BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Archaeal alcohol dehydrogenase active at increasedtemperatures and in the presence of organic solvents

Matthias Hess & Garabed Antranikian

Received: 26 August 2007 /Revised: 1 October 2007 /Accepted: 3 October 2007 / Published online: 8 November 2007# Springer-Verlag 2007

Abstract The adhA gene of the extreme thermoacidophilicArchaeon Picrophilus torridus was identified by the meansof genome analysis and was subsequently cloned inEscherichia coli. PTO 0846, encoding AdhA, consists of954 bp corresponding to 317 aa. Sequence comparisonrevealed that the novel biocatalyst has a low sequenceidentity (<26%) to previously characterized enzymes. Therecombinant alcohol dehydrogenase was purified usinghydroxyapatite, and alcohol oxidative activity of thepurified AdhA was measured over a wide pH andtemperature range with maximal activity at 83°C andpH 7.8. Detailed analysis suggests that the active AdhA isa multimer, consisting of 12 identical subunits, with amolecular mass of 35 kDa each. AdhA represents the firstdodecameric alcohol dehydrogenase characterized until todate. AdhA is able to oxidize primary and secondaryalcohols with ethanol and 1-phenylalcohol as preferredsubstrates and NAD+ as preferred cofactor. In addition,isopropanol, which has been used successfully as cosubstratein cofactor regeneration, is oxidized as well by AdhA.Besides being thermostable (t1/2=42 min at 70°C), AdhA isalso active in the presence of increased concentrations ofurea (up to 5 M) and in the presence of organic solvents [upto 50% (v/v)] commonly used for organic synthesis.

Keywords Alcohol dehydrogenase . Picrophilus torridus .

Thermoactive enzyme

Introduction

Alcohol dehydrogenases (ADHs; EC 1.1.1.1) belong to theoxidoreductase family and are important for the detoxifica-tion and metabolism of ethanol and other alcohols. Theycatalyze the cofactor-dependent oxidation of primary andsecondary alcohols to the corresponding aldehydes andketones (Ribas De Pouplana et al. 1991; Littlechild et al.2004). Since the three-dimensional structure of horse liveralcohol dehydrogenase (LADH) was reported in 1976(Branden et al. 1973), ADHs from various organisms havebeen studied intensively, and several sequence character-istics were identified. ADHs bind the required cofactorthrough a double βαβαβ fold, resulting in a six-strandedβ-sheet surrounded by α-helices, also known as finger-print motif or Rossmann fold (Rossmann and Argos1981). Most of the early characterized dehydrogenaseswere reported to contain a Gly-x-Gly-x-x-Gly pattern, with“x” indicating any amino acid (Lesk 1995). The first twoglycines of this fingerprint pattern are involved in thebinding of the cofactor, whereas the third Gly is involved inthe close packing of the β-strands and the α-helix(Wierenga et al. 1985). As new members containing theRossmann fold have been identified and characterized, thegeneral pattern is now being described as Gly-x-(x)-Gly-x-(x)-Gly (Kallberg and Persson 2006).

ADHs are ubiquitous enzymes within the three domainsof life, and numerous microbial ADHs with a variety ofsubstrate specificities and physiological functions havebeen identified and characterized until to date. Althoughethanol metabolism is a recognized physiological role ofADH, some ADH isozymes show higher activity towardother substrates. The ability to utilize a variety of differentsubstrates suggests alternate roles for ADHs, and it remainsdifficult to determine the physiological reaction and

Appl Microbiol Biotechnol (2008) 77:1003–1013DOI 10.1007/s00253-007-1238-8

M. Hess :G. Antranikian (*)Institute of Technical Microbiology,Hamburg University of Technology,Kasernenstr. 12,21073 Hamburg, Germanye-mail: antranikian@tuhh.de

significance of an ADH (Ismaiel et al. 1993; Radianingtyasand Wright 2003). Microorganisms living under extremeconditions represent a promising source for catalysts thatcan be employed in industrial bioconversions, and severalADHs from these extremophiles have been identified andproposed as promising biocatalysts for a temperature rangebetween 0 and 50°C (Keinan et al. 1986; Tsigos et al.1998). Despite the fact that several thermoactive andthermostable ADHs have been characterized from thermo-philic and thermoacidophilic Archaea (Ammendola et al.1992; Kube et al. 2006; Machielsen et al. 2006), thedemand for ADHs that are active and stable at temperaturesup to 90°C is enormous. Hitherto, three extremelythermostable and thermoactive ADHs (maximal activityat 95°C) were characterized from the thermoacidophilicArchaeon Sulfolobus solfataricus (Rella et al. 1987;Cannio et al. 1996). Picrophilus torridus is the mostacidophilic organism known to date with a pH optimum forgrowth at pH 0.7. In addition, this Archaeon whose genomehas been sequenced recently (Futterer et al. 2004) can growoptimally at elevated temperatures (optimal growth at 60°C).It is, therefore, very likely that enzymes from this thermoa-cidophile will be robust and active under extreme conditions.

