分支杆菌噬菌体D29 Lysin B的表达、纯化及酶学性质分析

Expression and purification of Lysin B in mycobacteriophage D29 and analysis of its enzymatic properties

克隆表达噬菌体D29 LysinB（LysB）并对其酶学性质进行研究。以噬菌体D29基因组为模板,用PCR方法扩增lysB基因,与表达载体pET22b连接,将重组质粒转化至Escherichiacoli BL21（DE3）中表达,镍柱亲和层析（Ni-NTA）纯化可溶性表达产物,并对重组蛋白的活性进行分析检测。结果表明：成功构建了pET22b-lysB表达载体,并从1L的LB培养物中获得了33.2mg高纯度重组蛋白（His-LysB）;His-LysB具有分解脂肪的能力,属于脂肪酶;生物化学特性分析表明：丁酸对硝基苯（pNPB）为水解底物,His-LysB热稳定性不佳,30℃以下比较稳定,随着温度的升高,稳定性逐渐降低;该蛋白具有较高的pH值适应性,pH5.0~9.5范围内稳定性较高;在23℃和pH7.5时酶活力最高,其比酶活为1.3U/mg;金属离子Zn2＋、Cu2＋、Mg2＋、Mn2＋和苯甲基磺酰氟（PMSF）抑制剂对酶活具有强烈的抑制作用。本研究为开发新的治疗结核药物提供了一个新的选择。

LysinB （LysB） in mycobacteriophage D29 was cloned and expressed and its enzymatic properties were analysed. The lysB gene was amplified by PCR from mycobacteriophage D29 genomic DNA and inserted into pET22b vector. The constructed recombinant plasmid was transformed into Escherichia coli BL21（DE3） to express fusion protein, which was purified by Ni-NTA column and enzymatic activity detected. The results showed that expression plasmid pET22b-lysB was constructed successfully. Highly purified recombination protein （His-LysB） was obtained 33.2 mg from 1 L LB culture medium. A screening for His-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. With p-nitrophenyl butyrate as substrate, the thermal stability of the enzyme was poor when the temperature was above 30oC. The enzyme exhibited higher stability at pH 5.0–9.5. The optimum temperature and pH for the lipolytic activity of His-LysB were 23oC and 7.5 respectively. Under theoptimum conditions, the specific activity of His-LysB was 1.3 U/mg. Zn2＋, Cu2＋, Mg2＋, Mn2＋and phenylmethane sulfonyl fruoride severely inhibited the lipolytic activity of His-LysB. The result provides a new option for tuberculosis drug research and development.