Journal
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
Volume 1814, Issue 5, Pages 622-629Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbapap.2011.03.013
Keywords
Recombinant production; tHBP-HA; Aldol condensation; Pseudomonas
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Funding
- MIUR-PRIN [20077MV8M9 004]
- Lombardia Regional Government
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The gene encoding trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) was isolated from Pseudomonas fluorescens N3, an environmental strain able to degrade naphthalene. This enzyme is an aldolase of class I that reversibly catalyzes the transformation of the trans-o-hydroxybenzylidenepyruvate (t-HBP), releasing pyruvate and salicylaldehyde. The enzyme was expressed in Escherichia coli as a recombinant protein of 38 kDa with a His6-Tag at its N-terminus. The recombinant protein His-tHBP-HA was purified by affinity chromatography and we present here the biochemical characterization of its activity in the aldol condensation reaction. The aldol condensation reaction parameters were determined using as acceptors both salicylaldehyde, which is the natural substrate taking part to the naphthalene degradative pathway, and benzaldehyde. In both cases, His-tHBP-HA shows similar apparent K(m) and apparent V(max) values. Further analyses showed that the optimal pH and temperature of His-tHBP-HA activity are 7.0 and 30 degrees C, respectively. The tHBP-HA catalytic rates and the availability of an efficient system to produce large amounts of purified protein are relevant from a biotechnological point of view. (C) 2011 Elsevier B.V. All rights reserved.
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