Archives of Microbiology

, Volume 193, Issue 8, pp 553–563 | Cite as

Purification and characterization of benzyl alcohol- and benzaldehyde- dehydrogenase from Pseudomonas putida CSV86

  • Rahul Shrivastava
  • Aditya Basu
  • Prashant S. Phale
Original Paper

Abstract

Pseudomonas putida CSV86 utilizes benzyl alcohol via catechol and methylnaphthalenes through detoxification pathway via hydroxymethylnaphthalenes and naphthaldehydes. Based on metabolic studies, benzyl alcohol dehydrogenase (BADH) and benzaldehyde dehydrogenase (BZDH) were hypothesized to be involved in the detoxification pathway. BADH and BZDH were purified to apparent homogeneity and were (1) homodimers with subunit molecular mass of 38 and 57 kDa, respectively, (2) NAD+ dependent, (3) broad substrate specific accepting mono- and di-aromatic alcohols and aldehydes but not aliphatic compounds, and (4) BADH contained iron and magnesium, while BZDH contained magnesium. BADH in the forward reaction converted alcohol to aldehyde and required NAD+, while in the reverse reaction it reduced aldehyde to alcohol in NADH-dependent manner. BZDH showed low Km value for benzaldehyde as compared to BADH reverse reaction. Chemical cross-linking studies revealed that BADH and BZDH do not form multi-enzyme complex. Thus, the conversion of aromatic alcohol to acid is due to low Km and high catalytic efficiency of BZDH. Phylogenetic analysis revealed that BADH is a novel enzyme and diverged during the evolution to gain the ability to utilize mono- and di-aromatic compounds. The wide substrate specificity of these enzymes enables strain to detoxify methylnaphthalenes to naphthoic acids efficiently.

Keywords

Benzyl alcohol dehydrogenase Benzaldehyde dehydrogenase Purification and kinetic constants Protein–protein interactions Substrate specificity Phylogenetic analysis 

Supplementary material

203_2011_697_MOESM1_ESM.doc (210 kb)
Supplementary material 1 (DOC 210 kb)

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Rahul Shrivastava
    • 1
  • Aditya Basu
    • 1
  • Prashant S. Phale
    • 1
  1. 1.Department of Biosciences and BioengineeringIndian Institute of Technology-BombayPowaiIndia

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