title: Purification, cloning, expression, and biochemical characterization of a monofunctional catalase, KatP, from Pigmentiphaga sp. DL-8.
abstract: Catalases are essential components of the cellular equipment used to cope with oxidative stress. The monofunctional catalase KatP was purified from Pigmentiphaga sp. using ammonium sulfate precipitation (ASP), diethylaminoethyl ion exchange chromatography (IEC), and hydrophobic interaction chromatography (HIC). The purified catalase formed polymer with an estimated monomer molecular mass of 54kDa, which were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis. KatP exhibited a specific catalytic activity of 73,000U/mg, which was higher than that of catalase-1 of Comamonas terrigena N3H (55,900U/mg). Seven short tryptic fragments of this catalase were obtained by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS/MS), and the gene, katP, was cloned by PCR amplification and overexpressed in Escherichia coli BL21 (DE3). Based on the complete amino acid sequence, KatP was identified as a clade 3 monofunctional catalase. The specific activities of recombinant KatP for hydrogen peroxide (690,000U/mg) increased 9-fold over that of the parent strain. The Km and Vmax of recombinant KatP were 9.48mM and 81.2mol/minmg, respectively. The optimal pH and temperature for KatP were 7.0 and 37°C, respectively, and the enzyme displayed abroad pH-stable range of 4.0-11.0. The enzyme was inhibited by Zn(2+), Cu(2+), Cr(2+), and Mn(2+), whereas Fe(3+) and Mg(2+) stimulated KatP enzymatic activity. Interestingly, the catalase activity of recombinant KatP displayed high stability under different temperature and pH conditions, suggesting that KatP is a potential candidate for the production of catalase.
authors: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors: Weiliang
authors: W
authors: Dong
authors: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China; College of Food and Bioengineering, Henan University of Science and Technology, 471003 Luoyang, China.
authors: Ying
authors: Y
authors: Hou
authors: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors: Shuhuan
authors: S
authors: Li
authors: College of Bioscience and Bioengineering, Jiangxi Agriculture University, 330045 Nanchang, China.
authors: Fei
authors: F
authors: Wang
authors: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors: Jie
authors: J
authors: Zhou
authors: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors: Zhoukun
authors: Z
authors: Li
authors: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors: Yicheng
authors: Y
authors: Wang
authors: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors: Fei
authors: F
authors: Huang
authors: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors: Lei
authors: L
authors: Fu
authors: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China.
authors: Yan
authors: Y
authors: Huang
authors: Key Lab of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agriculture University, 210095 Nanjing, China. Electronic address: czl@njau.edu.cn.
authors: Zhongli
authors: Z
authors: Cui
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D042521:Burkholderiaceae / Q000201:enzymology*; D002374:Catalase / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D002374:Catalase
keywords: Characterization; Enzyme purification; Monofunctional catalase; Pigmentiphaga sp.; Recombinant