FR2706906A1 - Alcohol dehydrogenase, microorganism producing it and its uses - Google Patents

Abstract

Thermostable and thermophilic alcohol dehydrogenase having an enantioselective oxidative activity. This enzyme advantageously has a mass of the order of 80 kD and an optimum activity at a pH of about 8.3. It is produced by a strain belonging to the Archaebacteria kingdom. This enzyme can be used for the preparation of alcohols, aldehydes and ketones.

Description

The subject of the present invention is an alcohol-

  thermostable dehydrogenase as well as a strain of

microorganism producing it.

  It further relates to uses of this enzyme. Alcohol dehydrogenases (ADH) catalyze the oxidation reaction of an alcohol to an aldehyde as well as the reduction of an aldehyde to alcohol. They are also able to reduce ketones. In general, they do not have strict specificity and can react with alcohols

  linear, branched aliphatic chain, aromatic.

  However, some DHAs have specificity for

primary or secondary alcohols.

  ADHs require a cofactor which can be NAD + / NADH, H + or NADP + / NADPH, H + or NADP + / NADPH, H + as well as analogs. Activity and structure may depend on

the presence of Zn2 +.

  The potential applications of ADH in industry are numerous: stereospecific synthesis, synthesis of aldehyde or ethers aromas used in the food industry,

  cosmetics and synthesis of various precursors.

  The use of mesophilic enzymes is limited by relatively narrow specificity, thermal instability and

  brittleness towards organic solvents.

  DHAs have been isolated from different sources: plants, animal tissues, microorganisms. Two thermostable DHAs, the DHA of Thermoanaerobium brockii and that of

  Sulfolobus solfataricus, have been studied.

  The ADH of Thermoanaerobium brockii is NADP dependent and was originally described by Lamed and Zeikus (1981, Biochem J. 195 (2), 183-190). The cellular extracts of T. brockii actually contain two ADH activities: one dependent on NAD, not very active and unstable with oxygen, the other

dependent on NADP.

  Lamed and Zeikus (1981-Bîochem J., 195 (2), 183-190) as well as Al-Kassim and Tasi (1990-Biochem. Cell. Biol., 68, 907-913) have purified and characterized this enzyme. It is active on linear primary alcohols (maximum activity for butanol), non-linear primary alcohols, cyclic secondary alcohols, aldehydes and ketones. It is more active on secondary alcohols and acts according to a

  bi-ordered mechanism with NADP + as the first reactant.

  It reduces asymmetrically acyclic aliphatic ketones, producing an optically chiral compound

  pure (Keinan et al., 1980, J. Am. Chem. Society, 108, 162-

169).

  Lamed et al (1981, Enzyme Microb. Technol., 3, 144-

  148) studied the properties making it possible to envisage applications for this enzyme: enantioselective reduction, regeneration of NADPH, immobilization, stability in organic solvent. Rella et al. (1987 Eur. J. Biochem., 167, 475-479) described the DHA of the Archaebacterium Sulfolobus solfataricus. This enzyme has a broad substrate specificity which includes primary and branched alcohols, linear and cyclic secondary alcohols as well as anisaldehyde. It is

specific to NAD.

  It has been studied for the regeneration of cofactors (Guagliardi et al., 1991, Biotech. Appl. Biochem., 13,25-35)

  and for the synthesis of odorous aldehydes in solid reactors-

  gas. The properties of thermostable enzymes allow them to be used under industrial conditions in which

  normal enzymes could not be used.

  Thus the use of high temperatures makes it possible to improve mass transfers by increasing the solubility and the diffusion, to decrease the viscosity of the media and therefore to be able to work in the presence of high concentrations of substrates, to reduce the risks of contamination. , increase conversion yields and use solid-gas reactors, which

  facilitates the recovery of volatile products.

  It appears from the analysis of the state of the art that, to the knowledge of the applicant, there are only two thermostable dehydrogenase alcohols and that these enzymes have specific selectivities making them usable only in

a reduced range of applications.

  To the knowledge of the applicant, they do not in particular have enantioselectivity with regard to their

oxidative activities.

  The applicant therefore set out to find an enzyme with significant stability at high temperature, a broad spectrum of action on different

  substrates and enantioselectivity.

