Phenol hydroxylase from Pseudomonas sp. KZNSA: Purification, characterization and prediction of three-dimensional structure

A 613 kDa Phenol hydroxylase (PheA) was purified and characterized from Pseudomonas sp. KZNSA (PKZNSA). Cell free extract of the isolate grown in mineral salt medium supplemented with 600 ppm phenol showed 21.58 U/mL of PheA activity with a specific activity of 7.67 U/mg of protein. The enzyme was purified to 1.6-fold with a total yield of 33.6%. The purified PheA was optimally active at pH 8 and temperature 30 degrees C, with approximate to 95% stability at pH 7.5 and temperature 30 degrees C after 2 h. The Lineweaver-Burk plot showed the v(max) and K-m values of 4.04 mu M/min and 4.03 mu M, respectively, for the substrate phenol. The ES-MS data generated from the tryptic digested fragments of pure protein and PCR amplification of a approximate to 600 bp gene from genomic DNA of PKZNSA lead to the determination of complete amino acid and nucleotide sequence of PheA Bioinformatics tools and homology modelling studies indicated that PheA from PKZNSA is likely a probable protein kinase UbiB (2-octaprenylphenol hydroxylase) involving Lys and Asp at positions 153 and 288 for binding and active site, respectively. Characterization and optimization of PheA activity may be useful for a better understanding of 2,4-dichlorophenol degradation by this organism and for potential industrial application of the enzyme. (C) 2019 Elsevier B.V. All rights reserved.