Carbonyl reductase of Candida parapsilosis - Stability analysis and stabilization strategy
Publikation: Beitrag in Fachzeitschrift › Forschungsartikel › Beigetragen › Begutachtung
Beitragende
Abstract
tThe homodimeric Candida parapsilosis carbonyl reductase 2 (CPCR2) is an industrially attractive biocata-lyst due to its broad substrate range and high stereoselectivity. In addition, CPCR2 is reasonably stable inmonophasic organic solvents (e.g. alcohols) but apparently instable in biphasic organic systems. Hence,we conducted first a thorough quantitative inactivation study of CPCR2, using both wild-type and stabil-ity improved variants, in an attempt to identify critical factors influencing the enzyme stability. Possibleinactivation phenomena including oxidation, shear forces, dissociation and adsorption at interfaces wereassessed on a microliter scale using quantitative kinetic assays. Our results demonstrate that interfaceinteractions and dimer dissociation are the main reasons for inactivation of CPCR2. Shear forces seems toenhance these inactivation processes whereas oxidation plays no role in CPCR2 inactivation. Secondly, anattempt was made to find suitable stabilization strategies to utilize CPCR2 in various reaction systems. Tominimize the inactivation, bovine serum albumin was used as traditional blocking and crowding agent.The residual activity of the wild-type was successfully increased up to 2.5-fold by addition of 1 g mL−1bovine serum albumin. To avoid dimer dissociation the cofactor concentration was successively increased.The residual activity was successfully enhanced up to 5-fold, 3-fold and 1.5-fold for the wild-type, singleand double mutant, respectively. Further, recently gained data from the enzyme crystal structure wereused to interpret the effects of stabilization. We propose conformational change of a flexible region inCPCR2 upon binding of the cofactor leading to internal stabilization of the enzyme. In conclusion, wepropose the addition of bovine serum albumin and the cofactor NADH as a suitable stabilization strategyto utilize CPCR2 in various reaction systems.
Details
Originalsprache | Englisch |
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Seiten (von - bis) | 45-53 |
Fachzeitschrift | Journal of Molecular Catalysis B: Enzymatic |
Jahrgang | 2015 |
Ausgabenummer | 112 |
Publikationsstatus | Veröffentlicht - 2015 |
Peer-Review-Status | Ja |
Externe IDs
Scopus | 84919949106 |
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ORCID | /0000-0002-2912-546X/work/171551938 |
Schlagworte
Schlagwörter
- alcohol dehydrogenase, CPCR2