufacturing process. Lipase, which is believed as one of the most promising industrial enzymes that could catalysis the synthesis of lipid, interesterification, alcoholysis as well as acidolysis, has been characterized with advantages in catalytic activity, variety of substrates, regioselectivity and corresponding selectivity. However, limited by the inherent property of biomacromolecule, requirements including the high enzymatic activity, repeatability, storage stability and the feasibility of separation might not be meet easily in the industrial biocatalysis process when applying free enzyme without optimization. To fulfill the need in industrial application of bio-catalysis, it is desired to develop valid, simple as well as effective enzymatic immobilization method for improving the practical application potential of lipase. Recently, self-assembly organic-inorganic hybrid nanoflowers has been developed and applied as a novel enzymatic immobilization method. Via this technology, the limitations for the application of free enzymes could be overcome as the stability, repeatability and storage stability of the enzyme would be highly improved after immobilization. Nevertheless, it should be noted that such immobilizing process would normally involve an incubation period of three days at the temperature of 4 ℃. This relative slow preparation duration will bring considerable limitations for its further scale-up application, though both the enzymatic activity and stability could be significantly enhanced by immobilizing lipase in the form of nanoflowers. In order to break through the above restriction, sonochemistry was introduced in the current study to have the free lipase hybridized with metal phosphate precipitation within 10 min. In consideration of the potential effect of reaction conditions on the activity of the hybrid nanoflowers, parameters including the difference in mental ions (Cu2+, Co2+, Ca2+, Mn2+ and Ni2+), concentration of different mental ions (1, 2 and 3 mmol/L), types of copper salt (CuSO4, CuCl2 and Cu(NO3)2), the concentration of immobilized lipase (0.01, 0.02, 0.05, 0.1, 0.15 and 0.2 mg/mL), ultrasonic duration (1, 5, 10 and 15 min) as well as the pH (6, 7, 7.3,7.5 ,8 and 9) of phosphate buffer saline (PBS) were investigated herein as to identify the most optimal immobilization condition. The results indicated that Cu2+ showed the best immobilization efficiency and the enzyme activity of Cu-Lip would be affected by the type of copper salts. The encapsulation ratio was measured as 100% when using CuSO4 for immobilization, while the introduce of CuCl2 was found to improve the relative enzyme activity to 145.7%. The catalytic efficiency (kcat/Km) of CuSO4-Lip and CuCl2-Lip was measured as 106% and 134% referring to that of free lipase. The current phenomenon might be contributed by the promotion effect on carry capacity of the Cu-Lip introduced by SO42- and the improvement of enzymatic activity by the participation of Cl-. Dependent on the type of copper salts, difference in the resulting catalytic efficiency was also suggested to be related with the variation in the final structure of the prepared Cu-LiP. In comparation of the relative activity of the immobilized enzyme between preparation conditions, immobilizing lipase by ultrasonic for 5 min using metal ions with the concentration of 3 mmol/L and lipase concentration of 0.05 mg/mL in PBS adjusted with pH of 7 was suggested as the optimal reaction condition. Compared with free enzyme, the optimal reaction temperature of both CuSO4-Lip and CuCl2-Lip was increased to some extent, whilst no significant difference in the optimal pH was observed herein. Scanning electron microscope (SEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) analysis were performed for the characterization of Cu-Lip hybrid nanoflowers. All the above analysis results further revealed that the lipase had been immobilized in Cu3(PO4)2·3H2O successfully with the observation of typical morphological features, reasonable crystallinity and the characteristic of PO43- peak of the hybrid nanoflowers, respectively. In addition, the current immobilization strategy was verified with the potential to improve the storage stability by maintaining 80% of relative activity after a storage of 30 days at room temperature. Moreover, the hybrid nanoflowers was found to exhibit 40% of relative activity after five consecutive reactions. Overall, with providing promising method for the improvement of the applicability of lipase in industry, the current result also lays the foundation for its further application in the field of biodiesel synthesis, detergent and industrial production requiring high temperature.
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