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WO1999021971A2 - Alcool deshydrogenases thermostables - Google Patents

Alcool deshydrogenases thermostables Download PDF

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Publication number
WO1999021971A2
WO1999021971A2 PCT/US1998/022607 US9822607W WO9921971A2 WO 1999021971 A2 WO1999021971 A2 WO 1999021971A2 US 9822607 W US9822607 W US 9822607W WO 9921971 A2 WO9921971 A2 WO 9921971A2
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Prior art keywords
die
enzymes
dehydrogenase
protein
activity
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PCT/US1998/022607
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English (en)
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WO1999021971A9 (fr
WO1999021971A3 (fr
Inventor
Larry Allen
Igor Brikun
John H. Alkens
David C. Demirjian
Ramesh Matur
Yuri Nikolsky
David Rozzell
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Thermogen, Inc.
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Priority to EP98957372A priority Critical patent/EP1027427A2/fr
Priority to CA002308095A priority patent/CA2308095A1/fr
Priority to AU13648/99A priority patent/AU1364899A/en
Publication of WO1999021971A2 publication Critical patent/WO1999021971A2/fr
Publication of WO1999021971A9 publication Critical patent/WO1999021971A9/fr
Publication of WO1999021971A3 publication Critical patent/WO1999021971A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)

Definitions

  • the present invention relates to the filed of catalytic reagents for use in chemical synthesis
  • the present invention relates to novel thermostable enzyme proteins with alcohol dehvdrogenase activity Background of the Invention
  • Dehydrogenase biocatalyst reagents efficiently reduce ketones to chiral alcohols
  • the reduction ot the carbonyl group is one of the most versatile transformations in organic chemistry It can be used to form carbon-carbon bonds, introduce heteroatoms, serve as masked functionality and can be the site at which chirality is installed (March, 1985)
  • Alcohol dehydrogenases are one of the principal enzymes that effectively operate on the carbonyl group
  • ⁇ ery few alcohol dehydrogenases are commercially available that can produce molecules of high enantioselectivity under conditions encountered in industrial applications New dehydrogenase biocatalysts need to be developed that meet the requirements of the industrial synthetic chemist Biocatalyst reagents offer methods to install chiral centers in high value chemicals.
  • Biocatalysts are increasingly being recognized as potential alternatives to traditional synthetic organic methods and are noted for their remarkable catalytic capacity (Faber, 1992). In spite of potential utility, biocatalysts and industrially important biotransformations remain to be discovered. Development of novel biocatalysts that are user friendly, economical and produce high yields of chiral chemicals are crucial to the future of industrial chemistry. Over 50% of existing therapeutic agents are chiral molecules and of those that are synthetic (528), 75% are prepared as racemic mixtures. Several companies have demonstrated that only one enantiomer of a racemate often produces the desired biological activity while the other antipode is ineffective or responsible for side effects.
  • Drugs such as the recently redeveloped antiasthma agent (R)-albuterol, and the nonsteroidal antiinflammatory compounds ibuprofen and naprosyn represent chiral drugs originally manufactured as racemates in which one enantiomer is the active species (1994).
  • Such agents have a huge market value each with sales in excess of one billion dollars worldwide(Stinson, 1994).
  • biocatalysts offer the potential to dramatically streamline synthetic processes by minimizing the total number of steps and or complex purification schemes (Crout and Christen, 1989). Shorter synthetic routes to chiral compounds also reduce waste streams and minimize environmental pressures that are beginning to force chemical makers to search for alternative synthetic schemes.
  • Alcohol dehydrogenases are a family of enzymes capable of formal reversible two electron chemistry in which alcohols are oxidized to the corresponding ketones (Table I (Faber, 1992)).
  • ketones can be reduced to the respective alcohols via a stereospecific delivery of a hyd ⁇ de equivalent catalyzed by the enzyme coupled to a bound cofactor (NADH or NADPH).
  • NADH or NADPH a bound cofactor
  • This system represents a mild, extremely selective route to valuable chiral intermediates that can be used particularly by the pharmaceutical industry for the preparation of chiral therapeutics.
  • Extensive investigations of alcohol dehydrogenases have shown that these proteins can be an efficient means for generanng a variety of compounds with capacity for industrial scale-up (See Drawing below) (Bradshaw, et al. 1992; Hummel, 1990; Seebach, et al., 1984)
  • the present invention provides for a protein with alcohol dehydrogenase activity selected from the group consisting of AD55.1, AD83.5, AD5.1, AD7.1, AD14.1, AD31.3, AD14. AD19. AD30, AD31, AD39, AD49.4, AD49.12, AD55, AD69, AD71, AD98, and XY (ADOl).
  • the present invention provides for a protein with chiral alcohol selective alcohol dehydrogenase activity selected from the group consisting of AD 19, AD30.
  • the present invention also provides for a protein with thermostable alcohol dehydrogenase activity selected from the group consisting of AD7 1, ADM 1. AD31 3. AD39 4. AD49 4. AD49.12. AD55.1, AD83.5, XY, and AD5.1 wherein said protein retains activity at temperatures above 32°C.
  • the present invention also provides for recombinant DNA constructs which encode for the proteins of the present invention.
  • DNA constructs which can be generated using recombinant DNA techniques, can be expression vector constructs whereby the nucleic acid sequence is transcribed and translated into the desired protein Said DNA constructs will be transformed into host cells which can express the proper protein.
  • the instant invention encompasses expressible recombinant DNA constructs which express the protein of the present invention, as well as host cells which are transformed with such constructs.
  • the present invention also provides for using the DNA sequences and constructs of the present invention to hybridize with DNA or RNA in target gene banks, gene libraries, or expression libraries, under stringent hybridization conditions, to detect substantially related or related protein encoding nucleic acids from a pool of nucleic acids.
  • the present invention provides for methods of producing recombinant proteins of the invention, as well as methods for detecting other related protein encoding nucleic acid sequences from pools of nucleic acid.
  • Figure 1 is a genetic map of the ⁇ pMYF construct
  • Figure 2 shows the DNA fragment analysis of cloned dehydrogenases
  • Figure 3 is a diagram of the SDS gel analysis of ADH candidates
  • Figure 4 are graphs of Gel Filtration Profiles and Molecular Weight Determination for cloned enzymes;
  • Figure 5 shows Native PAGE (Polyacrylamide Gel Electrophoresis) analysis of Crude Extracts;
  • Figure 6 are graphs of Optimal Temperature Charts for Several ADH Candidates
  • Figure 7 are graphs of Residual Temperature Charts for Several ADH Candidates. Wherein the optimal temperature charts for enzymes produced from strains AD 19, AD30, AD39, AD49.4,
  • AD49.12, AD55, AD69, AD71, AD98, XY, and HLADH containing the Horse Liver Alcohol Dehydrogenase control enzyme on plasmid pBPP. The data is presented as percent of original activity.
  • Dehydrogenases have been identified as useful biocatalysts for chemical synthesis applications, particularly in the reduction of carbonyl groups to alcohols. They allow simplification of reactions that are difficult by traditional synthetic methodology.
  • T e highly stable biocatalysts of the present invention add a variety of new dehydrogenase specificities to the synthetic chemist's toolbox.
  • the present invention describes new enzymes from thermophilic organisms suitable for use as stable off-the-shelf reagents for selectively and mildly installing chiral centers from corresponding carbonyl groups. These enzymes have been characterized and show a variety of substrate specificities and enantioselectivities which indicates that they are useful biocatalysts which can be used economically to prepare fine chemicals and intermediates.
  • the enzymes of the present invention are suitable for use in methods for large scale chemical reactions, and show an expanded range of substrate specficities.
  • Dehydrogenases from whole yeast cells are classic synthetic biocatalysts. Dehydrogenase catalyzed reactions have been performed by two principle methods. Most common to the organic chemist is the formation of chiral alcohols catalyzed by whole cell baker's yeast Baker's yeast has enjoyed reasonable notoriety as a biocatalyst because it can be used as an off the shelf reagent, requiring no special treatment such as refrigeration, purification or cofactors.
  • the active component of yeast is an alcohol dehydrogenase which has been extensively studied by X-ray crystallography, mechanistic biochemistry and organic chemistry.
  • Isolated dehydrogenase biocatalysts offer advantages over whole cell methods.
  • Isolated enzymes offer an alternative method to whole cell systems to effect biotransformations.
  • Chiral compounds with high enantiomeric excess have been prepared using purified dehydrogenase catalysts without contamination from undesired competing reactions.
  • Dehydrogenases have shown promise for commercial application in the preparation of unusual amino acids (Benoiton, et al., 1957), b- hydroxyketones (Casy, et al., 1992) and resolution of racemic alcohols (Jones and Jakovic, 1982).
  • thermophilic organisms could increase environmental tolerance although thermophiles have yet to be widely investigated for useful dehydrogenase catalysts.
  • the alcohol dehydrogenase isolated from Thermoanaerobium brockii is one of the few successfully isolated dehydrogenases from thermophilic organisms which show promise for industrial applications (Keinan, et al., 1986; Lamed and Ziek ⁇ s, 1981).
  • NAD(P)H Cofactors are Required for Dehydrogenase Based Synthetic Methods.
  • the role of the cofactor in the catalytic system needs to be addressed since NAD(P)H are relatively costly reagents (as much as $250,000/mole) and are used stoichiometrically during the reaction.
  • Cofactor cost currently limits the use of cofactor requiring enzymes to high value applications such as the preparation of pharmaceuticals.
  • cofactors can not be simply disposed of at the conclusion of the reaction but rather need to be recycled.
  • cofactors should function at catalytic concentrations, necessitating in situ regeneration of the active species.
  • Recycling systems offer the additional advantage of beginning the reaction with the oxidized form of the cofactor which is considerably less expensive then the reduced species.
  • NAD + molecules have been treated with vanadate to produce a less expensive analog to the highly cosdy NADP + molecule required for some dehydrogenase catalyzed reactions (Crans, et al., 1993).
  • Dehydrogenases reduce ketones stereoselectively following Prelog rules.
  • the dehydrogenase chemical mechanism begins by binding the substrate either at the carbonyl or alcohol oxidation state.
  • analysis of reaction kinetics and X-ray crystal structures of substrate bound dehydrogenases have shown that the pro-R hydrogen on the nicotinamide ring is positioned by the protein to efficiently transfer a formal hydride moiety to the carbonyl group.
  • Prelog developed a set of rules for reduction of carbonyl groups which predict that nucleophilic attack should occur at the face opposite the large substituent (shown in the drawing below)(Prelog, 1984).
  • ⁇ Dehydrogenise The Prelog Rule for reduction of ⁇ carbonyl groups.
  • Cubic Space model for alcohol dehydrogenase catalyzed reactions (Jones and Jakovac, 1982). Cubic surface model of the active site of horse liver alcohol dehydrogenase. Cubes with solid lines are forbidden spaces occupied by active site amino acid residues. Dotted lines are cubes with disfavored spaces originating from potential interactions with charged groups on active site residues. Open spaces are available for substrate access to the active site, which allows positioning of the substrate in such a way that the active carbonyl functionality is located just above the hydride equivalent of the cofactor at the Cl-Dl boundary.
  • the cubic surface model remains an effective stereoselectivity model since it evaluates catalysis based on boti a library of substrates with differing structure and high resolution X-ray crystal data instead of addressing specific interactions between substrate and protein.
  • Dehydrogenase catalysts will be synthetic reagents that are both mild and selective even in die presence of sensitive functionality to produce chiral building blocks for assembly of more complex compounds. Dehydrogenase catalyzed reactions are expected to be applied to the synthesis of complex therapeutic compounds such as antibiotics and antiviral agents. Few options are available for chemists who wish to develop biocatalytic methods using dehydrogenase enzymes. The two best studied commercially available biocatalytic methods are whole-cell yeast systems and horse liver alcohol dehydrogenase (HLADH).
  • HLADH horse liver alcohol dehydrogenase
  • Thermophilic organisms provide stable biocatalyst reagents for organic synthesis.
  • One of die greatest challenges still facing syndietic chemists is to develop inexpensive, efficient ways of producing compounds.
  • die problem is not only to find a system to effect a given transformation but also to implement the reaction on a large scale.
  • Proteins derived from tiiermophilic sources provide an opportunity to satisfy the demands of the industrial process chemist.
  • Thermophile derived biocatalysts maintain activity under relatively harsh conditions
  • Thermophile derived biocatalysts can reduce production and catalyst cost
  • the ability to function at elevated temperatures should not be confused widi a need to operate under such conditions.
  • thermophilic enzymes resulting from longer catalyst lifetime
  • