1.Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus.
Pennacchio A1, Pucci B, Secundo F, La Cara F, Rossi M, Raia CA. Appl Environ Microbiol. 2008 Jul;74(13):3949-58. doi: 10.1128/AEM.00217-08. Epub 2008 May 2.
The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adh(Tt)) was heterologously overexpressed in Escherichia coli, and the protein (ADH(Tt)) was purified to homogeneity and characterized. ADH(Tt) is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to approximately 73 degrees C and a 30-min half-inactivation temperature of approximately 90 degrees C, as well as good tolerance to common organic solvents. ADH(Tt) has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and alpha-keto esters, and poor activity on aromatic alcohols and aldehydes.
2.Characterization and further stabilization of a new anti-prelog specific alcohol dehydrogenase from Thermus thermophilus HB27 for asymmetric reduction of carbonyl compounds.
Rocha-Martín J1, Vega D, Bolivar JM, Hidalgo A, Berenguer J, Guisán JM, López-Gallego F. Bioresour Technol. 2012 Jan;103(1):343-50. doi: 10.1016/j.biortech.2011.10.018. Epub 2011 Oct 17.
The use of dehydrogenases in asymmetric chemistry has exponentially grown in the last decades facilitated by the genome mining. Here, a new short-chain alcohol dehydrogenase from Thermus thermophilus HB27 has been expressed, purified, characterized and stabilized by immobilization on solid supports. The enzyme catalyzes both oxidative and reductive reactions at neutral pH with a broad range of substrates. Its highest activity was found towards the reduction of 2,2',2″-trifluoroacetophenone (85 U/mg at 65 °C and pH 7). Moreover, the enzyme was stabilized more than 200-fold by multipoint covalent immobilization on agarose matrixes via glyoxyl chemistry. Such heterogeneous catalyst coupled to an immobilized cofactor recycling partner performed the quantitative asymmetric reduction of 2,2',2″-trifluoroacetophenone and rac-2-phenylpropanal to (S)-(+)-α-(trifluoromethyl)benzyl alcohol and (R)-2-phenyl-1-propanol with enantiomeric excesses of 96% and 71%, respectively.
