title: [Expression and purification of lysin B in mycobacteriophage D29 and analysis of its enzymatic properties].
abstract: 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.
authors.affiliation: College of Life Science, Qufu Normal University, Qufu 273165, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Hou
authors.forename: Limei
authors.initials: L
authors.lastname: Hao
authors.forename: Jiancheng
authors.initials: J
authors.lastname: Qi
authors.forename: Ge
authors.initials: G
authors.lastname: Yang
mesh_terms: D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D009160:Mycobacteriophages / Q000201:enzymology*; D009169:Mycobacterium tuberculosis / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D014764:Viral Proteins