载酶海藻酸钙复合微球稳定水包油型Pickering乳液及其强化界面酶催化反应

doi:10.11949/0438-1157.20190901

摘要/Abstract

摘要：

以疏水改性二氧化钛修饰的载酶海藻酸钙微球（E@Alg@s-TiO₂微球）稳定水包油（O/W）型Pickering乳液用于两相界面酶催化反应。与传统的两相游离酶体系相比，此体系以绿色、温和的方式将酶固定在乳液界面上，并强化了两相界面酶催化反应。研究成果归纳如下：油水比为1∶1.2时，Pickering乳液为O/W型；E@Alg@s-TiO₂微球浓度为3%（质量）时稳定效果最好，脂肪酶的负载量为15.8 mg·g^-1。以三丁酸甘油酯的水解反应为研究对象，该体系对油水体系的界面酶催化反应有很好的强化效果，具有96%转化率并提高酶活力7.8倍。重复使用5个批次能保留80%的酶活力。本研究进一步拓展了载酶海藻酸盐微球稳定的Pickering乳液体系的应用范围，有望为O/W体系的界面生物催化过程提供绿色平台。

关键词: 生物催化, Pickering乳液, 过程强化, 海藻酸钙, 酶, 固定化

Abstract:

Hydrophobically-modified titanium dioxide-modified calcium alginate microspheres (E@Alg@s-TiO₂ microspheres) stabilized oil-in-water (O/W) type Pickering emulsion is used for two-phase interfacial enzyme catalysis. Compared with the traditional two-phase free enzyme system, the system immobilizes the enzyme at the emulsion interface in a green and gentle manner, and strengthens the two-phase interface enzyme catalyzed reaction. The research results are summarized as follows: when the oil-water ratio is 1∶1.2, the O/W emulsion is obtained; the concentration of the microspheres is 3%(mass). The O/W system has a good enhancement effect on the biphasic enzyme-catalyzed hydrolysis reaction, with 96% conversion rate and 7.8 times higher enzyme activity than the free enzyme in conventional two-phase system, 5 batches cycles with 80% of residue enzyme activity. This study further expands the application range of the enzyme-loaded alginate microsphere-stabled Pickering emulsion system, which is expected to provide a green platform for the interfacial biocatalysis process.

Key words: biocatalysis, Pickering emulsion, process intensification, calcium alginate, enzyme, immobilization

中图分类号:

TQ 032.4

王伟浩, 杨鑫, 李飞, 孙梦梦, 王垚磊, 孟涛. 载酶海藻酸钙复合微球稳定水包油型Pickering乳液及其强化界面酶催化反应[J]. 化工学报, 2019, 70(12): 4777-4786.

Weihao WANG, Xin YANG, Fei LI, Mengmeng SUN, Yaolei WANG, Tao MENG. E@Alg@s-TiO₂ microsphere stabilized O/W Pickering emulsion and the enhancement of interfacial enzymatic catalysis[J]. CIESC Journal, 2019, 70(12): 4777-4786.

图/表 9

图1 E@Alg@s-TiO2微球稳定O/W型Pickering乳液体系及界面酶催化的机理

Fig.1 Mechanism of O/W Pickering emulsion stabilized by E@Alg@s-TiO2 microsphere and interfacial enzyme catalysis

图2 E@Alg[(a)~(c)]和E@Alg@C6-TiO2微球[(d)~(f)]的SEM照片及EDS元素分析

Fig.2 SEM photographs and EDS elemental analysis of E@Alg[(a)—(c)] and E@Alg@C6-TiO2 microspheres[(d)—(f)]

图3 E@Alg@C6-TiO2微球光学显微镜照片（a）和荧光显微镜照片（b），E@Alg@C6-TiO2微球（2%（质量））稳定的水中液体石蜡的Pickering乳液光学显微镜照片（c），（c）在不同波长下的荧光显微镜照片，绿色荧光是由FITC激发（d），蓝色荧光是由C6-TiO2激发（e），E@Alg@C6-TiO2微球稳定的Pickering乳液产生的荧光机理示意图（f）（油水比1∶1.2，乳液制备1 h后观察）

Fig.3 Photomicrographs of E@Alg@C6-TiO2 microspheres (a) and fluorescence micrographs (b), optical microscopy of paraffin-in-water Pickering emulsion stabilized by E@Alg@C6-TiO2 microspheres (2%(mass)) (c), fluorescence micrographs at different wavelengths, green fluorescence is excited by FITC (d), blue fluorescence is excited by C6-TiO2 (e), and schematic diagram of Pickering emulsion stabilized by E@Alg@C6-TiO2 microspheres (f)(oil-water ratio 1∶1.2, observed after 1 h of emulsion preparation)

图4 液体石蜡-水体系中E@Alg@C6-TiO2（a），E@Alg@C8-TiO2（b）和E@Alg@C10-TiO2（c）的三相接触角

Fig.4 Three-phase contact angle of E@Alg@C6-TiO2(a), E@Alg@C8-TiO2(b) and E@Alg@C10-TiO2(c) in paraffin oil-water system

图5 E@Alg@C6-TiO2微球在不同油水比下对液体石蜡/水Pickering乳液稳定情况（微球浓度3%（质量），乳液制备1 h后观察）

Fig.5 E@Alg@C6-TiO2 microspheres stabilized paraffin oil-water Pickering emulsion with different oil-water ratios(concentration of microspheres is 3%(mass), and emulsion is observed after 1 h)

图6 E@Alg@6C-TiO2浓度为1%(质量)（a），2%(质量)（b），3%(质量)（c）时的液体石蜡/水Pickering乳液的液滴光学显微镜照片及其对应的液滴粒径分布（d）（油水比1∶1.2，乳液制备1 h后观察）

Fig.6 Droplet optical micrograph of paraffin oil/water Pickering emulsion with E@Alg@6C-TiO2 concentration of 1%(mass) (a), 2%(mass) (b), 3%(mass) (c) and its corresponding droplets diameter distribution (d)(oil-water ratio 1∶1.2, observed after 1 h preparation of emulsion)

图7 不同底物浓度对反应速率（a）和转化率（b）的影响（所有测量重复3次）

Fig.7 Effect of different substrate concentrations on v (a) and conversion (b)（all measurements were repeated 3 times）

图8 不同体系中的酯水解反应转化率随时间变化关系（所有测量重复3次）

Fig.8 Conversion of ester hydrolysis reaction in different systems and time(all measurements repeated 3 times)

图9 E@Alg@s-TiO2微球稳定的O/W型Pickering乳液在7次反应循环里脂肪酶的相对酶活力比较（每次10 h）

Fig.9 Comparison of relative enzyme activities of lipase in E@Alg@s-TiO2 microsphere-stabilized O/W Pickering emulsion in 7 reaction cycles (10 h per cycle)

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