ADHs are employed efficiently in the synthesis ofoptical active alcohols, which are important buildingblocks in many chemical processes (i.e., synthesis ofpharmaceuticals, agrochemicals, and fragrances). In thereactions catalyzed by ADHs, the substrate and theproduct may be organic molecules (i.e., alcohols, alde-hydes, and ketones) which denature proteins, and theexpensive cofactors NAD+ and NADP+ are required foralcohol oxidation. Two procedures are feasible to recyclethe required cofactors. The first procedure, the “coupled-substrate system,” makes use of a single enzyme thatconverts substrate and cosubstrate simultaneously, whereasthe second, more complex “coupled-enzyme system”approach uses two distinct enzyme reactions. In thecoupled-substrate system, the equilibrium is shifted towardsthe desired produce by applying the cosubstrate in largeexcess (Cowan 1992; Kroutil et al. 2004; Edegger et al.2006). Hence, it is of great importance to identify newADHs stable and active in the presence of organic solventsand to evaluate their potential of improving the efficiencyof these processes (Cowan 1992; Egorova and Antranikian2005). Thermoacidophiles undoubtedly are a promisingsource for biocatalysts possessing those features of stabilityand activity under extreme reaction conditions (Egorova andAntranikian 2005).

In this work, we describe the cloning, expression, anddetailed biochemical characterization of AdhA, the firstADH from P. torridus, simultaneously representing the firstdodecameric ADH characterized until to date.

Materials and methods

Strains and growth conditions

P. torridus DSM 9790 was obtained from the DeutscheSammlung fuer Mikroorganismen und Zellkulturen(DSMZ) and was grown aerobically at 60°C and pH 0.7as described in 2002 (Serour and Antranikian 2002). Themedium contained (per liter): 1.32 g (NH4)2SO4, 0.28 gKH2PO4, 0.25 g MgSO4×7H2O, 0.07 g CaCl2×2H2O,0.02 g FeCl3×6H2O, 1.8 mg MnCl2×4H2O, 4.5 mgNa2B4O7×10H2O, 0.22 mg ZnSO4×7H2O, and 0.05 mgCuCl2×2H2O. The pH was adjusted to 0.9 with concen-trated H2SO4. Escherichia coli NovaBlue (Novagen,Madison, WI, USA) was used for blue/white screening oftransformants containing the ADH gene from P. torridus.For production of the recombinant ADH, E. coli Rosetta™(DE3) placI (Novagen) was used. The E. coli strains werecultivated aerobically in Luria–Bertani (LB; Sambrooket al. 1989). When necessary, antibiotics were supple-mented to the medium to maintain the plasmids (50 mg/lcarbenicillin, 50 mg/l chloramphenicol, and 50 mg/lkanamycin). Growth of all strains was analyzed bymeasuring the optical density (OD) at 600 nm.

DNA isolation and extraction

DNA from P. torridus was isolated by chemical lysis.Therefore, 0.1 g of P. torridus cells was resuspended in1 ml 1 M NaCl and incubated on ice for 1 h. Cells werecentrifuged, and the pellet was resuspended in 300 µl TE-saccharose buffer composed of 10 mM Tris–HCl, pH 8,1 mM EDTA, and 20% saccharose. Subsequently, 40 µlsodium dodecyl sulfate [SDS; 10% (w/v)], 5 µl RNAse(10 mg/ml), and 360 µl DNA extraction buffer composedof 100 mM Tris–HCl, pH 8, 100 mM Na2EDTA, 100 mMNa2HPO4, 1.5 mM NaCl, and 1% cetyltrimethyl ammo-niumbromide were added. The mixture was incubated at37°C for 4 h. Subsequently, 300 µl of a 5% sarcosylsolution and 10 µl proteinase K solution (10 mg/ml) wereadded. Samples were incubated at 37°C overnight, andDNA was extracted by phenol/chloroform standard proce-dure (Sambrook et al. 1989).

Cloning and expression of PTO 0846 (adhA)

Open reading frame (ORF) encoding for the ADH wasidentified in the whole genome sequence of P. torridus(Futterer et al. 2004) using the ERGO software package(Integrated Genomics, Chicago, IL, USA). Amplification ofthe genetic region was conducted in a thermocycler(Biometra, Germany) by means of polymerase chain

1004 Appl Microbiol Biotechnol (2008) 77:1003–1013

reaction (PCR) using Platinum®Taq DNA Polymerase HighFidelity (Invitrogen, Carlsbad, USA). The synthetic forwardand reverse primers used for amplification of adhAwere 5′-ATGAGGGCAATAGTTCTTGA-3′, 5′-TTAAGCTTTTAATAGTATTCTGCC-3′, respectively. Primers were pur-chased from MWG Biotech, Germany (HPSF grade, highpurity, salt free), and PCR products were analyzed at GATCBiotech (Germany) using the dideoxy chain terminationmethod (Sanger et al. 1977). Obtained PCR products werepurified using the NucleoSpin-Plasmid kit (Macherey-Nagel, Dueren, Germany) and sub-cloned into the vectorpETBlue-1 AccepTor (Novagen). The resulting plasmidspETadhA was transformed into E. coli NovaBlue, andpositive clones were identified by blue/white screeningfollowed by colony PCR. Plasmids, containing an insert ofthe correct size, were extracted from transformants andtransformed into the expression host E. coli Rosetta (DE3)placI. Successful uptake of the plasmid was confirmedusing selective LB-agar plates. Expression of adhA wasinduced with 5 mM isopropyl-thiogalactopyranoside(IPTG), after the cells reached an OD of 0.8 at 600 nm.

