TITLE

A STEREOSELECTIVE ENZYME ALCOHOL DEHYDROGENASE CONVERTING GLYCEROL TO D-GLYCERALDEHYDE: EXTRACTION, PURIFICATION AND CHARACTERIZATION.

TECHNICAL FIELD

This invention related to a stereoselective enzyme alcohol dehydrogenase having a property to oxidise glycerol to D-glyceraldehyde and process for extraction, purification and characterization of the same.

BACKGROUND OF INVENTION

Alcohol dehydrogenase (ADH) [EC 1.1.1.1] is present in all organisms. There are many ways to classify alcohol dehydrogenases. One way is to look at these enzymes in terms of their cofactor specificity towards (a) Nicotinamide adenine dinucleotide (NAD) or (b) Nicotinamide adenine dinucleotide phosphate (NADP) or (c) pyrrolo-quinoline quinone, heme or F ₄₂ o and (d) Flavin adenine dinucleotide (FAD) [Machielsen et al., 2006]. The classical NAD-dependent ADH from liver and Baker's yeast are called primary alcohol dehydrogenases although these enzymes do oxidize some secondary alcohols at much lower rate. NAD(P)- dependent ADH from some organisms like Escherichia coli or Pseudomonas are active only on long chain primary alcohols or hydroxyl fatty acids [Hou et al., 1981]. Peretz and Burstein (1989) have pointed out the classes of ADH based upon their quaternary structure and metal ion necessary for the oxidoreductase activity. Danielson et al. (1994) have discussed six classes of ADH in mammalian tissues in the context of evolutionary aspects. In the last few decades ADH from microorganisms have attracted considerable attention because of their valuable applications in biotransformations,/ of alcohols and corresponding carbonyl i

compounds. Such applications include synthesis or modification of chiral alcohols [Hummel and KuIa 1989; Xu, 2005].

US patent application no. 20030171544 deals with novel alcohol dehydrogenase (ADHF1) from Pseudomonas fluoresceins (DSM 50106) and its novel variants to reduce ketones to their respective alcohols.

US patent application no. 20040265978 teaches about reduction of organic keto compounds to the corresponding chiral hydroxy compounds. An alcohol dehydrogenase from Lactobacillus minor was used.

US patent application no. 20060177913 deals with alcohol dehydrogenase from yeast, equine liver, Rhodococcus erythropolis, Thermoanaerobium sp. Lactobacillus kefir or Lactobacillus brevis for preparing chiral secondary alcohols by reducing a keto compound.

US patent application no. 20060216801 teaches about regeneration of the cofactor (e.g. NADH or NADPH) which is used during reaction of the enzyme alcohol dehydrogenase by means of formate dehydrogenase.

US 4,352,885 disclosed a method for producing thermostable NADP linked alcohol, aldehyde/ketone oxidoreductases from a strain of thermophilic bacteria selected from the group consisting of Thermoanaerobium brockii and Clostridium thermohydrosulfuricum. The NADP specific thermostable alcohol aldehyde, ketone oxidoreductases disclosed could react at elevated temperatures with a wide range of alcohols, ketones and aldehydes. A unique preference for secondary over primary alcohols is observed according to the following decreasing order of activity: secondary alcohols, ketones and aldehydes, and

primary alcohols. No activity could be detected with NAD or NADH as coenzymes, L-lactic acid or glycerol did not react as substrates.

US5763236 disclosed a method of producing alcohol comprising reacting ketone or aldehyde with an alcohol dehydrogenase isolated from Candida parapsilosis that oxidizes an alcohol using NAD ⁺ . as a coenzyme to produce a ketone or aldehyde; using NADH as a coenzyme to produce an alcohol; has higher activity on secondary alcohols than primary alcohols; and preferentially oxidizes 2-butanol having an S-configuration; or with a microorganism producing said enzyme or a crude enzyme solution prepared by disrupting said microorganism and reducing said ketone or aldehyde to alcohol. US6255092 has disclosed similar enzyme from Candida parapsilosis as given in US5763236.

