Zhao Dongying, Pu Zhongji, Su Qiao, Zhang Yue, Sun Wenhui, Bao Yongming
School of Bioengineering, Dalian University of Technology, Dalian 116024, China; Ningbo institute, Dalian University of Technology, Ningbo 315016, China.
Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou 310058, China.
Enzyme Microb Technol. 2022 Jan;153:109957. doi: 10.1016/j.enzmictec.2021.109957. Epub 2021 Nov 25.
κ-Carrageenan oligosaccharides from κ-carrageenan hydrolysis are important biochemicals with more bioactivity. Enzyme engineering plays a key role in improving κ-carrageenase catalytic efficiency for production of κ-carrageenan oligosaccharides. Effect of metal ions on enzyme activity, especially stability and efficiency, is main factor in catalytic process, but metal ions addition leads to gelation of κ-carrageenan solution. In this study, molecular dynamics simulation was used to explore the interaction between κ-carrageenase CgkPZ and Ca, and Ca bonded to D164 and E167 in the catalytic center resulting in the catalytic efficiency increase. Circular dichroism analysis indicated that the secondary structure of κ-carrageenase could change in the presence of Ca. Therefore, a novel self-assembly κ-carrageenase-inorganic hybrid nanoflowers CaNF@CgkPZ was synthesized and systematically characterized. The catalytic efficiency (k/K) of CaNF@CgkPZ was 382.1 mL·mg·s, increased by 292% compared with free κ-carrageenase. Notably, the enzyme activity of CaNF@CgkPZ was not reduced significantly after 19 cycles use, and 70-100% relative activity was still retained when stored at 4-25 ℃ for 15 days. This work provides an efficient approach for κ-carrageenase immobilization with good storage stability, reusability and enhanced catalytic efficiency, which is of great significance in practical applications.
κ-卡拉胶水解产生的κ-卡拉胶寡糖是具有多种生物活性的重要生物化学物质。酶工程在提高κ-卡拉胶酶催化生产κ-卡拉胶寡糖的效率方面起着关键作用。金属离子对酶活性的影响，尤其是对稳定性和效率的影响，是催化过程中的主要因素，但添加金属离子会导致κ-卡拉胶溶液凝胶化。在本研究中，采用分子动力学模拟来探索κ-卡拉胶酶CgkPZ与Ca之间的相互作用，Ca与催化中心的D164和E167结合，导致催化效率提高。圆二色性分析表明，在Ca存在下κ-卡拉胶酶的二级结构会发生变化。因此，合成了一种新型的自组装κ-卡拉胶酶-无机杂化纳米花CaNF@CgkPZ，并对其进行了系统表征。CaNF@CgkPZ的催化效率（k/K）为382.1 mL·mg·s，与游离κ-卡拉胶酶相比提高了292%。值得注意的是，CaNF@CgkPZ在使用19个循环后酶活性没有显著降低，在4-25℃下储存15天后仍保留70-100%的相对活性。这项工作为κ-卡拉胶酶固定化提供了一种高效方法，具有良好的储存稳定性、可重复使用性和提高的催化效率，在实际应用中具有重要意义。