DNA sequence analysis

Expression plasmid pETadhA was purified from therecombinant E. coli strains and sequenced using theBigDye Terminator v3.1 Cycle Sequencing Kit (AppliedBiosystems, Foster City, CA, USA). Sequence analysis wasperformed using Artemis (Berriman and Rutherford 2003)and Vector NTI Advance (Invitrogen) under a privatelicense. Chromatograms were analyzed with Chromas(Technelysium Pty, Australia). Sequence homology wasinvestigated using NCBI-BLAST (Altschul and Lipman1990; Altschul et al. 1997) provided by the National Centerfor Biotechnlology Information (NCBI), Bethesda, MD,USA. Multiple alignments were done using ClustalW andClustalX (Thompson et al. 1994, 1997). Nucleotide andamino acid sequences were retrieved from The Institute ofGenomic Research (http://www.tigr.org), Genomes OnLineDatabase (GOLD 2006), and from nucleotide, genome,protein, and structure databases provided by NCBI (http://www.ncbi.nlm.nih.gov/). SignalP version 3 was used toanalyze proteins for the presence of potential signal peptidesequences (Bendtsen et al. 2004).

Purification of the recombinant alcohol dehydrogenase

Cells were grown overnight at 30°C, harvested bycentrifugation (24,000×g for 30 min at 4°C), and the pelletwas resuspended and washed in ice-cold 50 mM Tris–HClbuffer (pH 8). Supernatant was concentrated ×20, andprotein composition was examined. The cells were dis-

rupted at 4°C by ultrasonification (Branson sonifier 450,duty cycle 40%, and output control level 4). Cell lysis wasverified by light microscopy. Cell debris was removed bycentrifugation (60,000×g for 1 h at 4°C). The cell-freesupernatant, from now on referred to as crude extract, wasdialyzed overnight at 4°C against 10 mM Na2HPO4 buffer(pH 6.8). Three milliliters of dialyzed sample was appliedto a hydroxyapatite chromatography (HAC) column Type I(Bio-Rad, Hercules, CA, USA) equilibrated with 10 mMNa2HPO4 buffer (pH 6.8) using a low pressure Econosystem (Bio-Rad). The total bed volume of the column was35 ml, and loaded samples were eluted with a linearincreasing Na2HPO4 gradient (10–400 mM), after thecolumn had been washed twice with 10 mM Na2HPO4

buffer (pH 6.8). Salt concentration and elution of proteinwere monitored continuously. Fractions of 4.5 ml werecollected and analyzed for ADH activity and proteincomposition. The samples containing ADH activity werepooled and dialyzed overnight against 50 mM Tris–HCl(pH 7.8) at 4°C. Dialyzed samples were stored at 4°C andused for further characterization. After each run, the HACcolumn was regenerated with three to five column volumesof 400 mM Na2HPO4 buffer (pH 6.8) and washed with twovolumes of 10 mM Na2HPO4 buffer (pH 6.8) containing20% (v/v) ethanol.

Gel electrophoresis

SDS-polyacrylamide gel electrophoresis (PAGE) was per-formed to analyze the protein composition of the samples.SDS-PAGE was carried out with 4% polyacrylamide/12%polyacrylamide gels (stacking gel/separating gel) in a MiniProtean II electrophoresis system (Bio-Rad, Richmond,CA, USA; Laemmli 1970). Samples were incubated inreducing sample buffer with the following concentrations:63 mM Tris–HCl, pH 6.8, 10% (v/v) glycerol, 0.0025%(w/v) bromphenol blue, 4% (w/v) SDS, 1.25% (w/v)dithiothreitol (DTT) at 95°C for 5 min before loading.After electrophoresis, protein bands were stained withCoomassie brilliant blue R-250. Low-molecular-weight(LMW) marker (Amersham, Germany) was used as stan-dard. The LMW marker contains the standard proteins asfollows: phosphorylase b (97 kDa), bovine serum albumin(66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa),trypsin inhibitor (20.1 kDa), α-lactalbumin (14.4 kDa).

Non-denaturing PAGE was performed with gradient gels(Novex pre-cast gels, 4–20% polyacrylamide) purchasedfrom Invitrogen. Samples were mixed with non-reducingsample buffer [final concentration 63 mM Tris–HCl,pH 8.8, 0.0025% (w/v) bromphenol blue, 10% (v/v)glycerol] before loading. High-molecular-weight (HMW)marker (Amersham, Germany) was used as standard to

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estimate the subunit composition. The HMW markercontains the standard proteins as follows: thyroglobulin(669 kDa), ferritin (440 kDa), catalase (232 kDa), lactatedehydrogenase (140 kDa), and albumin (66 kDa).

