沙门菌噬菌体PSDA-2解聚酶Dpo32异源表达及生物学特性研究  

Heterologous Expression and Biological Characterization of Salmonella Phage PSDA-2 Depolymerase Dpo32

作　　者：刘亚茹 满都拉[1] 陈忠军[1] 孙子羽 LIU Yaru;MAN Dlaa;CHEN Zhongjun;SUN Ziyu(College of Food Science and Engineering,Inner Mongolia Agricultural University,Hohhot 010000,China)

机构地区：[1]内蒙古农业大学食品科学与工程学院,呼和浩特010000

出　　处：《中国生物工程杂志》2024年第8期69-77,共9页China Biotechnology

摘　　要：对前期分离得到的沙门菌噬菌体PSDA-2基因组进行基因功能分析,发现其尾丝蛋白与鼠伤寒沙门菌的内切鼠李糖苷酶有较高序列相似性,表明该蛋白质可能具有多糖解聚酶活性。对编码该尾丝蛋白的基因进行克隆、异源表达和纯化,得到多糖解聚酶Dpo32。利用苯酚硫酸法和双层平板法分析该多糖解聚酶活性,测定其酶谱及pH、温度、乙醇和金属离子对其稳定性的影响。结果显示多糖解聚酶Dpo32可在较宽温度范围内降解沙门菌表面多糖产生还原糖,在平板上使1株鼠伤寒沙门菌(CICC 21483)裂解,3株沙门菌产生晕圈。稳定性研究结果显示该酶在30~80℃、pH 2~11、≤60%乙醇溶液和≤10 mmol/L的K^(+)、Ca^(2+)、Mg^(2+)、Cu^(2+)、Zn^(2+)溶液中保持良好活性,表明其具有较高环境适应性。The gene function of phage PSDA-2 was analyzed,revealing a high sequence similarity between its tail fiber protein and the endorhamnosidase of Salmonella typhimurium,suggesting a potential polysaccharide depolymerase activity for this protein.The gene encoding the protein was cloned,heterologously expressed,and purified to obtain polysaccharide depolymerase Dpo32.The activity of the polysaccharide depolymerase was assessed using the phenol-sulfuric acid method and the double-layer agar plate method,and its stability in terms of enzyme spectrum,pH,temperature,alcohol,and metal ions was studied.The results demonstrated that Dpo32,a polysaccharide depolymerase,has the ability to enzymatically degrade Salmonella surface polysaccharides and generate reducing sugars over a broad temperature range.This enzymatic activity resulted in the lysis of one strain of Salmonella typhimurium(CICC 21483)and the formation of halos around three strains on double-layer agar plates.The results of the stability study showed that the enzyme exhibited robust activity over a wide range of conditions,including temperatures ranging from 30℃to 80℃ and pHs from 2 to 11.Furthermore,it can maintain good activity in ionic solutions containing≤60%alcohol and≤10 mmol/L of K^(+),Ca^(2+),Mg^(2+),Cu^(2+),Zn^(2+).These findings suggest that the enzyme possesses exceptional environmental adaptability.

关 键 词：噬菌体 解聚酶 沙门菌 生物学特性 

分 类 号：Q939.48[生物学—微生物学]