ZIF-8-戊二醛固定化细胞生产α-酮戊二酸

doi:10.16085/j.issn.1000-6613.2022-0385

摘要/Abstract

摘要：

为了提高表达L-谷氨酸氧化酶（LGOX）重组大肠杆菌的催化稳定性，本研究采用了沸石咪唑框架（ZIF-8）涂层和戊二醛（GA）交联的组合固定技术。确定了ZIF-8和GA的工艺参数以及固定化细胞的催化性能，并对固定化重组大肠杆菌催化L-谷氨酸生产α-酮戊二酸（α-KG）的稳定性展开了研究。当2-甲基咪唑、醋酸锌和戊二醛的添加量分别为240mmol/L、80mmol/L和50mmol/L时，固定化细胞（E.coli@ZIF-8-GA）的比活性和活性回收率分别达到33.4U/g和95.83%。结果表明，在重复使用10个批次后，E.coli@ZIF-8-GA转化生产α-酮戊二酸的产量仍能达到70.03g/L。与游离细胞相比，固定化细胞对温度和pH的耐受性均发生显著提高。

关键词: 固定化, 沸石咪唑框架, L-谷氨酸氧化酶, α-酮戊二酸, 稳定性

Abstract:

In order to improve the catalytic stability of recombinant Escherichia coli (E.coli) containing L-glutamate oxidase (LGOX), a combined immobilization technique of zeolite imidazole framework (ZIF-8) coating and glutaraldehyde (GA) cross-linking was used in this study. The process parameters of immobilized cells were determined and the stability of α-ketoglutarate (α-KG) produced by immobilized cells was studied. The specific activity and activity recovery of immobilized cells (E.coli@ZIF-8-GA) were reached 33.4U/g and 95.83%, respectively, when 2-methylimidazole, zinc acetate and glutaraldehyde were added at 240mmol/L, 80mmol/L, and 50mmol/L, respectively. The results showed that the yield of α-KG could still reached 70.03g/L after reusing immobilized cells for 10 batches, which meant the stability of free cells in the reaction was enhanced. Compared with free cells, the tolerance to temperature and pH of immobilized cells was significantly improved, which laid a solid foundation for the application of immobilized catalyst to producing α-KG.

Key words: immobilization, ZIF-8, L-glutamic acid oxidase, α-ketoglutaric acid, stability

中图分类号:

Q814

代静新, 宋伟, 陈修来, 刘立明, 吴静. ZIF-8-戊二醛固定化细胞生产α-酮戊二酸[J]. 化工进展, 2022, 41(12): 6522-6530.

DAI Jingxin, SONG Wei, CHEN Xiulai, LIU Liming, WU Jing. ZIF-8-glutaraldehyde-immobilized cells to produce α-ketoglutaric acid[J]. Chemical Industry and Engineering Progress, 2022, 41(12): 6522-6530.

图/表 11

图1 不同载体对细胞的比活力和活性回收率的影响

图2 固定化参数的优化

图3 固定化细胞PXRD图和X-射线能谱图

图4 固定化细胞的红外谱图

图5 游离细胞和固定化细胞的扫描电镜图谱

图6 固定化细胞和游离细胞的最适温度和耐热性

图7 固定化细胞和游离细胞的最适pH和pH耐受性

图8 固定化细胞和游离细胞的操作稳定性

图9 固定化细胞和游离细胞的储藏稳定性

图10 PASP的添加对固定化酶吸附率和活性的影响

图11 ZIF-8对LGOX的保护效果和固定化酶的操作稳定性

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