Activity detection and measurement

ADH activity was determined spectrophotometrically at340 nm by measuring the amount of produced NADHaccording to a method published previously (Lamed andZeikus 1980; Lamed and Zeikus 1981). The alcohol/NAD+

mixture [1 mM alcohol, 1.5 mM NAD+, 50 mM Tris–HCl(pH 7.8)] was pre-warmed, and the reaction was initiated byaddition of 11 mU/ml AdhA. NAD+ was replaced byNADP+ to determine the cofactor preference of AdhA.Oxidation of alcohols was carried out at 83°C for 30 minand was terminated by placing the samples on ice. Sampleswere centrifuged for 2 min at 9,400×g. The amount ofNADH was measured photometrically at 340 nm. For thestandard assay, the substrate used was 1 mM ethanol.Substrate specificity towards 1° and 2° alcohols wasinvestigated with ethanol, methanol, 1-propanol, pentanol,2-aminoethanol, 2-propanol, racemic phenylethanol, andracemic 2,3-butanediol as substrate. One unit of enzymewas defined as the amount of enzyme resulting in therelease of 1 µmol of NADH per min. If not statedotherwise, the values are the mean of triplicates. Theextinction coefficient (ɛ340) was 6,220 M−1 cm−1. Measure-ments were corrected for autohydrolysis of the substrate.Enzymatic assays to determinate the kinetic values weredone at different ethanol concentrations, in three indepen-dent trials. The corresponding values were computed usingthe Michaelis–Menten equation and the BioDataFit pro-gram (Chang Bioscience, Castro Valley, CA, USA), and thevalues given represent the calculated mean.

Influence of temperature and pH

Enzyme activity was determined over a temperature rangefrom 30 to 100°C. The assays were performed under theassay standard conditions at pH 7.8. Effect of pH on ADHactivity was determined over a pH range of 2–9 in 40 mMuniversal buffer (Britton and Robinson 1931) at 83°C.Thermostability of the enzymes was evaluated by incuba-tion of AdhA at temperatures between 30–90°C for up to3 h. Standard assays were conducted to determine theresidual enzyme activity.

Effect of diverse substances

The influence of metal ions was investigated by addingselectively 10 mM of Al3+, Ca2+, Co2+, Cr3+, Cu2+, Fe3+,K+, Mg2+, Mn2+, Na+, Ni2+, or Zn2+ directly to the standard

assay mixture. Enzyme activity determined in the absenceof additional metal ions was defined as 100% activity.

The influence of various other substances such asinhibitors, detergents, and organic solvents was investigatedusing a slightly modified protocol. Before the standardactivity assay, the enzyme was incubated for 1 h at RT in apre-incubation mixture, containing selectively an inhibitor,detergent, or organic solvent. The reaction was performedunder standard assay conditions and was initiated byaddition of the NAD+/ethanol mixture. The concentrationof the additive during the standard assay was as follows:10 mM inhibitor, 10% detergent, and 50% (v/v) or 90% (v/v)organic solvent. Concentration of urea was 1, 3, or 5 M.Enzyme activity determined for samples without additivewas defined as 100% activity.

Sequence accession number

The adhtA sequence is available in the GenBank databaseunder the accession number AAT43431.

Results

Identification and sequence analysis of AdhA

Analysis of the complete genome of P. torridus for thepresence of conserved regions distinctive for ADHsrevealed the presence of an ORF, namely, PTO 0846,possessing sequence homology to previously reportedADHs. PTO 0846 consists of 954 bp corresponding to317 aa. Multiple sequence alignment of the AdhA sequencewith the amino acid sequences of the previously character-ized Acinetobacter sp. strain M-1 AlrA (Tani et al. 2000),Aeropyrum pernix K1 AePxADHI (Guy et al. 2003), S.solfataricus SsADH (Ammendola et al. 1992), Sulfolobussp. RC3ADH (Cannio et al. 1996), and Pyrococcus furiosus

Fig. 1 Sequence comparison of AdhA. Sequence comparison of theP. torridus alcohol dehydrogenase AdhA with sequences frompreviously characterized ADHs [Acinetobacter sp. strain M-1 AlrA(Tani et al. 2000), A. pernix K1 AePxADHI (Guy et al. 2003), S.solfataricus SsADH (Ammendola et al. 1992), Sulfolobus sp.RC3ADH (Cannio et al. 1996), and P. furiosus ADHC (Kube et al.2006)]. Identical amino acids are white letters on black background.Conserved amino acids are white letters on dark grey background.Weakly similar and non-similar amino acids are black letters on whitebackground. The structural zinc-binding site is located in Box I, andthe conserved Cys residues at position 90, 93, and 101 are indicatedby triangles. The conserved Gly-x-Gly-x-x-Gly hexapeptide atposition 167 to 172 is underlined. The short hydrophobic region isindicated by circles, and the conserved Lys and Asp at position 153and 194, respectively, characteristic for the Rossmann fold, areindicated by an asterisk. Position of residues refers to P. torridusAdhA sequence

b

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ADHC (Kube et al. 2006) suggests a conserved Gly-x-Gly-x-x-Gly hexapeptide from position 167 to 172. In addition,a short hydrophobic region located just N-terminal of thehexapeptide and a Rossmann fold consisting of a conservedLys at position 153 and a conserved Asp at position 194were identified in the AdhA sequence (Fig. 1). Sequenceanalysis indicated that P. torridus AdhA possesses anextremely low sequence identity with other describedADHs, and the highest sequence identity was found forSsADH from S. solfataricus (Ammendola et al. 1992) witha sequence identity of 25%. Sequence identity of AdhAwith previously characterized enzymes was determined asfollows: 22, 9, 7, 7, 20, 24, 22, 16, and 16% withAcinetobacter sp. strain M-1 AlrA (Tani et al. 2000),Thermoanaerobacter ethanolicus lAdhA (Holt et al. 2000),Thermococcus hydrothermalis ADHA (Antoine et al.1999), Thermococcus strain AN1 ADH (Li and Stevenson1997), A. pernix K1 AePxADHI (Guy et al. 2003),Sulfolobus sp. RC3ADH (Cannio et al. 1996), P. furiosusADHC (Kube et al. 2006), T. ethanolicus 39E ADHB(Burdette et al. 1996), and T. brockii TBAD (Peretz andBurstein 1989), respectively.