  The Applicant has surprisingly found that an enzyme produced by a strain of thermophilic microorganisms responds to its. criteria. He also showed that this

  enzyme could be produced by this microorganism.

  The present invention therefore relates to a thermostable and thermophilic alcohol dehydrogenase characterized in that it has an enantioselective oxidative activity. Such an enzyme can have an isoelectric weight of approximately

  4.5. It advantageously has an optimal activity at pH 8.3.

  Preferably, such an enzyme has a mass

of the order of 80 kD.

  It can be activated by NaCl and be inhibited separately by benzaldehyde, CuSO4, Sodium Dodecyl Sulfate

(SDS), ZnC12, and i'EDTA ..

  Preferably, such an enzyme uses NADP +

as a co-factor.

  Advantageously, this enzyme is specific for unbranched primary alcohols. He can present an activity

thermostable aldehyde reductase.

  The present invention also relates to a

  microorganism producing such an enzyme.

  This microorganism preferentially belongs to the reign of Archaebacteria. It can thus be the strain deposited with the National Collection of Culture of Microorganisms of

  the Pasteur Institute under the N'I-1319.

  The strain 1-1319 was obtained from fragments of polymetallic sulphides originating from an active hydrothermal structure. According to observations made by scanning electron microscopy, the cells are irregular spheres 0.8 to 1.2 μm in diameter. No flagella or pili were observed; no mobility was seen on

  fresh conditions, under an optical microscope.

  Resistant to antibiotics such as vancomycin, penicillin, kanamycin, streptomycin, rifampicin and chloramphenicol, this strain is part of the kingdom of

  Archaebacteria. She is strict anaerobic chemo-

  organoheterotrophic and uses elemental sulfur as an electron acceptor. No growth was detected autotrophically on H2 / C02. 1-1319 develops little in the absence of sulfur. Although its growth is higher on complex media and a mixture of 20 amino acids, the strain can use simple substrates (glucose, pyruvate). Some physiological characteristics have been determined. Thus, the strain develops in a range of temperatures from 65'C to 100 'C with maximum growth - & 80 * C. It is able to grow in a wide range of pH between 2.5 and 9.5. The optimal pH for its growth is between 5.5 and 6.5. This strain is difficult to develop in the absence of sea salts; optimal growth is detected for a sea salt concentration of 30 g / l (sea water contains 40 g / l). She

is therefore moderate halophile.

  Its DNA has a proportion of guanine and cytosine bases (% G + C) of around 58%. The present invention further relates to the products derived from these microorganisms, such as thermostable enzymes and in particular the vital enzymes of the

  cellular machinery (polymerases, ligases .....).

  Another object of the present invention is

  the use of the enzymes or microorganisms described above

  above for the preparation of alcohols, aldehydes, or ketones. The alcohol dehydrogenase which is the subject of the present invention can be obtained by any method of preparation known to those skilled in the art and in particular by chromatography, on an ion exchange column, of crude extracts of the microorganisms producing it, which allows purification

partial of this enzyme.

  It can also be obtained by genetic engineering methods. The present invention is illustrated without however being limited by the following examples, in which: FIGS. 1A and 1B respectively represent the evolution of the oxidative and reducing activities of the enzyme (on the ordinate) as a function of the pH (on the abscissa) ) in different buffers, at 70 ° C; - Figure 2 illustrates the evolution of oxidative and reducing activities (on the ordinate) as a function of

  temperature (abscissa) at pH 7.5.

  - Figures 3A and 3B respectively represent the resistance of the alcohol dehydrogenase and aldehyde reductase activities of the enzyme (on the ordinate) and as a function of time

  incubation (on the abscissa) at different temperatures.

  - Figures 4A and 4B represent the alcohol dehydrogenase activity (on the ordinate) as a function of the concentration

  in NADP + and in benzyl alcohol (on the abscissa).

  - Figures 5A to 5E respectively illustrate the effects of NaCi, ZnC12, CuSO4, ethylene diamine tetracetic acid EDTA and Sodium Dodecyl Sulfate (SDS) (concentrations on the abscissa) on the activity of the enzyme (in ordered). - Figures 6A, 6B and 6C are photographs of electrophoresis gels respectively in native condition and in

denaturing condition.

