Hyaluronate lyases (HA lyases) have been proved to distribute widely among microorganisms, with large potential in hyaluronan processing. Here, a highly active HA lyase HylC from Citrobacter freundii strain Cf1 is reported. HylC was expressed in Escherichia coli BL21(DE3) under the regulation of T7 promoter, and purified to electrophoretic homogeneity for enzymatic characterization, which suggested its suitable thermo- and pH stability under 45 °C and pH rang of 4-8, and high halotolerancy in 1.5 M NaCl. The enzyme exhibited the optimal activity under 37 °C and pH 5.5, and was activated by Ca2+, K+, Zn2+, Ni2+ and Li+. Analysis of degradation product proved it cleave HA in endolytic manner, releasing unsaturated disaccharides as final product. Then, through optimization of promoter and construction of dual promoter, expression level of HylC improved from 1.10 × 104 U/mL to 2.64 × 104 U/mL on shake-flask level. Finally, through batch fermentation, a highest activity of 2.65×105 U/mL was achieved in a 5-L fermenter. Taken together, this work demonstrates the potential of HylC and its recombinant strain in industrial applications. To our knowledge, the HA lyase production reported in this study was the highest level in literatures to date.

中文翻译：

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在透明质酸寡糖制备中具有显着潜力的高活性透明质酸裂解酶 HylC 的高水平表达和表征。

透明质酸裂解酶 (HA lyases) 已被证明在微生物中广泛分布，在透明质酸加工中具有巨大潜力。在这里，报道了一种来自弗氏柠檬酸杆菌菌株 Cf1 的高活性 HA 裂解酶 HylC。HylC 在 T7 启动子的调控下在大肠杆菌 BL21(DE3) 中表达，并纯化至电泳同质性以进行酶学表征，这表明其在 45 °C 和 4-8 的 pH 范围内具有合适的热稳定性和 pH 稳定性，并且具有高耐盐性在 1.5 M NaCl 中。该酶在 37 °C 和 pH 5.5 下表现出最佳活性，并被 Ca2+、K+、Zn2+、Ni2+ 和 Li+ 激活。降解产物分析证明它以内切方式裂解 HA，释放不饱和二糖作为最终产物。然后，通过启动子的优化和双重启动子的构建，在摇瓶水平上，HylC 的表达水平从 1.10 × 104 U/mL 提高到 2.64 × 104 U/mL。最后，通过分批发酵，在5L发酵罐中达到了2.65×105U/mL的最高活性。总之，这项工作展示了 HylC 及其重组菌株在工业应用中的潜力。据我们所知，本研究报告的 HA 裂解酶产量是迄今为止文献中的最高水平。