WO03078615 has disclosed a biocatalyst having alcohol dehydrogenase activity which can be obtained from Rhodococcus which shows stereospecific alcohol dehydrogenase activity in the oxidation of secondary alcohols or the reduction of ketones, comprising Zn2+ bound to the molecule.

JP patent no. 54113491 describes a universal method for purifying water soluble enzymes like alcohol dehydrogenase wherein water soluble enzyme solution along with salt when brought into contact with water insoluble polyhydroxy carrier free of ion exchange group like cellulose and starch. The adsorbed enzyme is eluted by lowering the concentration of the salt.

JP patent no. 54113489 deals with a method of separation of the water soluble enzyme wherein the enzyme like alcohol dehydrogenase with a high

concentration of the salt is brought into contact with polyhydroxy compound forming a hydrogen bond between them and further the enzyme is eluted by high concentrated salt solution containing eluant such as methanol; diols; diols, sugars such as fructose; sugar acids such as gluconic acid; uronic acids such as glucuronic acid; and aminosaccharides such as glucosamine.

JP patent no. 54113490 describes a method for purifying the water soluble enzyme as described in a method as given in JP 54113489 wherein urea is used as eluant along with the salt.

While aldehydes, ketones and alcohols have always been useful in food, pharmaceutical and fine chemical industries, chiral compounds are becoming increasingly important for obtaining chirally pure pharmaceutical products. In this regard, enzymes in particular ADH from thermophiles are of considerable value because of their higher stability during the industrial process.

SUMMARY OF INVENTION

This invention discloses an Isolated alcohol dehydrogenase enzyme having high stereospecific activity isolated from Thermus thermophilus. The strain used for this isolation is Thermus thermophilus strain (MTCC 1494). Other strains of Thermus thermophilus may also give essentially same enzyme. The Alcohol dehydrogenase this invention discloses higher activity towards glycerol and butanol as compared to short chain and secondary alcohols. Alcohol dehydrogenase of this invention has relative activity of 0% for methanol and 100% for Glycerol and Butanol. Alcohol dehydrogenase of this invention has been shown to be capable of converting glycerol to D-glyceraldehyde in

enantiomeric excess. It is also likely that it may convert butanol ito D- butanaldehyde in enantiomeric axcess which is around 90% or more.

The invention also discloses an Isolated NAD dependent alcohol dehydrogenase enzyme extracted from Thermus thermophilus and characterized by having a Zn+2 binding site, a molecular weight on denaturing SDS- polyacrylamide electrophoresis from 39 to 47 kDa, especially of about 43 kDa, a molecular mass of 166 to 174 kDa, especially of about 170 kDa by analytical ultracentrifugation method, optimum temperature range of 7O.degree.C. to 9O.degree.C, especially of about δO.degree.C. for substantially purified form, optimum pH range of 7 to 10, especially of about pH 8.8 for substantially purified form and having N terminal amino acid sequence M KAAWH KARXI RVE.

The invention also discloses a process for producing thermostable alcohol dehydrogense enzyme, said method comprising the steps of (a) inoculating a culture medium with a strain of thermophilic bacterium Thermus species (b) incubating said bacteria at an elevated temperature upto about 75 ⁰ C (c) isolating the bacterial cell mass, and (d) isolating alcohol dehydrogenase from the cell mass.

A process of isolating an alcohol dehydrogenase enzyme from a cell debris free alcohol dehydrogenase enzyme containing cell lysate of Thermus thermophilus, the said process comprising steps of (a) adding a salt to the supernatant for salting out a portion of impurities, (b) removing the impurities by centrifugation, (c) bringing in contact the supernatant of the centrifugation with an adsorbent by applying the same to an adsorbentwherein the said adsorbent is designed for capture or removal of relatively strong hydrophobic molecules at low salt concentration, (d) eluting the adsorbent with a buffer containing stepwise

reduction of the said salt and pooling the fractions containing alcohol dehydrogenase activity. The salt used in this process is ammonium chloride., adsorbent used is Butyl-Sepharose and the said buffer comprised of 5OmM Tris- HCI, pH 7.0 at 25 ⁰ C, 10 mM MgCI ₂ , 1 mM β-mercaptoethanol, 100 mM PMSF, 5% (v v ¹ ) glycerol and 30% ammonium sulphate; although any other equivalent alternative capable of achieving same objectives may be used without departing with the essential elements of the invention.

