纳米氧化锌的生物法合成及固定脂肪酶的研究

doi:10.11949/0438-1157.20191375

摘要/Abstract

摘要：

采用植物乳杆菌合成了纳米氧化锌粒子并成功用于脂肪酶的固定化。将筛选得到的高耐硫酸锌的植物乳杆菌株LP4用于纳米氧化锌的合成，采用扫描电镜、透射电镜和X射线衍射等一系列分析测试手段对得到的产物进行了表征。结果显示合成的材料为直径9~35 nm球形颗粒，在359 nm处有最大吸收峰，晶体呈六方体纤锌矿结构，X射线衍射峰与标准纳米氧化锌对比结果一致，这些结果表明植物乳杆菌株LP4成功合成了纳米氧化锌。然后将合成得到的纳米氧化锌粒子用于固定假丝酵母（Candida sp.）脂肪酶，与普通氧化锌以及传统法合成的纳米氧化锌粒子相比，生物法合成的纳米氧化锌固定效果最好，固定化酶的酶活收率分别比普通氧化锌和传统纳米氧化锌提高114.2%和20.5%。论文还对该生物纳米固定化酶的pH和热稳定性以及重复使用性能进行了测定，结果表明酶固定化后稳定性明显提高，而且具有较好的重复使用性能。

关键词: 纳米粒子, 脂肪酶, 固定化, 生物合成, 氧化锌

Abstract:

Zinc oxide nanoparticles (ZnO-NPs) were synthesized by a strain of L. plantarum and used as carriers for the immobilization of Candida sp. lipase. Firstly, a strain of L. plantarum with high tolerance to zinc sulfate was screened and used to synthesize ZnO-NPs. The synthesized particles were characterized by SEM, TEM and XRD and UV-vis spectroscopy tests. The ZnO-NPs are found to be almost spherical in shape, with a radius of 9—35 nm. XRD analysis indicated the biosynthesized particles had a hexagonal wurtzite structure and the corresponding X-ray diffraction peaks were consistent with the standard ZnO-NPs. UV-vis spectroscopy revealed that the biosynthesized ZnO-NPs displayed a clear absorption peak at 359 nm. These obtained results proved that ZnO-NPs were successfully synthesized. Furthermore, the biosynthesized ZnO-NPs, native ZnO and traditional ZnO-NPs were used as carriers for the immobilization of Candida sp. lipase, respectively. The results showed that the biosynthesized ZnO-NPs were the most suitable for the lipase immobilization, and the recovered activity was 114.2% and 20.5% higher than that of the native ZnO and traditional ZnO-NPs, respectively. The pH and thermal stabilities of the lipase immobilized on the biosynthesized ZnO-NPs showed a considerable increase in stability compared to the free lipase. Moreover, the immobilized lipase showed good reusability.

Key words: nanoparticles, lipase, immobilization, biosynthesis, ZnO

中图分类号:

TQ 645.6

尹春华, 彭思雨, 马垒珍, 张海洋, 闫海. 纳米氧化锌的生物法合成及固定脂肪酶的研究[J]. 化工学报, 2020, 71(5): 2248-2255.

Chunhua YIN, Siyu PENG, Leizhen MA, Haiyang ZHANG, Hai YAN. Biosynthesis of ZnO nanoparticles and their application in lipase immobilization[J]. CIESC Journal, 2020, 71(5): 2248-2255.

图/表 8

表1 植物乳杆菌在不同ZnSO4浓度MRS培养基中的生长情况(OD600)

Table 1 Growth of L. plantarum in MRS medium of different ZnSO4 concentration (maximum OD600)

ZnSO₄ /(mmol/L)	LP2	LP3	LP1	LP4
10	5.70±0.21	6.82±0.29	7.23±0.20	7.41±0.30
20	5.61±0.25	6.31±0.27	6.41±0.31	7.52±0.31
30	—	—	4.62±0.22	7.33±0.29
40	—	—	3.74±0.16	7.32±0.34
50	—	—	—	6.13±0.28
60	—	—	—	4.03±0.19
70	—	—	—	—

图1 植物乳杆菌LP4细胞（a）、植物乳杆菌合成的粗ZnO-NPs（b）和煅烧后ZnO-NPs（c）扫描电镜图

Fig.1 SEM micrograph of L. plantarum cells (a), crude ZnO-NPs synthesized by L. plantarum (b), and ZnO-NPs after calcination (c)

图2 植物乳杆菌合成ZnO-NPs（a）与煅烧后（b）透射电镜图

Fig.2 TEM micrograph of crude ZnO-NPs synthesized by L. plantarum (a) and ZnO-NPs after calcination (b)

图3 植物乳杆菌合成ZnO-NPs X射线衍射谱图

Fig.3 X-ray diffraction pattern of ZnO-NPs synthesized by L. plantarum

图4 植物乳杆菌合成ZnO-NPs的化学机理

Fig.4 Chemical mechanism of synthesis of ZnO-NPs by L. plantarum

图5 不同ZnO固定Candida sp.脂肪酶的比较

Fig.5 Comparison of immobilized Candida sp. lipase on various types of ZnOA—native ZnO; B—traditional ZnO-NPs; C—ZnO-NPs synthesized by L. plantarum

图6 生物法ZnO-NPs固定化酶与游离酶的pH和热稳定性比较

Fig.6 Stabilities of free lipase and immobilized lipase prepared by biosynthesized ZnO-NPs(a) pH stability; (b) thermal stability

图7 生物ZnO-NPs固定化酶重复使用性能

Fig.7 Reusability of immobilized lipase on biosynthesized ZnO-NPs

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