title: Characterization of the aldol condensation activity of the trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) cloned from Pseudomonas fluorescens N3.
abstract: 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 38kDa 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°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.
authors: Department of Environmental Sciences, University of Milano-Bicocca, Piazza della Scienza 1, 20126 Milano, Italy.
authors: Silvia
authors: S
authors: Ferrara
authors: Erika
authors: E
authors: Mapelli
authors: Guido
authors: G
authors: Sello
authors: Patrizia
authors: P
authors: Di Gennaro
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006836:Hydro-Lyases / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D016133:Polymerase Chain Reaction; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D006836:Hydro-Lyases; C078380:trans-2-hydroxybenzylidenepyruvate hydratase-aldolase