The illustrated process of invention comprised of steps of (a) washing the cells with 5OmM Tris-HCI, pH 7.0 at 25 ⁰ C, (b) suspending in four volumes of 5OmM Tris-HCI, pH 7 containing 5 mM EDTA, 1 mM β-mercaptoethanol, 100 mM PMSF, 5% (v v ¹ ) glycerol, Lysozyme (85 μgmL ^"1 ) and stirring the solution for 2 h at 4 ⁰ C, (c) adding MgCI ₂ to make the lysate to 25 mM, (d) treating with Deoxyribonuclease at 7 μgmL ^"1 , stirred for 20 min at 4 ⁰ C, (e) achieving complete cell breakage by sonication on an ice bath, (f) centrifuging at 15,000 g for 30 min at 4 ⁰ C, (g) collecting the supernatant and adding ammonium sulphate to make 30% wv ^"1 , (h) centrifuging at 15,000 g for 15 min, (i) applying the supernatant to Butyl-Sepharose Fast Flow column equilibrated with 5OmM Tris- HCI, pH 7.0 at 25 ⁰ C, 10 mM MgCI ₂ , 1 mM β-mercaptoethanol, 100 mM PMSF, 5% (v v ¹ ) glycerol and 30% ammonium sulphate, (j) eluted with an eluent that is equilibration solution of (i) with step-wise reduction of ammonium sulphate from 30% to 0% ammonium sulphate, (k) pooling the fractions iwith alcohol dehydrogenase activity.

This invention also discloses a method of producing optically active aldehyde comprising reacting alcohol with alcohol dehydrogenase isolated from Thermus

thermophilus. The said alcohol is glycerol and the said aldehyde is glyceraldehyde

DETAILED DESCRIPTION OF INVENTION

In the present work, we describe purification and characterization of an NAD- dependent and zinc containing ADH from Thermus thermophilus (TTHADH). The cell lysate of Thermus thermophilus containing alcohol dehydrogenase with 30% ammonium sulphate was adsorbed on Butyl-Sepharose Fast Flow column equilibrated with a buffer containing glycerol and 30 % (wv ^~1 ) ammonium sulfate bulk of the protein bound to the column by hydrophobic interactions and hence elution was performed using step-wise reduction of ammonium sulfate from 30 % to 0 ammonium sulfate. The enzyme showed much higher activity towards glycerol as compared to short chain primary and secondary alcohols. This thermostable enzyme is also highly stereospecific and converted glycerol into D- glyceraldehyde. In this respect, it differs from glycerol dehydrogenases, which produce dihydroxyacetone [Lee and Whitesides, 1986]. This invention also deals with use of NAD repeatedly for at least 5 times in the reaction. This factor contributes significantly to the overall economics of the reaction.

One embodiment of this invention comprises an isolated and purified NAD- dependent and zinc containing ADH (TTHADH).

In another embodiment of this invention comprises an isolated and purified NAD- dependent and zinc containing ADH (TTHADH) that has much higher activity towards glycerol as compared to short chain primary and secondary alcohols; relative to the activity with 2-Butanol and Glycerol, the activity of ADH (TTHADH)

with short chain primary and secondary alcohols ranged from 0 % for methanol to 69% for 2-Propanol .

In another embodiment, this invention comprises a process of isolation, purification and characterization of the said NAD-dependent and zinc containing TTHADH.

In a further embodiment of this invention, the said TTHADH is prepared for the first time form Thermus thermophilus strain (MTCC 1494).