  Wells 1 and 2 in FIG. 6A correspond to the crude extract, well 3 with albumin, while wells 4, 5 and 8 in FIG. 6B correspond to eluates from the ion exchange column and well 7 to a crude extract

from strain 1-1319.

  In FIG. 6B, wells 1 and 2 correspond to crude extracts of strain 1-1319, well 3 to the eluate from the ion exchange column and well 4 is a marker for

molecular weight.

EXAMPLES:

  Materials and Methods 1. Preparation for the measurements of enzymatic activities The enzymatic activities were measured on crude extracts (intracellular proteins) or

  partially purified preparations.

  1.1- Preparation of the crude extracts The crude extracts were prepared from microbial cultures of various volumes by centrifugation and then breaking of the cell pellet by ultrasound in the presence of

  phenylmethylsulfonyl fluoride (PMSF) and Triton X100.

  1.2 Variation in pH with temperature To study the evolution of activity as a function of temperature, it is necessary to know the variation in pH of the buffers and if possible to choose one that is

stable.

  The pH was adjusted at room temperature but the enzymatic kinetics were carried out at elevated temperature. We therefore estimated the actual pH values at this temperature. The pHs of the citrate-phosphate and phosphate buffers do not vary with the temperature (tested between and 70 C). On the other hand, the pH of the 100 mM KCl / boric acid / NaOH buffer adjusted to 8.65 at 25 ° C., loses 0.031 units every 5 ° C. up to 70 ° C. The 100 mM Tris / HC1 buffer adjusted to

  8.94 at 25'C loses 0.107 pH units every 5 C.

  2. Measurement of the alcohol-dehydrogenase activity The alcohol-dehydrogenase (ADH) activity is easily reversible and can therefore be measured according to the following two reactions: alcohol + oxidized co-enzyme - aldehyde + reduced co-enzyme The co- NAD + and NADP + enzymes whose form

reduced is detected at 340 nm.

  At high temperature, the reduced co-enzyme degrades very quickly and it is impossible to determine the molar extinction coefficient at high temperature. He has

  therefore been measured at room temperature.

  3. Measurement of thermostability The enzymatic thermostability was determined by measuring the residual activity after incubation of the sample at high temperature. For this, the enzymatic solution was distributed in small volumes in ampoules of lyophilization of 1 or 2 ml which were sealed before incubation. Each time the vial is taken out of the bath, immersed in ice to stop the denaturation,

then immediately frozen.

  When thermodenaturation can be modeled by first order kinetics during the first minutes, the half-life time (tl / 2) or time after which 50% of the initial activity remains is determined according to the following model: A = Ao ekt and ti / 2 = (Ln 2) / ko A is the residual activity at time t Ao is the initial activity

  k is the denaturation rate constant.

4. Purification.

  All purification steps are carried out at 4-C. The material used (Pharmacia) is a system of

standard chromatography.

  4.1 - Gel filtration The column (2.6 x 70 cm) was filled with Sephacryl S300 gel (Pharmacia). Standard proteins come from

  of Pharmacia and Touzart and Matignon calibration kits.

  4.2 - Ion exchange column The column was filled with DEAE Sepharose CL6B gel from Pharmacia (2.6 x 36 cm), equilibrated with 100 mM phosphate buffer pH 7.5 and eluted with this same buffer containing

  increasing concentrations of NaCl.

  Each chromatography was followed by detection of the enzymatic activity in the fractions collected during the elution. For this, each well of a microplate was filled with 250 μl of substrate and 10 or 20 μl of the fraction studied. The microplates were incubated for 20 minutes at 70 ° C. and then read on a plate reader. The enzymatic activity was expressed in arbitrary units (AU). The protein concentration in each fraction was evaluated by measuring

  absorbance at 280 nm in quartz tanks.

  At each step, the purification rate and the yield were evaluated by measuring the protein concentration as well as

  the enzymatic activity on the recovered fractions.

  Electrophoresis made it possible to visualize the

progress of purification.

  5. Protein assay methods Protein assays were carried out according to two different methods depending on the desired sensitivity range: - Lowry method (Lowry et al., 1952 J. Biol. Chem., 265-275) for a range of protein concentrations

between O, O1 and O, 1 g 1-1.

