Hou Lili, Hao Limei, Qi Jiancheng, Yang Ge
College of Life Science, Qufu Normal University, Qufu 273165, China.
Sheng Wu Gong Cheng Xue Bao. 2010 Apr;26(4):517-22.
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 30 degrees C. The enzyme exhibited higher stability at pH 5.0-9.5. The optimum temperature and pH for the lipolytic activity of His-LysB were 23 degrees C and 7.5 respectively. Under the optimum 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.
对分枝杆菌噬菌体D29中的溶菌酶B（LysB）进行了克隆、表达及酶学性质分析。通过PCR从分枝杆菌噬菌体D29基因组DNA中扩增出lysB基因，并将其插入pET22b载体。将构建好的重组质粒转化到大肠杆菌BL21(DE3)中表达融合蛋白，该融合蛋白经镍-氮三乙酸（Ni-NTA）柱纯化并检测酶活性。结果表明成功构建了表达质粒pET22b-lysB。从1 L LB培养基中获得了33.2 mg高度纯化的重组蛋白（His-LysB）。对His-LysB在酯酶和脂肪酶底物上的活性进行筛选，证实了其脂解活性。以对硝基苯丁酸为底物，当温度高于30℃时，该酶的热稳定性较差。该酶在pH 5.0 - 9.5时表现出较高的稳定性。His-LysB脂解活性的最适温度和pH分别为23℃和7.5。在最适条件下，His-LysB的比活性为1.3 U/mg。锌离子、铜离子、镁离子、锰离子和苯甲基磺酰氟严重抑制His-LysB的脂解活性。该结果为结核病药物研发提供了新的选择。
