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

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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 K m 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 K m 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.

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Acknowledgments

PP and RS would like to thank Sophisticated Analytical Instrument Facility (SAIF), IIT-B, for ICP–AES analysis. Senior research fellowship to RS from IIT-B is gratefully acknowledged.

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Authors and Affiliations

  1. Department of Biosciences and Bioengineering, Indian Institute of Technology-Bombay, Powai, Mumbai, 400076, India

    Rahul Shrivastava, Aditya Basu & Prashant S. Phale

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  1. Rahul Shrivastava
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  2. Aditya Basu
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  3. Prashant S. Phale
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Corresponding author

Correspondence to Prashant S. Phale.

Additional information

Communicated by Jan Roelof van der Meer.

R. Shrivastava and A. Basu contributed equally.

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Shrivastava, R., Basu, A. & Phale, P.S. Purification and characterization of benzyl alcohol- and benzaldehyde- dehydrogenase from Pseudomonas putida CSV86. Arch Microbiol 193, 553–563 (2011). https://doi.org/10.1007/s00203-011-0697-6

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  • DOI: https://doi.org/10.1007/s00203-011-0697-6

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