A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii

Timpson, Leanne M.; Liliensiek, Ann-Kathrin; Alsafadi, Diya; Cassidy, Jennifer; Sharkey, Michael A.; Liddell, Susan; Allers, Thorsten; Paradisi, Francesca

doi:10.1007/s00253-012-4074-4

Cited by 38 publications

(48 citation statements)

References 39 publications

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“…The expression vector is based on the pHV2 origin which maintains the plasmid at a copy number of approximately 6 per genome equivalent (Allers 2010 ). To date, the H. volcanii expression system has been successfully applied for heterologous protein expressions on a shaker-flask scale (Karan et al 2013 ;Timpson et al 2012Timpson et al , 2013.…”

Section: Introductionmentioning

confidence: 99%

Production of halophilic proteins using Haloferax volcanii H1895 in a stirred-tank bioreactor

Strillinger

Grötzinger

Allers

et al. 2015

Appl Microbiol Biotechnol

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The success of biotechnological processes is based on the availability of efficient and highly specific biocatalysts, which can satisfy industrial demands. Extreme and remote environments like the deep brine pools of the Red Sea represent highly interesting habitats for the discovery of novel halophilic and thermophilic enzymes. Haloferax volcanii constitutes a suitable expression system for halophilic enzymes obtained from such brine pools. We developed a batch process for the cultivation of H. volcanii H1895 in controlled stirred-tank bioreactors utilising knockouts of components of the flagella assembly system. The standard medium Hv-YPC was supplemented to reach a higher cell density. Without protein expression, cell dry weight reaches 10 g L . Two halophilic alcohol dehydrogenases were expressed under the control of the tryptophanase promoter p.tna with 16.8 and 3.2 mg g , respectively, at a maximum cell dry weight of 6.5 g L . Protein expression was induced by the addition of L-tryptophan. Investigation of various expression strategies leads to an optimised two-step induction protocol introducing 6 mM L-tryptophan at an OD of 0.4 followed by incubation for 16 h and a second induction step with 3 mM L-tryptophan followed by a final incubation time of 4 h. Compared with the uncontrolled shaker-flask cultivations used until date, dry cell mass concentrations were improved by a factor of more than 5 and cell-specific enzyme activities showed an up to 28-fold increased yield of the heterologous proteins.

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Section: Introductionmentioning

confidence: 99%

Production of halophilic proteins using Haloferax volcanii H1895 in a stirred-tank bioreactor

Strillinger

Grötzinger

Allers

et al. 2015

Appl Microbiol Biotechnol

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“…These characterized enzymes are maximally active at ambient temperatures and are inhibited by their own substrates. This limits their usefulness because industrial biocatalysts must tolerate harsh environmental conditions, such as high temperatures and high concentrations of substrates (18). Thus, more-robust enzymes are needed to meet the requirements of biotechnological applications.…”

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confidence: 99%

Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

Ying

Wang

Xiong

et al. 2014

Appl Environ Microbiol

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A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the CAO bond of allylic aldehydes/ketones to the corresponding ␣,␤-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg ؊1 for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg ؊1 using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower K m values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP ؉ , suggesting the nature of being an aldehyde reductase.

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“…A rchaea represent an evolutionarily distinct class of organisms that include many extremophiles that function under conditions of high temperature, acidity, or salinity (1). These properties prompt interest in the potential use of their enzymes for biotechnology applications (2).…”

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Expression in Haloferax volcanii of 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Facilitates Isolation and Characterization of the Active Form of a Key Enzyme Required for Polyisoprenoid Cell Membrane Biosynthesis in Halophilic Archaea

VanNice¹,

Skaff²,

Wyckoff³

et al. 2013

Journal of Bacteriology

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Enzymes of the isoprenoid biosynthetic pathway in halophilic archaea remain poorly characterized, and parts of the pathway remain cryptic. This situation may be explained, in part, by the difficulty of expressing active, functional recombinant forms of these enzymes. The use of newly available expression plasmids and hosts has allowed the expression and isolation of catalytically active Haloferax volcanii 3-hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (EC 2.3.310). This accomplishment has permitted studies that represent, to the best of our knowledge, the first characterization of an archaeal hydroxymethylglutaryl CoA synthase. Kinetic characterization indicates that, under optimal assay conditions, which include 4 M KCl, the enzyme exhibits catalytic efficiency and substrate saturation at metabolite levels comparable to those reported for the enzyme from nonhalophilic organisms. This enzyme is unique in that it is the first hydroxymethylglutaryl CoA synthase that is insensitive to feedback substrate inhibition by acetoacetyl-CoA. The enzyme supports reaction catalysis in the presence of various organic solvents. Haloferax 3-hydroxy-3-methylglutaryl CoA synthase is sensitive to inactivation by hymeglusin, a specific inhibitor known to affect prokaryotic and eukaryotic forms of the enzyme, with experimentally determined K i and k inact values of 570 ؎ 120 nM and 17 ؎ 3 min ؊1 , respectively. In in vivo experiments, hymeglusin blocks the propagation of H. volcanii cells, indicating the critical role that the mevalonate pathway plays in isoprenoid biosynthesis by these archaea

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A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii

Cited by 38 publications

References 39 publications

Production of halophilic proteins using Haloferax volcanii H1895 in a stirred-tank bioreactor

Production of halophilic proteins using Haloferax volcanii H1895 in a stirred-tank bioreactor

Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

Expression in Haloferax volcanii of 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Facilitates Isolation and Characterization of the Active Form of a Key Enzyme Required for Polyisoprenoid Cell Membrane Biosynthesis in Halophilic Archaea

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