College of Light Industry and Food Engineering, Guangxi University, Daxue East Road No. 100, Nanning, 530004, People's Republic of China.
Key Laboratory of Carbohydrate Chemistry and Biotechnology, School of Life Sciences and Health Engineering, Ministry of Education, Jiangnan University, Lihu Avenue No. 1800, Wuxi, 214122, People's Republic of China.
Appl Biochem Biotechnol. 2024 Oct;196(10):6782-6801. doi: 10.1007/s12010-024-04883-w. Epub 2024 Feb 27.
Hyaluronate lyase (HA lyase) has potential in the industrial processing of hyaluronan. In this study, HylP, an HA lyase from Streptococcus pyogenes phage (SPB) was successfully expressed in Bacillus subtilis. To improve the extracellular enzyme activity of HylP in B. subtilis, signal peptide engineering systematic optimization was carried out, and cultured it from shake flasks and fermenters, followed by purification, characterization, and analysis of degradation products. The results showed that the replacement of the signal peptide increased the extracellular enzyme activity of HylP from 1.0 × 10 U/mL to 1.86 × 10 U/mL in the shake flask assay, and using a 20 L fermenter in a batch fermentation process, the extracellular enzyme activity achieved the level of 1.07 × 10 U/mL. HylP exhibited significant thermal and pH stability in the temperature range of 40 °C and pH range of 4-8, respectively. The enzyme showed optimum activity at 40 °C and pH 6, with significant activity in the presence of Na, Mg, and Co ions. Degradation analysis showed that HylP efficiently degraded hyaluronan as an endonuclease, releasing unsaturated disaccharides. These comprehensive findings underscore the substantial industrial potential of HylP for hyaluronan processing applications, offering valuable insights into enzyme characterization and optimization of expression for potential industrial utilization.
透明质酸裂解酶（HA 裂解酶）在透明质酸的工业加工中有很大的潜力。在这项研究中，来源于化脓性链球菌噬菌体（SPB）的 HylP 成功在枯草芽孢杆菌中表达。为了提高 HylP 在枯草芽孢杆菌中的胞外酶活性，我们对其信号肽进行了系统优化，并在摇瓶和发酵罐中进行培养，然后进行了纯化、表征和降解产物分析。结果表明，信号肽的替换使 HylP 的胞外酶活性在摇瓶试验中从 1.0×10 U/mL 增加到 1.86×10 U/mL，并且在 20 L 发酵罐的分批发酵过程中，胞外酶活性达到了 1.07×10 U/mL。HylP 在 40°C 的温度范围内和 pH4-8 的范围内具有显著的热稳定性和 pH 稳定性。该酶在 40°C 和 pH6 下表现出最佳活性，在 Na+、Mg2+和 Co2+离子存在下具有显著的活性。降解分析表明，HylP 作为内切酶有效地降解透明质酸，释放不饱和二糖。这些综合发现突显了 HylP 在透明质酸加工应用方面的巨大工业潜力，为酶的特性分析和潜在工业应用的表达优化提供了有价值的见解。
