RT Journal Article
T1 Purification, immobilization and stabilization of a highly enantioselective alcohol dehydrogenase from Thermus thermophilus HB27 cloned in E. coli
A1 Rocha Martín, Javier
A1 Vega, Daniel
A1 Cabrera, Zaida
A1 Bolívar Bolívar, Juan Manuel
A1 Fernández-Lafuente, Roberto
A1 Berenguer, José
A1 Guisán, José
AB This paper shows the purification and immobilization of a very interesting thermophilic alcohol dehydrogenase from Thermus thermophilus HB27 cloned in Escherichia coli. The purification was based on a first thermal treatment of the crude extract, that leaves the target enzyme in the supernatant, followed by the adsorption of most contaminant proteins in a IMAC column (the target protein did not adsorb on these columns due to the poorness of His residues). Final purification factor was around a 9-fold factor (no other protein bands were detected in SDS-PAGE gels) with an overall yield around 80%. Covalent immobilization of the enzyme on very different supports only permitted to improve the enzyme stability by a 5–10-fold factor, very similarly to the results obtained by the adsorption of the enzyme on polyethyleneimine coated supports. This enzyme adsorbed by ionic exchange maintained the activity unaltered during immobilization which was a very rapid process, and was more stable than the covalent preparations in the presence of organic solvents, and the enzyme was quite strongly adsorbed on the support. Therefore, it was proposed as a good option to prepare industrial biocatalysts of the enzyme. This preparation was utilized in the asymmetric reduction of acetophenone to produce (S)-(−)-1-phenylethanol, with an enantiomeric excess of more than 99%.
PB Elsevier
SN 1359-5113
YR 2009
FD 2009
LK https://hdl.handle.net/20.500.14352/93698
UL https://hdl.handle.net/20.500.14352/93698
LA eng
NO Rocha-Martin, Javier, et al. «Purification, Immobilization and Stabilization of a Highly Enantioselective Alcohol Dehydrogenase from Thermus Thermophilus HB27 Cloned in E. Coli». Process Biochemistry, vol. 44, n.o 9, septiembre de 2009, pp. 1004-12.  https://doi.org/10.1016/j.procbio.2009.04.026.
NO FundingThis work has been supported by CAM (grants S0505/PPQ/0344) and MEC (grants BIO2004-02671 and CTQ2005-02420/PPQ). An institutional grant from Fundación Ramón Areces to CBMSO is also acknowledged. J.M. Bolívar and J. Rocha-Martín are holders of a PhD fellowship from CAM. Dr. Ángel Berenguer (Universidad de Alicante) is gratefully acknowledged for his kind help during the writing of this paper.
NO Comunidad de Madrid
NO Ministerio de Educación y Ciencia (España)
NO Fundación Ramón Areces
DS Docta Complutense
RD 8 abr 2025