Neural networks and hidden Markov models, trained onboth eukaryotes and prokaryotes, were applied on theamino acid sequence of the putative ADH from P. torridusto predict potential signal peptide sequences. No potentialsignal peptide sequence was identified. Furthermore, aGroES-like domain between residue 28 and 129 and astructural zinc-binding domain between Cys90 and Cys101were identified by comparing the AdhA sequence withpreviously published 3D protein structures.

Cloning and expression of adhA

Protein-free DNA from P. torridus was used as template forPCR-based amplification of ORF PTO 0846. The PCR-obtained product (designated adhAPCR) was purified andcloned successfully, and the constructed plasmid (pET-BadhA) was transformed into electrocompetent E. coliNovaBlue cells with a transformation efficiency of 1.2×107

transformants/µg. Subsequently, transformants carrying theDNA fragment of interest were identified by blue/whitescreening, and the correct orientation of the insert wasconfirmed by colony PCR. The purified pETBadhA wastransformed into E. coli Rossetta DE3 placI with atransformation efficiency of 1.6×107 transformants/µg.Successful transformation of pETBadhA in the expressionhost E. coli Rossetta DE3 placI was confirmed by theacquired resistance to ampicillin/carbenicillin, and colonyPCR of selected transformants.

Production of soluble AdhA was achieved by inducingadhA expression with 5 mM IPTG, and ADH activity wasdetected after cell harvest and preparation of the crudeextract derived from E. coli Rosetta (DE3) placI pET-BadhA. Alcohol oxidation was detected with the spectro-photometrical standard assay, and measurements of NADHproduction were corrected with E. coli Rosetta DE3pETBlue-1 crude extract as negative control. SpecificADH activity of 11 U/g was determined for E. coli Rosetta(DE3) placI pETBadhA crude extract and SDS-PAGE wasused to analyze the protein pattern of supernatant and crudeextract prepared from E. coli Rosetta (DE3) placI pET-BadhA and E. coli Rosetta DE3 pETBlue-1. An additional

Fig. 2 SDS PAGE and Native PAGE of AdhA. Protein pattern of E.coli Rosetta DE3 placI pETBlue-1 and E. coli Rosetta DE3 placIpETBadhA were analyzed using SDS-PAGE (a). When compared tothe protein pattern of the negative control E. coli Rosetta DE3 placIpETBlue-1 (lane 2), a prominent band at 35 kDa was detected in theprotein pattern of E. coli Rosetta DE3 placI pETBadhA (lane 3) afterthe gel had been loaded with 6 µg protein and stained with Coomassieblue R250. Lane 1 contains the LMW marker (Amersham, Germany).In addition, molecular mass of purified AdhA under denaturing and

non-denaturing condition was determined by SDS-PAGE (b) andNative-PAGE (c). Samples containing 4 µg purified AdhA wereloaded to lane 2 of both gels. Lane 1 (b) contains the LMW marker(Amersham, Germany). Lane 1 (c) contains the HMW native marker(Amersham, Germany). After electrophoresis under denaturing condi-tion and subsequent protein staining, a single band of 35 kDa wasvisible in lane 2 (b), whereas a band of 420 kDa was visible in lane2 of the gel exposed to electrophoresis under non-denaturingcondition (c)

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band was detected in the E. coli Rosetta (DE3) placIpETBadhA crude extract (Fig. 2), and a molecular weightof 35 kDa was calculated using the migration length Rf.

Purification of AdhA and kinetic studies

The recombinant ADH was purified in an extremelyefficient one-step purification procedure, during whichHAC was applied. AdhA was eluted between 270 and300 mM Na2HPO4, and obtained results of the AdhApurification are summarized in Table 1. Sixty-three percentof the initial ADH activity previously determined in the E.coli Rosetta DE3 placI pETBadhA crude extract and aspecific activity of 225 U/g were measured for the purifiedAdhA after the samples had been pooled and dialyzed,commensurating to a 21-fold purification factor.

SDS-PAGE and native-PAGE of purified AdhA wereperformed to confirm the homogeneous purification and todetermine the molecular mass of the protein underdenaturing and under non-denaturing condition. Resultsobtained from both SDS-PAGE and native-PAGE verifiedthat HAC was used successfully to purify AdhA tohomogeneity. Furthermore, the results suggest that activeAdhA is a homododecamer composed of 12 subunits with amolecular mass of 420 kDa (35 kDa/subunit; Fig. 2). Aturnover rate of 8.6×10−4 s−1 and a Km of 56 µM/min werecalculated for AdhA with ethanol as substrate.