  - Bradford microdosage (Bradford, 1976, Analytic Biochem., 72,248-254) for a range of concentrations

  proteins between 1 and 25 mg 1-1.

  6. Electrophoretic analyzes The electrophoreses were carried out in a cold room (4'C) in tanks for vertical electrophoresis of two different dimensions (Bio-Rad). The gels after revelation of the proteins were dried under vacuum at 80 ° C.

for 1 hour.

  6.1- Preparation of gels and samples The compositions of the gels and buffers are

mentioned in appendix 3.

  Before deposition, the sample was prepared by adding half a volume of sample buffer to the protein solution. The migration was carried out at 100 V in the gel

  alignment and 180 V in the separation gel.

  Pre-molded gels in 4-20% acrylamide gradient have

  also been used (Bio-Rad).

  6.2 Revelation: staining of proteins As soon as the migration front of the dye reached the end of the gel, the migration was stopped and the gel

  colored with Coomassie blue for one hour then discolored.

  Coloring solution: Coomassie Blue bleaching solution O, 1% Methanol 30% Methanol 50% Acetic acid 10% Acetic acid 10% Water 60% Water 40% 6.3- Revelation of alcohol dehydrozenase activity ADE activity has been detected directly on the gel by precipitation of insoluble formazan dye: - preparation of an alcohol (50mM) -coenzyme (lmM) mixture in 150 ml of buffer - incubation of the gel in this mixture for one hour at "C" - elimination of the mixture reactive before adding

  a solution containing 0.25 mg m-1 of MTT (bromide of 3- [4,5-

  dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) and 0.05 mg ml-1 of PMS (phenazine methosulfate) - incubation at 60 ° C for exactly 15 minutes then rinsing of the gel with water. The active bands appear purple. 6.4 - Denaturing electroDhoreses These electrophoreses were carried out using the Laemmli method (1970, Nature, 227,680-685) on pre-molded gels sold by Touzart and Matignon at 10% T. The samples were prepared and denatured according to the recommended method by SIGMA for the molecular weight markers MS-SDS-200: ovalbumin (45000), bovine albumin (66000), phosphorylase B (97400), 3-galactosidase (116000),

myosin (205000).

  The gels have been colored like native blue gels

  of Coomassie then discolored and dried in the same way.

ANNEX 1

  Composition of culture media Medium 2216S (2S) (Belkin and Jannasch, 1985, Arch. Microbiol., 141, 181-186) Yeast extract 0.5 g Peptone 1 g Sea salts 30 g Sulfur 5 g Acid -2- [N-morpholino] ethane sulfonic acid il (MES) 3.09 g for pH 5, 5 Réazurine Distilled water qs 1000 The pHs were adjusted to room temperature. The anaerobic media were reduced by adding sterile Na2S & 2.5% in an anaerobic enclosure after tendalisation (2 times 30 minutes at 100 C) if they contained elemental sulfur or sterilization (30 minutes at 1200C) otherwise .

APPENDIX 2

  Calculation of alcohol dehvdrogenase activity The enzymatic activity expressed in number of pmol of co-enzyme (or substrate) reduced or oxidized per minute and per unit volume of enzyme (U ml-1) was calculated by the formula following: Act = 16 DO x V / (E x Ve) o Act is the activity expressed in U ml-1 16 DOI is the absolute value of the variation in optical density obtained per minute E is the molar extinction coefficient of reduced co-enzyme V is the total reaction volume in ml

Ve is the volume of the enzyme in ml.

APPENDIX 3

  Preparation of qels and electrophoresis buffers - alignment gel (6% T): Acrylide / bisacrylamide solution at, 8% T and 2.6% C 2 ml Tris HC1 buffer 0.5 M pH 6.8 2.5 ml Water -1 5.3 ml Ammonium persulfate 100 g 1 0.1 ml N, N, N ', N'-tetramethylenediamine (TEMED) diluted to 1/2 0.01 ml - Separation gel ( 10% T) 30.8% T and 2.6% C acrylamide / bisacrylamide solution 20 ml Tris HC1 buffer 1.5 M pH 8.8 15 ml Water 24 ml Ammonium persulfate 100 g 1-1 0, 6 ml 1/2 TEMED 0.06 ml - Sample buffer: Tris HCl 0.5 M pH 6.8 2.5 ml Glycerol 2 ml Water 3 ml Bromophenol blue 0, 1% - Migration buffer: Tris 6 g Wisteria 28.8 g Water 1 1

  EXAMPLE 1 Characterization of the alcoholic enzyme activity

  dehydroaenase in oxidation and reduction.

