Uria, A.R., R. Machielsen, B.E. Dutilh, M.A. Huynen, and J. van der Oost, 2006. Alcohol Dehydrogenases from Marine Hyperthermophilic Microorganisms and Their Importance to the Pharmaceutical Industry. Presented orally in International Seminar and Workshop on Marine Biodiversity & their Potential for Developing Bio-Pharmaceutical Industry, on the 17-18th of May 2006 at Jakarta.

 

Abstract:
Alcohol dehydrogenases (ADHs) are enzymes capable of catalyzing the intercoversion of alcohols to their corresponding aldehydes or ketones by using different redox-mediating cofactors. They are valuable tools for the biocatalytic synthesis of various chiral compounds, which are potential building blocks or intermediates in the organic synthesis of therapeutics in the pharmaceutical industry. In particular, ADHs from marine hyperthermophilic microorganisms have attracted special attention as catalysts in several industrial processes over the last two decades, mainly due to some unique properties, including their relatively broad specificity towards various carbonyl substrates, their high thermal and chemical stability, as well as their suitability for cofactor regeneration. Therefore, this review article describes ADHs from marine hyperthermophiles, including their activity screening, cloning, heterologous production, and characterisation. Their potential for application in the pharmaceutical industry is discussed. (Click here for the full text)

Machielsen R., A. R. Uria, S.W.M. Kengen and J. van der Oost, 2006. Production and Characterization of a Thermostable Alcohol Dehydrogenase that belongs to Aldo-Keto Reductase Superfamily. Journal of Applied and Environmental Microbiology 72(1):233-238. 

Abstract:

The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily, has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The  gene,  referred  to  as adhD,  was  functionally  expressed  in  Escherichia coli  and subsequently purified to homogeneity. The enzyme has a monomeric conformation with a  molecular mass of 32 kDa. The catalytic activity of  the enzyme  increases up  to 100°C, and  a  half  life  value  of  130 min  at  this  temperature  indicates  its  high  thermostability. AdhD exhibits a broad substrate specificity, with in general a preference for the reduction of ketones (pH optimum 6.1), and the oxidation of secondary alcohols (pH optimum 8.8). Maximal specific activities were detected with 2,3-butanediol (108.3 U/mg) and diacetyl /acetoin  (22.5 U/mg)  in  the  oxidative  and  reductive  reaction,  respectively. GC-analysis indicated  that AdhD mainly  produced  (S)-2-pentanol  (ee  89%) when  2-pentanone was used as substrate. The physiological role of AdhD is discussed. (Click here for the full text)

Uria, A.R., 2004. Heterologous Production, Purification and Characterization of a NAD(P)-dependent Alcohol Dehydrogenase from the Marine Hyperthermophilic Archaebacterium, Pyrococcus furiosus and Generation of a DNA Construct for Its Overexpression in the Deep-Sea Hyperthermophile, Pyrococcus abysii . MSc Thesis in Molecular Microbiology. Supervisors: Dr Ronnie Machielsen and Prof John van der Oost (Bacterial Genetics Group, Lab Microbiology, Wageningen University).

 

Abstract:

Alcohol dehydrogenases (ADHs) originated from hyperthermophilic microorganisms have attracted much scientific attention for the use in various industrial processes, mainly due to their thermal stability, tolerance towards common denaturing reagents, capability of producing high-value chiral alcohols, and suitability for being used in the regeneration of cofactors, such as NAD+ and NADP+. In our research work, a NAD(P)-dependent alcohol dehydrogenase of the marine hyperthermophilic archaebacterium, Pyrococcus furiosus was recombinantly overproduced by cloning of the expression vector harbouring adh D gene, pWUR85, into Escherichia coli BL21 (DE3). The resulting expression product, designated as ADH D, was purified to homogeneity and then characterized. Results of the enzyme characterisation indicated that ADH D catalysed the interconversion of alcohols into the corresponding aldehydes or ketones with the preference for NAD+ instead of NADP+ as the electron acceptor. This enzyme existed in a monomeric form with the molecular size of 32 kDa and showed no requirement of metal for its activity as found on the majority of AKR. It was much more efficient in catalyzing ketone reduction than alcohol oxidation based on the kinetics data. The bioinformatic analysis revealed that the cofactor- and substrate-binding sites of this enzyme resembled those from the members of the aldo-keto reductase (AKR). It was found that the optimal pH for its oxidative activity was around 8.8, which was much different from that for its reductive activity (pH of 6.12). The isoelectric point (PI) of the enzyme was estimated to be 5.49. This enzyme showed an extremely high activity at 100oC (approximately 39 U/mg) with the half-life (T1/2) at 100oC of 130.75 minutes. It seemed to be more specific for aliphatic mediate-chain secondary alcohols, especially those which contain a hydroxyl group at their secondary carbon and two hydrophobic groups on their both sides. The activity towards diacetyl was extremely high, which was approximately 1.5-fold higher than that towards acetoin. All these properties above place this NAD(P)-dependent enzyme as a new member of the AKR superfamily. As an initial step for overexpressing ADH D in the deep-sea hyperthermophile P. abysii, the Psip promoter, in which its function is strongly controlled by elemental sulfur, was isolated from P. furiosus genome. Then the promoter was successfully combined with the adh D gene resulting in a Psip-adh D hybrid. This hybrid will be used further for constructing an archaebacterial shuttle vector. (Click here for the full text)

 
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