溶藻弧菌噬菌体裂解酶lysV208的重组表达和溶菌活性

Recombinant expression and activity analysis of the lyase lysV208 from a phage of Vibrio alginolyticus

摘要:

目的 挖掘并开发高活性、高稳定性且可异源重组表达的噬菌体源裂解酶，确定其最优作用条件。方法 采用浊度法验证重组表达裂解酶的溶菌活性及其最适作用条件。结果 对溶藻弧菌噬菌体phiV208全基因组进行注释和蛋白预测，结果显示ORF30编码的蛋白具备裂解酶功能，将其命名为lysV208。lysV208可在大肠杆菌BL21(DE3)中可溶性高效表达，经0.25 mmol/L IPTG诱导16 h后，纯化浓度达204 μg/mL。与0.5 mmol/L EDTA协同作用时，其溶菌活性(浊度降低率)可从24.2%大幅提高至68.0%。酶学特征研究表明，lysV208的最适作用温度为45 ℃ (溶菌活性75.6%)，在水生动物乃至陆生动物常见的细菌性疾病流行温度范围(25-37 ℃)内，也具有较高溶菌活性(52.8%-71.9%)。lysV208的最适作用pH为7.0，在弱碱性条件(pH 7.0-9.0)下可保持较高的溶菌活性(44.0%-63.2%)，显示出对海淡水养殖环境的适应性。此外，二价金属离子(Zn²⁺、Mg²⁺、Mn²⁺、Fe²⁺)在0.1-1.0 mmol/L浓度范围内对lysV208的溶菌活性有一定促进作用，但高浓度(10.0 mmol/L)离子会极显著抑制酶活性(P<0.01)。裂解谱检测表明，重组酶lysV208不仅能高效裂解源噬菌体的宿主菌V208，还对非宿主菌株表现出广谱溶菌能力，对溶藻弧菌V039、创伤弧菌H1、副溶血弧菌GH32、哈维氏弧菌TY13和G1菌株的溶菌活性分别达到了59.7%、68.9%、65.8%、38.0%和65.6%。结论 重组裂解酶lysV208具有显著且稳定的体外溶菌活性，其裂解谱比源噬菌体phiV208更广。该酶在病原菌感染的生物防控领域，以及噬菌体-裂解酶协同制剂的开发中具有良好的应用前景。

Abstract:

Objective To identify and develop a phage-derived lyase that can be heterologously expressed with high activity and stability and determine its optimal working conditions.Methods We employed the turbidity reduction assay to evaluate the bacteriolytic activity and identify the optimal parameters.Results Genome annotation and protein prediction of the Vibrio alginolyticus phage phiV208 showed that ORF30 encoded a lyase, named lysV208. This enzyme demonstrated soluble expression in Escherichia coli BL21(DE3), reaching a purified concentration of 204 μg/mL after 16 h induction with 0.25 mmol/L IPTG. Its bacteriolytic activity (turbidity reduction rate) increased from 24.2% to 68.0% in the presence of 0.5 mmol/L EDTA. Enzymatic characterization revealed that lysV208 exhibited the maximum bacteriolytic activity (75.6%) at 45 ℃ while maintaining high activity (52.8%-71.9%) within the temperature range of 25-37 ℃, which is typical for bacterial disease outbreaks in aquatic and terrestrial animals. The enzyme showed the maximum activity at pH 7.0 and retained substantial bacteriolytic activity (44.0%-63.2%) under alkalescence conditions (pH 7.0-9.0), demonstrating adaptability to marine and freshwater aquaculture environments. Divalent metal ions including Zn²⁺, Mg²⁺, Mn²⁺, and Fe²⁺ at 0.1-1.0 mmol/L moderately enhanced the bacteriolytic activity of lysV208, whereas those at 10.0 mmol/L reduced the activity (P<0.01). In addition, lysV208 displayed broad-spectrum lytic effects, showing the bacteriolytic activity of 59.7% against V. alginolyticus V039, 68.9% against Vibrio vulnificus H1, 65.8% against Vibrio parahaemolyticus GH32, and 38.0% and 65.6% against Vibrio harveyi TY13 and G1, respectively.Conclusion The recombinant lyase lysV208 demonstrates robust and stable in vitro bacteriolytic activity and a broader spectrum than its source phage. These findings highlight its potential for the control of bacterial infections and the development of phage-lyase synergistic agents.