Key Laboratory of Marine Drugs, Ministry of Education; Shandong Provincial Key Laboratory of Glycoscience and Glycoengineering; School of Medicine and Pharmacy, Ocean University of China, Qingdao, 266003, China; Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China.
Key Laboratory of Marine Drugs, Ministry of Education; Shandong Provincial Key Laboratory of Glycoscience and Glycoengineering; School of Medicine and Pharmacy, Ocean University of China, Qingdao, 266003, China; Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China.
Protein Expr Purif. 2021 Jun;182:105840. doi: 10.1016/j.pep.2021.105840. Epub 2021 Feb 7.
Hyaluronate lyases have received extensive attention due to their applications in medical science, drug and biochemical engineering. However, few thermotolerant and pH-stable hyaluronate lyases have been found. In this study, hyaluronate lyase TcHly8B from Thermasporomyces composti DSM22891 was expressed in Escherichia coli BL21(DE3), purified, and characterized. Phylogenetic analysis revealed that TcHly8B belonged to a new subfamily in PL8. The molecular mass of recombinant TcHly8B determined by SDS-PAGE was approximately 86 kDa. The optimal temperature of TcHly8B was 70 °C, which was higher than that of previously reported hyaluronate lyases. TcHly8B was very stable at temperatures from 0 to 60 °C. The optimal pH of TcHly8B was 6.6. It could retain more than 80% of its original enzyme activity after incubation for 12 h in the pH range of 3.0-10.6. TcHly8B degraded hyaluronic acid into unsaturated disaccharides as the end products. The amino acid sequence and structure analysis of TcHly8B demonstrated that the amino acid composition and salt bridges might contribute to the thermostability of TcHly8B. Overall, this study provides an excellent example for the discovery of thermotolerant hyaluronate lyases and can be applied to the industrialized production and basic research of hyaluronate oligosaccharides.
透明质酸裂解酶因其在医学、药物和生化工程中的应用而受到广泛关注。然而，很少发现耐热和 pH 稳定的透明质酸裂解酶。本研究在嗜热真菌 Thermasporomyces composti DSM22891 中表达了透明质酸裂解酶 TcHly8B，并对其进行了表达、纯化和特性分析。系统发育分析表明 TcHly8B 属于 PL8 家族的一个新亚家族。SDS-PAGE 测定的重组 TcHly8B 的分子质量约为 86 kDa。TcHly8B 的最适温度为 70°C，高于先前报道的透明质酸裂解酶。TcHly8B 在 0-60°C 温度范围内非常稳定。TcHly8B 的最适 pH 为 6.6。在 pH 3.0-10.6 范围内孵育 12 h 后，仍保留 80%以上的原始酶活。TcHly8B 将透明质酸降解为不饱和二糖作为终产物。对 TcHly8B 的氨基酸序列和结构分析表明，氨基酸组成和盐桥可能有助于 TcHly8B 的耐热性。总之，本研究为耐热透明质酸裂解酶的发现提供了一个极好的范例，并可应用于透明质酸寡糖的工业化生产和基础研究。
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