In a further embodiment of this invention, the said TTHADH is highly stereospecific and converted glycerol into D-glyceraldehyde.

The specificity of the enzyme towards glycerol has considerable industrial importance. A large amount of glycerol is being produced as a byproduct of biodiesel production. The global glycerol market was 800,000 tons in 2005 as compared to 60,000 tons in 2001 [Pagliaro et a/., 2007]. This has led to search for conversions/ biotransformations which can convert glycerol into value added products [Pagliaro et a/., 2007]. The present enzyme, which is capable of converting glycerol into a chiral molecule like D-glyceraldehyde opens up several synthetic opportunities.

In the following are described experiments conducted that serve as non limiting illustrations of how the invention is performed. Any modifications or variations in the parameters including but not limited to organisms used, enzymes isolated, chemicals and their concentrations used, isolation and purification steps used are merely illustrative and any equivalents of them that are obvious to a person skilled in the art and that are capable of achieving the same objective if used in

their place shall be considered as included in the content / scope of this specification.

FIGURES AND THEIR DESCRIPTION

Fig. 1. Polyacrylamide gel electrophoresis. A. Aliquots of TTHADH at each stage of purification were subjected to SDS-PAGE using a 12% acrylamide slab gel. Lanes contained the following: M, Bio-rad high molecular weight marker; 1, cell-free extract (30 μg); 2, pooled Butyl-Sepharose fractions (20 μg). B. Aliquots of purified TTHADH were subjected to PAGE using a 8% acrylamide slab gel for in situ activity staining using nitro blue tetrazolium and phenazonium methosulfate. Lanes contained the following: 3, activity with glycerol; 4, activity with ethanol.

Fig. 2. Comparison of N-terminal amino acid sequence of TTHADH and ADHs from other organisms. TTHADH, Thermus thermophilus; TRADH ₁ Thermococcus roseus; TBADH, Thermoanaerobacter brockii; THADH, Thermus litoris; TEADH, Thermococcus essas; ECADH, Escherichia coli; ZMADH, Zymomonas mobilis

Fig. 3. pH versus activity profiles. Activity of TTHADH at various pH values. Sodium phosphate buffer was used (100 mM) at pH range 5.4 to 7.9 and a glycine-NaOH buffer (50 mM) was used at pH range 7.9-10.3. The percentage of the maximal activity at each pH is plotted.

Fig. 4. Temperature dependence of TTHADH. The enzyme activities were determined between 25 and 90 ⁰ C under the conditions described under "Experimental Procedures". The percentage of the maximal activity at each temperature is plotted.

Fig. 5. Thermal denaturation by CD.

Fig. 6. HPLC profile of D-glyceraldehyde. A. Commercial DL-Glyceraldehyde. B. Reaction mixture at the end of 14 h.

MATERIALS

The thermophilic Thermus thermophilus strain (MTCC 1494) was procured from IMTECH, Chandigarh, India. All chromatography media and Poly (vinylidene difluoride) membranes for electroblotting were obtained from Pharmacia, GmbH. β-NAD ⁺ was procured from Sigma Chemical Co., St. Louis, USA. Nitro-blue tetrazolium and phenazine methosulfate were obtained from Sisco Research Laboratories, Mumbai, India. All other chemicals were of analytical grade.

MICROORGANISM CULTIVATION

Thermus thermophilus cells were grown at 70 °C for 48 h in Castenholz medium (Castenholz, 1969). The cells can, of course be grown at any other temperature at which cells would normally grow, although these inventors found 70 ⁰ C as optimum. Cells were then pelleted (7,00Og, 10 min) and frozen immediately at - 70 °C. The cultivation can be done at other temperatures too at which Thermus thermophilus cells can be ordinarily grown including temperature elevated up to 75 °C. Cell mass can also be isolated by other means such as flocculation.

TTHADH ISOLATION AND PURIFICATION PROCEDURE.