  The enzymatic and physicochemical characteristics of the alcooldéshydrogénase of the strain 1-1319 were determined in the crude extract. This was obtained from 7.5 l of culture. It contains 2.404 g / l of

  proteins, concentration determined by the Lowry assay.

  Both reactions - oxidation of alcohol and reduction of

aldehyde - have been studied.

  With few exceptions, the alcohol used as substrate is benzyl alcohol, less volatile than ethanol, at a final concentration in the reaction medium of 40 mM; the aldehyde used is 10 mM benzaldehyde; co-enzymes

  are NADP + at 1 mM and NADPH at 0.6 mM in final concentration.

  1. Effects of PH and temperature on the speed of

reaction.

  The optimal pH for the reaction rate at 70 ° C. is 8.3 in the 100 mM KCl / Borate / NaOH buffer (pH adjusted to 8.6 room temperature). The oxidation rate is equivalent in the phosphate buffer and in the Tris HC1 buffer. It is higher in these buffers than in the KC1 / Borate / NaOH buffer, at identical pH values, at 70 ° C.

(Figures 1A and 1B).

  The reaction rate was studied as a function of the temperature in the phosphate buffer pH 7.5 in order to free itself from variations in pH of the buffer with temperature. The maximum temperature studied was 90 ° C, the co-enzyme being very unstable at high temperature. The two activities, alcohol dehydrogenase and aldehyde reductase, are maximum at 75 ° C (Figure 2). The curve drawn according to the Arrhenius equation representing the logarithm of the activity as a function of the inverse of the absolute temperature made it possible to calculate the activation energies at pH 7.5: the activation energy of l oxidative activity is 66.7 KJ mol-1 and that of the reducing activity is 48.1 KJ mol-1 2. Thermostability The resistance of the enzyme to thermal denaturation was determined by incubation in phosphate buffer & pH 7.5 (Figures 3A and 3B). Reductase activity is more thermostable than dehydrogenase activity. The denaturation curves obtained can be modeled by reaction kinetics of order i and the half-life times have been determined at

  different temperatures tested.

  For the dehydrogenase activity, the half-life times are respectively 87 minutes, 56 minutes, 36 minutes and 7 minutes at 52'C, 62'C, 69'C, and 83.5 C. For the reductase activity , at 69 ° C and 83.5 C, the half-life times at these temperatures are 6 h and

minutes.

  At 52 ° C. and 62 ° C., this activity remains stable for 1 h 30 min. 3. Activity as a function of the concentrations of the substrates The study of the variation of the reaction rate of the dehydrogenase activity was carried out according to different benzyl alcohol and NADP + concentrations at pH 7.5 in the 100 mM phosphate buffer (Figures 4A and 4B). The kinetics were carried out at 75 ° C. 4. Effect of effectors on the rate of alcohol oxidation. Enzymatic kinetics in the presence of different additives (NaCl, CuSO4, SDS, ZnC12, EDTA) were carried out

(Figures 5A to 5E).

  All the substances tested inhibit the activity except for sodium chloride for which the reaction rate is maximum for a concentration of 0.2 M (Figure A). However, NaCl concentrations above 1 M

inhibit activity.

  NaCl was not added to the samples so that their ionic strength was not too high and did not interfere with the

  fixation of the enzyme on chromatography columns.

  Benzaldehyde has also been shown to inhibit

  alcohol dehydrogenase activity.

  EXAMPLE 2 Partial purification of the enzyme.

  By ion exchange column chromatography, DEAE Sepharose CL6B, a partial purification of the enzyme was carried out from a crude extract from a 27 liter culture. When the chromatography is carried out at pH 7.5 (100 mM phosphate buffer), the protein is eluted at a concentration of 0.25 M in NaCl. After this step, the rate

  of purification obtained is 2.5 and the yield 92%.

  It is therefore an efficient purification step without loss

significant activity.

  The active fractions recovered were then applied to a column containing an affinity gel onto which is grafted Cibacron blue, a substance having

  a structural analogy with the co-enzymes NAD + and NADP +.

  For some unknown reason the protein did not bind to

this column.

  On the other hand, other proteins were fixed since the purification rate increased: the specific activity in the recovered fractions was 81 times higher than in the initial crude extract. This preparation was used for further studies. It contains 0.078 g 1-1 of protein (Bradford) and 1.10 U ml-1. The specific activity of alcohol dehydrogenase in this preparation is 14.2 U

  mg-1 (70 ° C, pH 7.5 phosphate buffer).

  Following this purification, electrophoresis was carried out under native conditions (FIG. 6A) and denaturing conditions (FIG. 6B). The revelation with Coomassie blue on two gels (a native electrophoresis and a denaturing electrophoresis) shows that the preparation is not pure and the revelation of the active bands on the mixture

  ethanol-NADP + also shows the presence of four bands.

  Physico-chemical characteristics By chromatofocusing on PBE94 gel, the isoelectric point of the protein was determined; it is 4.5. A single peak of active fractions was identified during elution. Gel filtration on Sephacryl S300 determined that the protein size was around 80,000 daltons. A single peak of activity in the recovered fractions

during elution was observed.

  EXAMPLE 3 Specificities of substrates of the purified enzyme The oxidative and reducing activities of the enzyme were tested on various substrates in the presence of the co-enzyme NADP + at 1 mM or NADPH at 0.5 mM. The results are summarized in Tables 1 and 2. For reasons of solubility, certain alcohols were prepared at the concentration of 0.8 mM. Where possible, activities were measured for a

40 mM substrate.

  The alcohol dehydrogenase of 1-i319 is more active on primary alcohols than on branched alcohols. It is likely that the activities detected on secondary alcohols, cyclohexanol, and tertiary butanol are of the order of the sensitivity of the assay and are not significant. It should be noted that the activity increases with the length of the chain. primary alcohol

  The activity on polyols such as glycerol is low.

  Finally, this alcohol dehydrogenase is enantio-

  selective; in fact, it is more than ten times active on the

  geraniol only on nerol which are two enantiomers.

  There is also a strong activity on cinnamic alcohol.

CONCLUSION:

  The enzyme according to the invention has properties and characteristics which distinguish it from the two ADHs

thermostable already known.

  Table 3 summarizes these differences.

  It will be noted in particular that the enzyme according to the present invention has a broad specificity of substrates; it is different from those of Sulfolobus alcohol dehydrogenases

  solfataricus (Rella et al., 1987, Eur. J. Biochem. 167,475-

  479), and Thermoanaerobium brockii, two other alcohol-

thermostable dehydrogenases.

  The alcohol dehydrogenase according to the invention is specific for the co-enzyme NADP +, while that of T. brockii (denoted TAADH) uses NADP% as well as NAD +, even if it prefers NADP + and that of S. solfataricus (denoted SSADH )

is specific to NAD +.

  The three enzymes have a broad specificity of substrates: they hydrolyze various primary, secondary, cyclic alcohols and various polyols. TAADH however prefers secondary alcohols just like SSADH. The enzyme according to the present invention is distinguished because it has an activity

  preferential over primary alcohols.

  Even if the specificity for primary alcohols is very widespread among DHAs, the enzyme according to the invention has the advantage of being thermostable. Aromatic alcohols as well as ring-containing aldehydes are

  very good substrates for this enzyme. The enantioselectivity of the enzyme according to the invention

  appears by comparing the hydrolysis of geraniol and nerol.

  This characteristic is particularly interesting for stereospecific synthesis (flavors for the food and cosmetic industries, applications in the pharmaceutical industry). Indeed, two enantiomers of the same compound may have different properties and it is always

  difficult to separate a mixture of enantiomers.

  Its thermostability combined with its enantioselectivity

  allows its use in industrial processes.

BOARD.

