华西医学

华西医学

带 His 标签重组产朊假丝酵母尿酸酶的表达、纯化与部分酶学性质研究

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目的研究重组产朊假丝酵母尿酸酶的高效表达条件、分离纯化和部分酶学性质。方法选择在菌体对数生长末期加入诱导剂,通过摇瓶培养对异丙基-β-D-1-硫代半乳糖苷(isopropyl β-D-1-thiogalactopyranoside,IPTG)和乳糖诱导产酶的效果进行比较;利用 50 L 发酵罐研究诱导培养时间;通过 Ni-琼脂糖凝胶柱亲和层析、Sephacryl S-200 HR 层析纯化目的蛋白并研究其最适 pH 值和酸碱稳定性、最适温度及热稳定性。结果摇瓶培养结果显示乳糖诱导效果优于 IPTG。发酵罐培养工程菌,在对数生长末期添加乳糖诱导 7 h 后酶活力和生物量均达到最大值,分别为 164 U/g 菌体和 23 g 菌体/L 发酵液,即每升发酵液含酶活性 3 772 U。纯化的尿酸酶比活性为 4.5 U/mg,其最适 pH 值为 7.5,最适温度 40℃,酸碱稳定范围在 pH 值 6.0~10.0,温度低于 45℃ 时热稳定性较好。结论以乳糖作为诱导剂培养效果更佳;带有 His 标签的重组尿酸酶经 Ni-琼脂糖凝胶柱亲和层析和 Sephacryl S-200HR 分子层析后即可获得纯品,纯化流程更为简单;该纯化酶的最适反应温度、最适 pH 值、酸碱稳定性和热稳定性的确定为其结构与功能的关系以及未来的实际应用提供了一定的实验依据。

ObjectiveTo study the efficient expression conditions, purification, and partial enzymatic properties of His-tagged recombinant Candida utilis uricase.MethodsThe effects of isopropyl β-D-1-thiogalactopyranoside (IPTG) and lactose as inducers which were added in the end logarithmic phase were compared by the method of shake flask culture. The induction culturing time was studied with 50 L fermentor. The protein of interest was purified by Ni-Sepharose and Sephacryl S-200 HR chromatographies, and the optimal pH value, temperature, and thermal stability were also studied.ResultsThe shake flask culturing experiment results showed that IPTG was better than lactose as an inducer. In the fermentor culturing and at the end of the logarithmic growth stage, the enzyme activity of 164 U per gram bacteria and biomass of 23 grams per litter fermented solution were maximal after adding lactose for 7 hours as an inducer, i.e. the enzyme activity could be collected at 3 772 U per liter. The specific activity of purified uricase was 4.5 U/mg and the optimal pH value and temperature were 7.5 and 40℃. Additionally, the enzyme was stable at pH 6.0–10.0 and the thermal stability was below 45℃.ConclusionsIt is better to use lactose as an inducer in the process of culture. The recombinant uricase with His label can be purified by Ni-column affinity chromatography and Sephacryl S-200HR molecular sieve chromatography, and the purification process is easier than ever before. The optimum temperature, pH value, acid-base stability and thermal stability of the purified enzyme have been established to provide some experimental basis for the relationship between structure and function and practical application in the future.

关键词: 尿酸酶; His 标签; 产朊假丝酵母; 诱导发酵

Key words: Uricase; His-tag; Candida utilis; Inducing fermentation

引用本文: 王颖, 肖雪, 李丹, 钟春燕, 范翔, 李刚锐, 孟尧. 带 His 标签重组产朊假丝酵母尿酸酶的表达、纯化与部分酶学性质研究. 华西医学, 2018, 33(8): 989-993. doi: 10.7507/1002-0179.201604005 复制

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