Substrate specificity

ADH activity of the recombinant enzyme towards primary(1°), secondary (2°), and aromatic alcohols was quantifiedby measuring the formation of NADH spectrophotometri-cally at 340 nm. The recombinant ADH was found tohydrolyze both 1° and 2° alcohols to the correspondingaldehydes and ketones with NAD+ as cofactor. Ethanol and1-phenylethanol were the most favored substrates and wereoxidized by AdhA with a specific activity of 225 and234 U/g, respectively. Specific activity towards methanol,1-propanol, 2-propanol, pentanol, 2-aminoethanol, and 2,3-butanediol were 77, 110, 86, 101, 142, and 115 U/g,respectively. Relative activity of AdhA with ethanol as

substrate and NADP+ as cofactor was only 8.5%, whencompared to AdhA activity with ethanol as substrate andNAD+ as cofactor.

Influence of temperature and pH

Alcohol oxidation catalyzed by recombinant AdhA wasinvestigated over a temperature range from 30 to 100°Cusing the spectrophotometrical standard assay with 1 mMethanol as substrate and 1.5 mM NAD+ as cofactor. Thederived results (Fig. 3) indicate that the recombinant ADHis active over a broad temperature range, and ethanoloxidation is maximal at 83°C. In the temperature rangebetween 50 and 65°C, corresponding to the temperaturefound in the habitat of P. torridus, the measured alcoholoxidation was suboptimal (<30%). Despite a significantdecrease in temperatures above 83°C, a relative activity of9% was determined at 95°C for AdhA. AdhA was found tobe moderately stable at temperatures as high as 90°C.Residual activity of AdhA came to 41, 31, 24, and 20%after 2 h of preincubation and to 12, 6, 3, and 1% after 3 hof preincubation at 60, 70, 80, and 90°C, respectively. Thedetermined half-life of AdhA was 114, 110, 96, 55, 42, 18,and 4 min at 30, 40, 50, 60, 70, 80, and 90°C, respectively.

Enzyme activity was analyzed between pH 2 and pH 9,using the standard assay and 40 mM universal buffercontaining phosphoric, acetic, and boric acid. Maximalactivity of AdhA was measured at a slightly alkalic pHvalue of 7.8. AdhA was found to be active in the rangebetween pH 3 and pH 8.5. Forty-one and 49% of maximalactivity were measured at pH 7 and pH 8, respectively.AdhA activity decreased significantly when the pH value ofthe assay was adjusted to values below pH 7 or above pH 8.AdhA was found to retain 10% of its maximal activityunder acidic conditions (pH 3–6).

Table 1 Purification of recombinant AdhA from Picrophilus torridus

Purificationstep

Totalprotein(mg)

Totalactivity(mU)

Specificactivity(mU/mg)

Yield(%)

Purificationfactor(fold)

Crudeextract

43.7 470 11 100 1

HAcolumn

1.3 290 225 63 21

HA column Hydroxyapatite column

Fig. 3 Temperature profile of AdhA. Enzyme activity of AdhA wasdetermined over a temperature range from 30 to 100°C using thestandard assay. The ethanol/NAD+ mixture [1 mM ethanol, 1.5 mMNAD+, 50 mM Tris–HCl (pH 7.8)] was prewarmed, and the reactionwas initiated by addition of 11 mU AdhA. Oxidation of ethanol wascarried out in 1 ml for 30 min and was terminated by placing thesamples on ice. Samples were centrifuged for 2 min at 9,400×g. Theamount of NADH was measured photometrically at 340 nm. Measure-ments were corrected for autohydrolysis of the substrate

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Effect of metal ions, denaturing agents, and organicsolvents

The effect of various metal ions was investigated byperforming the standard ADH assay in the presence of either10 mM Al3+, Ca2+, Co2+, Cr3+, Cu2+, Fe3+, K+, Mg2+, Mn2+,Na+, Ni2+, or Zn2+. With the exception of Al3+, K+, and Ni2+,the supplemented ions did not affect the alcohol oxidationsignificantly (Table 2). Addition of 10 mM Al3+, K+, andNi2+ decreased the activity of AdhA by 30, 29, and 46%,respectively. AdhA activity in the presence of 10 mM Fe3+,Zn2+, Ca2+, Co2+, Cr3+, Cu2+, Mg2+, Mn2+, and Na+ was101, 103, 94, 94, 92, 98, 89, 91, and 87% correspondingly,when compared to AdhA activity measured in the absence ofadditional metal ions.

The effect of non-ionic (Tween20, Tween80, Triton-X100), zwitterionic (CHAPS), ionic (SDS) detergents,

various inhibitors, and denaturing agents on ADH activitywas investigated by preincubation of AdhA in the presenceof a sole reagent, before conducting the standard assay. Theresults are summarized in Table 2. All detergents usedduring this study reduced AdhA activity significantly.Zwitterionic CHAPS reduced the activity by 59%, whereasthe ionic SDS and the non-ionic Tween20, Tween80, andTriton-X100 reduced AdhA activities even by 82, 89, 78,and 95%, respectively. The addition of 10 mM sulfidereducing DTT led to a slight increase in ADH activity(114%). The serine modifying agents PMSF and Pefablocreduced the AdhA activity to 92 and 68%, respectively,whereas the cation-chelating agent EDTA had no effect onthe catalytic activity of AdhA. AdhA activity was notaffected significantly by 1 M urea, but decreased to 83 and59% in the presence of 3 and 5 M urea, respectively.