  Oxidative activity of the enzyme according to the invention

SUBSTRATE CONCENTRATION ACTIVITY

RELATIVE

  Ethanol 40 mM 100 Propanol 1 40 mM 139.1 Isopropanol 40 mM 1.8 Butanol 1 40 mM 152.9 Butanol 2 40 mM 6.2 Tertiary butanol 40 mM 4.6 Pentanol 1 40 mM 232.6 Hexanol 1 40 mM 240 , 1 Cyclohexanol 40 mM 5.0 Benzyl alcohol 40 mM 180.9 Glycerol 40 mM 4.4 D. sorbitol 40 mM 3.0 Ethylene glycol 40 mM 7.0 Propylene glycol 40 mM 14.2 1.3- propanediol 40 mM 20.4 Ethanol 0.8 mM 100 Cinnamic alcohol 0.8 mM 749.0 Furfuryl alcohol 0.8 mM 216.0 Geraniol 0.8 mM 780.0 Nerol 0.8 imM 66.7

TABLE 2

  Reducing activity of the enzyme according to the invention

SUBSTRATE CONCENTRATION ACTIVITY

RELATIVE

  Acetaldehyde 0.8 mM 100, O Benzaldehyde 0.8 mM 87.9 DL-Glyceraldehyde 0.8 mM 15.1 m-Anisaldehyde 0.8 mM 61.8 p- Anisaldehyde 0.8 mM 78.2 Laurinaldehyde 0.8 mm 9.5 2-furaldehyde 0.8 mM 107.8 Citral 0.8 mM 51.2

TABLE 3

  Comparison of the ADH according to the invention and of two thermostable ADHs according to T '. brockl t S. solfatarleus the invention the invention molecular weight. (D) 150,000 70,000 80,000 subunits (D) 40,000 37,000 4o ooo 37 0OO tetramer dimer optimal fold 7.8 8. 5 / 7.5 # 8.6 TC optimal ≥95C 75C

PI 5.2 I1.5

  thermostability 20 '91-C, 20h 60'C, 5h 70C lh 62C ADII tl / 2 and. 70 '65C 6h 69 Creductase KM mM butanol 2 0.31 ethanol 0.26 acetaldehyde 0.7 benzol 0.013 specificity alc. 2nd alc. 2nd alc. their first NADP + dependent NAD prefers enantioselective NADP effector Zn2 + and EDTA EDTA inhibits NaC1 active without effect Zn2 * active Zn2 + inhibits remarks aggregates At high concentration *: depending on the substrate benzyl alcohol / anisaldehyde alc. 2nd = secondary alcohols alc. lrs = primary alcohols nI = isoelectric point KM = Michaelarts-Menten constarts

Claims (15)

  1. Thermostable and thermophilic alcohol dehydrogenase characterized in that it has an enantioselective oxidative activity. 2. Alcohol dehydrogenase according to claim 1, characterized in that it has an isoelectric point about 4.5.   3. Alcohol dehydrogenase according to one of claims   1 or 2, characterized in that it has an activity optimal at pH 8.3.   4. Alcohol dehydrogenase according to one of claims   1 to 3, characterized in that it has a mass of around 80 kD.   5. Alcohol dehydrogenase according to one of claims   1 and 4, characterized in that it is activated by NaCl.   6. Alcohol dehydrogenase according to one of claims   1 to 5 characterized in that it is inhibited by the   benzaldehyde, CuSO4, SDS, ZnC12 and EDTA.   7. Alcohol dehydrogenase according to one of claims   1 to 6, characterized in that it uses NADP + as co- postman.   8. Alcohol dehydrogenase according to one of claims   1 to 7, characterized in that it has an activity   specific with respect to unbranched primary alcohols.   9. Alcohol dehydrogenase according to one of claims   1 to 8, characterized in that it has an activity thermostable aldehyde reductase.   10. Microorganism characterized in that it produces a   alcohol dehydrogenase according to one of claims 1 to 9.   11. Microorganism according to claim 10, characterized in that it belongs to the reign of Archaebacteria. 12. Microorganism according to claim 11, characterized in that it is a strain deposited with the National Collection of Culture of Microorganisms   the Pasteur Institute under ne I-1319.   13. Products derived from microorganisms according to one of claims 10 to 12.   14. Product according to claim 13, characterized in   what it is a thermostable enzyme.   15. Use of the enzyme according to one of the   claims 1 to 9 or the microorganism according to one of   claims 10 to 12 for the preparation of alcohols, aldehydes or ketones.