FAD为辅基的葡萄糖脱氢酶发酵、纯化及酶学性质

Enzyme Characterization，Fermentation and Purification for FAD-Conjugated Glucose Dehydrogenase

DOI：10.3969/j.issn.1673-1689.2020.02.014

中文关键词: FAD 葡萄糖脱氢酶 乳糖诱导 分离纯化 酶学性质

英文关键词: FAD,glucose dehydrogenase,lactose induction,purification,enzyme properties

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中文摘要:

细菌来源的FAD为辅基的葡萄糖脱氢酶（FAD-GDH）较为罕见，作者利用乳糖为诱导剂，发酵E. coli BL21（DE3）生产FAD-GDH。7.5 L发酵罐中培养该菌，通过分批补料，分阶段温度控制，酶活达到1 341 U/L，菌体量达 12.4 g/L。通过镍柱、脱盐柱、阴离子交换柱三步层析对粗酶液分离纯化，获得比酶活109 U/mg的重组酶。SDS-PAGE凝胶电泳显示，重组蛋白质纯化后呈单一条带，相对分子质量约60 000。等电聚焦电泳检测酶的等电点pI 为5.3。酶标仪全波长扫描吸收光谱表明，该酶的辅基为FAD，与酶非共价键结合。相比其它糖类，以葡萄糖为底物酶的Km最小，为2.56 mmol/L，k_cat/K_m为 3 487.7 L/（mmol·s）。该酶在pH 5.5～8.0内稳定，最适反应pH 7.0，当pH高于8.0时酶活迅速降低。差式扫描量热分析（DSC）测得酶的Tm值为77 ℃，热稳定性良好。本研究为该酶的进一步工业化生产应用提供参考与借鉴。

英文摘要:

Bacterial-derived FAD-conjugated glucose dehydrogenase is rare. Using lactose as an inducer，fed-batch fermentation ofE. coliBL21（DE3） was carried out to produce FAD-GDH in 7.5 L fermenter under stepped temperature control strategy. Enzyme activity and dry cell weight reached 1 341 U/L and 12.4 g/L respectively. By using Histrap HP，HiPre TM 26/10 Desalting and DEAE anion exchange chromatography，a purified enzyme was obtained with a specific enzyme activity of 109 U/mg.The molecular weight conformed to the theory about approximately 60 000 as determined by SDS-PAGE. The isoelectric point of the enzyme was 5.3. Absorption spectroscopy of full wavelength scanning by microplate reader showed that the enzyme was FAD conjugated. Compared with other substances，the Km of the enzyme was lowest of 2.56 mmol/L when glucose was used as substrate，andk_cat/K_mwas 3 487.7 L/（mmol·s）. The enzyme kept relatively stable when the pH ranged from 5.5 to 8.0，with optimal pH of 7.0. When the pH is higher than 8.0， the enzyme activity decreases rapidly. The Tm of the enzyme measured by differential scanning calorimetry（DSC） was 77 ℃ showing high thermal stability. This experiment provides reference for the further industrial production and application of the enzyme.

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