The process of isolating alcohol dehydrogenase from the cell mass of Thermus thermophylus comprises steps of washing and lysing the cells, getting a cell lysate, treating the cell lysate to get rid of deoxyribonucleic acids preferably, achieving substantially complete cell breakage, removing cell debris by

centrif ligation, adding a salt to the supernatant for salting out a portion of impurities, removing the impurities by centrif ugation, bringing in contact with or by applying to an adsorbent, the said adsorbent being designed for capture or removal of relatively strong hydrophobic molecules at low salt concentration, eluting the column with a buffer containing stepwise reduction of the said salt and pooling the fractions containing alcohol dehydrogenase activity.

In an illustrative isolation, the cells were washed twice with 50 mM Tris-HCI, pH 7.0 (25 ° C) and suspended in four volumes of 50 mM Tris-HCI, pH 7.0, containing 5 mM EDTA, 1 mM β-mercaptoethanol, 100 mM PMSF, 5% (w ^~1 ) glycerol. Lysozyme (85 μgmL ^"1 ) was added and the suspension was stirred for 2 h at 4 ⁰ C. Any other method alternative to lysozyme treatment can be used to achieve cell wall lysis including mechanical disintegration, osmotic lysis. The resulting cell lysate was made 25 mM in MgCI ₂ , treated with DNase (7 μgmL ^"1 ) and stirred for 20 min at 4 °C. Complete cell breakage was performed by sonication on an ice bath after Dnase treatment. Sonication may be replaced by any other emthod commonly known in the art for cell breakage. To remove cell debris, the cell lysate was centrifuged at 15,00Og for 30 min at 4 °C. The supernatant was collected and ammonium sulfate was added to the supernatant. Preferred addition was to a concentration of 30 %, wv ^"1 . After mixing the ammonium sulfate, the sample was centrifuged again at 15,00Og for 15 min. The clear supernatant was applied to Butyl-Sepharose Fast Flow column equilibrated with 50 mM Tris-HCI, pH 7.0 (25 °C), 10 mM MgCI ₂ , 1 mM β-mercaptoethanol, 20 μM PMSF, 5 % (W ¹ ) glycerol, and 30 % (wv ^'1 ) ammonium sulfate. In place of Butyl-Sepharose, any other adsorbent may be used that is designed for capture or removal of relatively strong hydrophobic molecules at low salt concentration.

Bulk of the protein bound to the column. Elution was performed using step-wise reduction of ammonium sulfate from 30 % to 0 ammonium sulfate. Fractions with ADH activity were pooled and stored frozen at -70 °C.

ENZYME ASSAY

The alcohol dehydrogenase activity was measured in a buffer containing 200 mM Tris-HCI (pH 8.8), 8 mM NAD and purified enzyme. The reaction was started by adding the alcohol at a final concentration of 32 mM. The reaction was monitored spectrophotometrically by monitoring NADH at 340 nm. All assays were performed at 70 ⁰ C in a Beckman DU 640 spectrophotometer equipped with a thermostated circulating water bath. One unit of ADH was defined as the reduction of 1 μmole of NAD ⁺ per minute under initial velocity at the above mentioned conditions. Protein concentrations were determined using Bradford reagent with bovine serum albumin as a standard (Bradford, 1976).

pH OPTIMUM

The pH optimum for alcohol oxidation was determined in a sodium phosphate buffer (100 mM, pH range 5.4 to 7.9) and a glycine-NaOH buffer (50 mM, pH range 7.9-10.3).

TEMPERATURE OPTIMUM

The temperature optimum was determined in 50 mM glycine buffer, pH 8.8, by analysis of initial rates of glycerol oxidation in the range of 30-100 °C.

SDS-PAGE

SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) was performed on 12 % slab gel and 5 % stacking gel [Hames, 1986], using a Bangalore Genei electrophoresis system. The gels were stained with Coomassie Brilliant Blue.

