Characterization of the aldol condensation activity of the trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) cloned from Pseudomonas fluorescens N3
Articolo
Data di Pubblicazione:
2011
Citazione:
Characterization of the aldol condensation activity of the trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) cloned from Pseudomonas fluorescens N3 / S. Ferrara, E. Mapelli, G. Sello, P. Di Gennaro. - In: BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS. - ISSN 1570-9639. - 1814:5(2011), pp. 622-629.
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 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 Km and apparent Vmax 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.
Tipologia IRIS:
01 - Articolo su periodico
Keywords:
Aldol condensation; Pseudomonas; Recombinant production; tHBP-HA
Elenco autori:
S. Ferrara, E. Mapelli, G. Sello, P. Di Gennaro
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