title: [Purification and characterization of a pH-stable and thermostable biphenyl hydrolase from Rhodococcus sp. R04].
abstract: OBJECTIVE
  Biphenyl hydrolase (BphD) is an enzyme of the biphenyl biodegradation pathway in Rhodococcus sp. R04. Expression, purification and properties of BphD, together with chemical modification were investigated.
  
  
  METHODS
  Using heat-induced expression vector pBV220, we expressed bphD heterologously in E. coli BL21 (DE3). The products were purified by chromatography, and the purified enzyme was used to study its functions and properties by circular dichroism.
  
  
  RESULTS
  BphD has been purified to homogeneity with a final purification fold of 5.02. With 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) as a substrate, the optimum pH and temperature of BphD are 9 and 80 degrees C, respectively and the Km is 0.44 micromol/L. The enzyme had remaining 90% of activity incubating for 1 hour at 60 degrees C, and had a half-life of 1 hour at 70 degreesC, making it the most thermostable biphenyl hydrolase reported in Rhodococcus. Secondary structure of BphD has undergone great changes with the temperature up to 70 degrees C, and has been severely damaged at 80 degrees C. The sequence alignment between BphD and other homologous hydrolases as well as chemical modification of BphD indicated that Ser, His and Asp play important roles in the hydrolysis of HOPDA. In addition, Trp residue may be near the active center of enzyme as the key amino acid involved in substrate hydrolysis, which is not previously reported in the literature.
  
  
  CONCLUSIONS
  BphD with pH stability and thermostability is quite rare in the homologous hydrolases in Rhodococcus. The key status of Ser, His, Asp and Trp will build a basis for more learnings of the transformation mechanism of BphD.
authors.affiliation: Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, China. xiuqyang@sxu.edu.cn
authors.forename: Xiuqing
authors.initials: X
authors.lastname: Yang
authors.forename: Pengli
authors.initials: P
authors.lastname: Li
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zheng
authors.forename: Chong
authors.initials: C
authors.lastname: Shen
mesh_terms: D000595:Amino Acid Sequence; D001713:Biphenyl Compounds / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D012240:Rhodococcus / Q000201:enzymology*; D013696:Temperature
chemical_list: D001713:Biphenyl Compounds; D006867:Hydrolases; C010574:biphenyl