Furthermore, the effect of 50% (v/v) polar and 90% (v/v)non-polar solvents on the activity of the recombinant ADHwas investigated. In general, both polar and non-polarsolvents reduced the catalytic activity of AdhA. Addition ofisooctane, heptane, and toluene reduced the activity to 2, 1,and 3%, respectively. A reduction of about 30% wasmeasured in the presence of 50% (v/v) DMSO andmethanol, whereas 50% (v/v; 8.6 M) of the substrateethanol reduced the activity of the recombinant enzyme to41%. Ninety percent (v/v) hexadecane reduced the activityof the recombinant AdhA to 29% (Table 3). Inhibition athigh substrate concentrations has been reported for numer-ous ADHs, and the reduction in AdhA activity at highsubstrate concentrations is not peculiar (Kube et al. 2006).

Table 2 Influence of metal ions and various reagents on AdhA

Compound Concentration Relative activityof AdhA (%)

None – 100Al3+ 10 mM 70Ca2+ 10 mM 94Co2+ 10 mM 94Cr3+ 10 mM 92Cu2+ 10 mM 98Fe3+ 10 mM 101K+ 10 mM 71Mg2+ 10 mM 89Mn2+ 10 mM 91Na+ 10 mM 87Ni2+ 10 mM 54Zn2+ 10 mM 103CHAPS 10% (w/v) 41SDS 10% (w/v) 5Triton-X100 10% (v/v) 22Tween20 10% (v/v) 18Tween80 10% (v/v) 11DTT 10 mM 114EDTA 10 mM 105Pefabloc 10 mM 68PMSF 10 mM 92Urea 1 M 105

3 M 835 M 59

Influence of metal ions and various reagents was investigated usingthe standard assay. Before the standard activity assay, the enzyme(11 mU) was incubated for 1 h at RT in a preincubation mixture,containing selectively a metal salt, a detergent, an inhibitor, or adenaturing agent. The reaction was initiated by addition of 1.5 mMNAD+ and 1 mM ethanol. Oxidation of ethanol was carried out in1 ml at 83°C for 30 min (50 mM Tris–HCl, pH 7.8) and wasterminated by placing the samples on ice. Samples were centrifugedfor 2 min at 9,400×g. Enzyme activity determined in the absence ofdetergents, inhibitors, and denaturing agents was defined as 100%activity.

Table 3 Influence of various solvents on AdhA

Compound Concentration Relative activityof AdhA (%)

None – 100Acetone 50% (v/v) 18DMSO 50% (v/v) 71Ethanol 50% (v/v) 41Isopropanol 50% (v/v) 12Methanol 50% (v/v) 70Tert. Butanol 50% (v/v) 8Heptane 90% (v/v) 1Hexadecane 90% (v/v) 29Isooctane 90% (v/v) 2Toluene 90% (v/v) 3

Influence of various solvents was investigated using the standardassay. Before the standard activity assay, the enzyme (11 mU) wasincubated for 1 h at RT in a preincubation mixture, containing onesolvent. The reaction was initiated by addition of 1.5 mM NAD+ and1 mM ethanol. Oxidation of ethanol was carried out in 1 ml at 83°Cfor 30 min (50 mM Tris–HCl, pH 7.8). Enzyme activity determined inthe absence of solvents was defined as 100% activity, whichcorresponds to 225 U/g.

1010 Appl Microbiol Biotechnol (2008) 77:1003–1013

In addition, AdhA was also active in the presence of 50%(v/v) acetone, isopropanol, and tertiary butanol.

Discussion

Analysis of the complete P. torridus genome revealed thepresence of adhA containing regions typical for the so-called Rossmann fold, which has been reported as structuralprerequisite for nicotinamide binding (Kallberg and Persson2006). Despite the conserved Rossmann fold and specificdetectable motifs, a sequence identity of only 15–30% istypically observed for members within one of the threedistinct ADH types (Persson et al. 2003). This observationwas in accordance with the sequence identities derivedfrom the sequence comparison conducted during thiswork. Highest sequence identity of AdhA was found withS. solfataricus SsAdh (Ammendola et al. 1992) andSulfolobus sp. RC3 Adh (Cannio et al. 1996) with 25 and24%, respectively.

Whereas an MW of 35 kDa per subunit is within therange of the molecular mass of previously reported ADHmonomers, data from non-denaturing PAGE suggest that thenative AdhA is a dodecamer. Analysis of the currentliterature revealed that dodecamers are common for gluta-mine synthetases (Bhatnagar et al. 1986) and formation ofdecamers and dodecamers has been observed for esterases,ornithine carbamoyltransferases, and DNA-binding proteinsfrom starved cells (Villeret et al. 1998; Honda et al. 2002;Ceci et al. 2005). On the contrary, no ADH consisting ofmore than six subunits has been reported so far (Ma andAdams 1999). Early evidence for protein stabilization viaoligomerization was demonstrated by fragmentation andmodeling studies on a lactate dehydrogenase and triosephos-phate isomerases, respectively (Opitz et al. 1987; Kohlhoffet al. 1996). Recent studies provide additional evidence thatoligomerization is an efficient mechanism to stabilizeproteins under various hostile conditions such as extremelyhigh temperatures (Villeret et al. 1998) or high concen-trations of chemical agents (Ceci et al. 2005).

