Bioaffinity purification of NADP+-dependent dehydrogenases: Studies with alcohol dehydrogenase from Thermoanaerobacter brockii

• Published in: Biotechnology and bioengineering 2002-03, Vol.77 (5), p.517-527
• Main Authors: McMahon, Mary, Mulcahy, Patricia
• Format: Article
• Language: English
• Subjects: 8′azo-linked immobilized NADP; alcohol dehydrogenase; Alcohol Dehydrogenase - isolation & purification; Azo Compounds - chemistry; Bacteria, Anaerobic - enzymology; Biological and medical sciences; Biotechnology; C8-linked immobilized NADP; Chromatography, Affinity - methods; Enzyme engineering; Enzymes, Immobilized; Fundamental and applied biological sciences. Psychology; Improved methods for extraction and purification of enzymes; kinetic locking-on tactic; Ligands; Methods. Procedures. Technologies; N1-linked immobilized NADP; N6-linked immobilized NADP; NADP - chemistry; NADP - metabolism; Protein Binding; stripping ligand tactic; thermoanaerobacter brockii
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.10079

This study describes the development and application of a bioaffinity chromatographic system for the one‐step purification of an NADP+‐dependent secondary alcohol dehydrogenase from the obligate anaerobe, Thermoanaerobacter brockii (TBADH, EC 1.1.1.2). The general approach is based upon improving the selectivity of immobilized cofactor derivatives (general ligand approach to bioaffinity chromatography) through using soluble enzyme‐specific substrate analogues in irrigants to promote biospecific adsorption (the kinetic locking‐on tactic). Specifically, the following is described: Evaluation of 8′‐azo‐linked, C8‐linked, N1‐linked, and N6‐linked immobilized NADP+ derivatives for use with the kinetic locking‐on strategy for bioaffinity purification of TBADH; evaluation of 2′, 5′‐ADP as a stripping ligand for TBADH bioaffinity purifications using an 8′‐azo‐linked immobilized NADP+ derivative in the locking‐on mode; and application of the developed bioaffinity chromatographic system to the purification of TBADH from a crude cellular extract. Surprizingly, of the four immobilized NADP+ derivatives investigated, only the 8′‐azo‐linked immobilized NADP+ derivative proved effective for TBADH affinity purification when used in conjunction with pyrazole (a competitive inhibitor of TBADH activity) as the locking‐on ligand. This is in contrast to other NADP+‐dependent dehydrogenases where the immobilized N6‐linked cofactor proved to be suitable. While the one‐step purification of TBADH to electrophoretic homogeneity is described in the present study (92% yield), results from the model chromatographic studies point to improvements that could be made to the immobilized cofactor derivative to improve its suitability for TBADH bioaffinity purification and to facilitate future large scale protein purification operations. © 2002 John Wiley and Sons, Inc. Biotechnol Bioeng 77: 517–527, 2002; DOI 10.1002/bit.10079