Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China.
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China.
J Biotechnol. 2023 Mar 20;366:35-45. doi: 10.1016/j.jbiotec.2023.03.003. Epub 2023 Mar 15.
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 Ca, K, Zn, Ni 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 × 10 U/mL to 2.64 × 10 U/mL on shake-flask level. Finally, through batch fermentation, a highest activity of 2.65×10 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.
透明质酸裂解酶（HA 裂解酶）已被证明广泛分布于微生物中，在透明质酸处理方面具有巨大的潜力。本文报道了来自弗氏柠檬酸杆菌 Cf1 的一种高活性 HA 裂解酶 HylC。HylC 在大肠杆菌 BL21(DE3)中受 T7 启动子调控表达，并通过电泳纯化为酶学特征分析进行了纯化，这表明其在 45°C 和 pH4-8 的范围内具有良好的热稳定性和 pH 稳定性，并且在 1.5 M NaCl 中有很高的耐盐性。该酶在 37°C 和 pH5.5 下表现出最佳活性，并且被 Ca、K、Zn、Ni 和 Li 激活。降解产物分析表明，它以内切方式切割 HA，释放不饱和二糖作为最终产物。然后，通过启动子优化和双启动子构建，在摇瓶水平上，HylC 的表达水平从 1.10×10 U/mL 提高到 2.64×10 U/mL。最后，通过分批发酵，在 5L 发酵罐中实现了 2.65×10 U/mL 的最高酶活。总之，这项工作证明了 HylC 及其重组菌株在工业应用中的潜力。据我们所知，本研究中报道的 HA 裂解酶的产量是迄今为止文献中最高的水平。