Regardless of the identified structural zinc-binding site,AdhA activity was not affected by ZnCl2 or the metal-chelating EDTA. Identical results were obtained for thetetrameric ADH from T. brockii grown on complex media(Lamed and Zeikus 1981). A possible explanation wasprovided recently by the observation that the tetramericADH from A. pernix forms a stabilizing disulfide boundwithin the structural zinc-binding site, hence maintainingthe structural integrity when zinc is absent or limited (Guyet al. 2003). In addition, extreme tight binding of theavailable zinc ions might promote the stability and theinsensibility to metal-chelating agents (Lamed and Zeikus1981). It was also shown that Asn249 is important for theaffinity of SsAdh towards both substrate and cofactor.

Replacement of Asn249 with Tyr reduced the affinity butsimultaneously enhanced the low activity of SsAdh due to afaster release of the cosubstrate (Giordano et al. 2005;Secundo et al. 2005). Within AdhA, an Asn249 equivalentis found at position 217, and it is likely that the relativelylow specific activity is partially caused by a slow release ofthe cosubstrate. The fingerprint motif was highly conservedwithin the studied ADHs, and the three characteristic Glyresidues were identified at position 167, 169, and 172 of theAdhA sequence (Fig. 1). Based on these findings, AdhAcan be categorized as medium-chain (Type I) ADH.

Based on its broad temperature profile, AdhA can beemployed efficiently at operating temperatures between 60and 95°C. The demand for biocatalysts like SsAdh andAdhA that can be employed at temperatures up to 90°C isenormous (Rella et al. 1987). Interestingly, the type IIIADH from T. hydrothermalicus has a similar temperatureprofile (Tempopt, 80°C) but is considerably less thermosta-ble than AdhA, emphasizing the significance of AdhA forlarge-scale industrial processes. Some ADHs require addi-tional activation before they catalyze the oxidation ofalcohols, and it was suggested that they might be inhibitedor simply be inactive until structural rearrangements occur(Antoine et al. 1999; Kube et al. 2006). It remains unknownif AdhA requires such extrinsic activators or if the activityof AdhA is simply limited by the mesophilic expressionhost. Until recently, no alternatives for the expression ofthermophilic proteins from Archaea were available, but newhigh-temperature expression systems such as S. solfataricusare being developed and represent promising tools toimprove catalytic activity of otherwise inactive biocatalysts(Albers et al. 2006).

Irrespective of the necessity to determine the optimalconditions for any biocatalyst to evaluate its industrialvalue, investigation of the effect of various agents andmetal ions is of particular importance for novel ADHs, asthe ADH-classification system distinguishes between zinc-dependent, short-chain, and iron-activated ADHs. BesidesFe3+ and Zn2+, the cations Ca2+, Mg2+, Mn2+, and Na+ havebeen reported as activators and stabilizer for dehydro-genases, and their effect on novel ADHs is of specialinterest (Tsigos et al. 1998; Chinnawirotpisan et al. 2003;Kuznetsova et al. 2005). AdhA remains active in thepresence of any metal used during this study and in thepresence of urea to concentrations up to 5 M, strengthen-ing once again the catalytic potential of AdhA. NeitherEDTA nor Zn2+ affects AdhA, despite the structural zinc-binding site identified by sequence analysis. Similarresults were obtained for the Adh from T. brockii, ADHI from A. pasteurianus SKU1108, and for SsAdh from S.solfataricus (Ammendola et al. 1992; Chinnawirotpisanet al. 2003). The activity of type I ADH from Moraxellasp. that contains one zinc atom per subunit was reduced

Appl Microbiol Biotechnol (2008) 77:1003–1013 1011

after EDTA treatment and in the presence of 0.1 mMZnCl2, but almost doubled its activity in the presence ofCaCl2 (Tsigos et al. 1998).

Given the tremendous potential of bioconversion inorganic solvents, it is not surprising that considerableefforts are made to identify novel biocatalysts, which areactive and stable in pure solvents and in aqueous-organicsolvent mixtures (Klibanov 2001). Hence, it was of greatinterest to study the ability of AdhA to perform bioconver-sion under enhanced concentrations of organic solvents.Interestingly, the recombinant ADH retained approximately70% of its activity in the presence of 50% (v/v) methanoland DMSO and 41% in the presence of 50% (v/v) ethanol,which are hydrophilic organic solvents commonly used fororganic synthesis (Iwase et al. 2006). Furthermore, AdhAmaintained 12% of its activity in 50% (v/v) isopropanol,which can be utilized as substrate (0.11 U/mg) and has beenused successfully as cosubstrate in cofactor regeneration(Keinan et al. 1986; Bradshaw et al. 1992; Itoh et al. 2002;Stampfer et al. 2003).

Acknowledgment This work was supported by grant 04-008202131 from the German BMBF. We would like to thank MoritzKatzer for his support.

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