ELECTROPHORESIS AND IN SITU ACTIVITY STAINING

The purified TTHADH was subjected to native polyacrylamide gel electrophoresis (PAGE) on 8.0 % polyacrylamide. The gel was cut into slices and incubated at 70 °C in 15 ml_ of glycine-NaOH, pH 10, containing 8 mM NAD ⁺ , and alcohol at 0.5 M concentrations. After 10 min, 0.5 mL of 4 mgmL ^"1 , nitro-blue tetrazolium and 0.5 mL of 0.5 mgmL ^"1 phenazine methosulfate, both dissolved in glycine-NaOH, pH 10, were added and incubations were continued for 5-20 min until formazane bands (dark blue) became visible.

ATOMIC ABSORPTION SPECTROSCOPY

Metals were analyzed by atomic absorption spectroscopy using Perkin Elmer A Analyst 100 Atomic Absorption Spectrometer. Glassware were rigorously made metal free as described [Riordan and Vallee, 1988]. Analysis was done after extensive dialysis of the protein samples against three changes of 100 volumes of 10 mM Tris-HCI (pH 7.0). Aiiquots were aspirated directly into an air-acetylene flame, and determinations were made on each sample. The system was calibrated using metal calibration standard solutions in the range of 0-2 ppm. Metal determinations were performed at two dilutions, each in duplicate.

CD MEASUREMENT

CD (Circular Dichroism) spectra were recorded on a Jasco J-815 spectropolarimeter (Japan) using solutions with protein concentration of about 0.15 mgmL ¹ . Results are expressed as molar ellipticity.

T _m MEASUREMENT

Thermal denaturation curves were determined by monitoring the CD values at 222 nm with a Jasco J-815 spectropolarimeter (Japan) using a 1 cm cuvette. Samples with concentration of 0.2 mgmL ^"1 were used. The temperature of sample solution was raised linearly by 1 °Cmin ^"1 . The heating curves were corrected for an instrumental baseline obtained by heating the buffer (100 mM sodium phosphate buffer, pH 7.8) alone. T _m was measured by determining the first derivative of relevant heating curves.

MOLECULAR WEIGHT DETERMINATION BY ANALYTICAL ULTRACENTRIFUGATION

Velocity sedimentation experiments were carried out at 20 ⁰ C in a Beckman Coulter Optima XL-I analytical ultracentrifuge. The protein was centrifuged at 40,000 rpm for 6 h at 20 ⁰ C in a standard cell. Data acquisition and analysis were done by SEDFIT analysis software that yielded S ₂ o,w for each set [Harding, 1997].

N-TERMINAL AMINO ACID SEQUENCE ANALYSIS

The amino-terminal sequence of TTHADH was determined after electroblotting purified enzyme preparation (approx. 500 pmol) onto a poly (vinylidene difluoride) membrane. Sequence analysis was performed by the Structural Biology Unit,

National Institute of Immunology, New Delhi, India, on Procise cLC sequenator (Applied Biosystems).

ENANTIOSELECTIVITY

The enantioselectivity was determined by oxidation of glycerol at 37 ⁰ C for 14 h. The reaction mixture contained 20 mM NAD and 32 mM glycerol. The product analysis was done in chiral column (Chiralcel OD RH) ₁ fitted with Agilent 1100 series HPLC system, using refractive index detector taking phosphate buffer (pH 5.8, 10 mM) as the eluent. The enantiomeric peaks of D,L-glyceraldehyde were separated by a programmed flow rate 0-4 min at 0.5 ml/ min; 4-6 min at 0.1 ml /min; 6-8 at 0.5 ml/min) and identified (after running authentic optically pure enantiomers) as: D-glyceraldehyde (4.566 min), L-glyceraldehyde (5.024 min). More than 90 % enantiomeric excess of D-gleceraldehyde was observed after 14 h.

Table 1 : Purification of alcohol dehydrogenase (TTHADH)

aOne unit of activity is defined as 1 μmol of NADH produced/min/ml.

Table 1 shows the purification of TTHADH by Butyl-Sepharose. The enzyme could be purified with 25-fold purification and 68% yield using a single step purification protocol. The SDS-PAGE showed a single band of molecular weight 43 kDa (Fig 1A). Activity staining was performed to confirm the presence of alcohol dehydrogenase using ethanol and glycerol as substrates (Fig 1 B). Analytical ultracentrifugation showed the molecular mass to be about 170 kDa. This observation (combined with molecular weight as estimated by SDS-PAGE) indicated the tetrameric nature of enzyme. The N-terminal amino acid sequence of TTHADH appeared to be different from sequences of ADHs from some of the other thermophiles like Thermoanaerobacter brockii, Thermococcus roseus, Thermus litoris, and Thermococcus essas (Fig 2). The pH optimum of the purified enzyme was 8.8 (Fig 3) and the temperature optimum was found to be 80 ⁰ C (Fig 4). Thermal denaturation curves were determined by monitoring the CD values at 222 nm and the T _m was found to be 89°C (Fig 5B). The enzyme showed much higher activity towards glycerol as compared to short chain primary and secondary alcohols (Table 2). This thermostable enzyme was also highly stereospecific in oxidation of glycerol and converted glycerol into D- glyceraldehyde (Fig 6).

Table 2: Substrate specificity of T. thermophilus ADH

b Values were normalized by setting activity assayed with glycerol to 100.

REFERENCES

1. R. Machielsen, A.R. Uria, S.W.M. Kengen, J. van der Oost (2006) Production and Characterization of a Thermostable Alcohol Dehydrogenase That Belongs to the Aldo-Keto Reductase Superfamily, Appl. Environ. Microbiol., 72, 233-238.

2. CT. Hou, R. Patel, A.I. Laskin, N. Barnabe, I. Marczak (1981) Substrate specificity and stereospecificity of nicotinamide adenine dinucleotide-linked alcohol dehydrogenases from methanol-grown yeasts, Appl. Environ. Microbiol., 41, 829-832.

3. M. Peretz, Y. Burstein (1989) Amino acid sequence of alcohol dehydrogenase from the thermophilic bacterium Thermoanaerobium brockii, Biochemistry, 28, 6549-6555.

4. O. Danielssoon, S. Atrian, T. Luque, L. Hjelmqvist, R. Gonsalez-Duarte, H. Jornvall (1994) Fundamental molecular differences between alcohol dehydrogenase clases, Proc. Natl Acad. Sci. USA, 91 , 4980-4984.

5. W. Hummel, M.-R. KuIa, Dehydrogenases for the synthesis of chiral compounds, Eur. J. Biochem. 184, 1-13.

6. F. Xu. (2005) Applications of oxidoreductases: Recent progress, Ind. Biotechnol., 1, 38-50.

7. L.G. Lee, G. M. Whitesides (1986) Preparation of Optically Active 1,2-Diols and &-Hydroxy Ketones Using Glycerol Dehydrogenase as Catalyst: Limits to Enzyme-Catalyzed Synthesis due to Noncompetitive and Mixed Inhibition by Product, J. Org. Chem., 51 , 25-36.

8. M. Pagliaro, R. Ciriminna, H. Kimura, M. Rossi, C. Delia Pina (2007) From Glycerol to Value-Added Products, Angew. Chem. Int. Ed. 46, 4434 - 4440.

9. R.W. Castenholz, R. W. 1969. Thermophilic blue-green algae and the thermal environment, Bact. Rev., 33, 476-504.

10. M. M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal.

Biochem., 72, 248-254.

11. B.D. Hames, in: B. D. Hames, D. Rickwood (Eds.), Gel electrophoresis of proteins, A practical approach, 2nd ed., IRL press, Oxford, 1986, pp. 1-86.

12. S. E. Harding, Hydrodynamic properties of proteins. In: T.E. Creighton, Editor, Protein Structure: A Practical Approach (2nd ed.), IRL Press, New York (1997), pp. 2419-2499.

13. J. F. Riordan, B.L. Vallee (1988) Preparation of metal-free water, Methods Enzymol., 158, 1583-1586.