Purification, immobilization and stabilization of a highly enantioselective alcohol dehydrogenase from Thermus thermophilus HB27 cloned in E. coli

• Published in: Process biochemistry (1991) Vol. 44; no. 9; pp. 1004 - 1012
• Main Authors: Rocha-Martin, Javier, Vega, Daniel E., Cabrera, Zaida, Bolivar, Juan M., Fernandez-Lafuente, Roberto, Berenguer, Jose, Guisan, Jose M.
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 01-09-2009
• Subjects: Asymmetric reductions; Dehydrogenases; Escherichia coli; PEI-coated supports; Phenyl ethanol; Reversible immobilization; Thermophylic enzymes; Thermus thermophilus; Phenyl ethanol; Reversible immobilization; Dehydrogenases; Thermophylic enzymes; Asymmetric reductions; PEI-coated supports
• ISSN: 1359-5113; 1873-3298
• DOI: 10.1016/j.procbio.2009.04.026

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%.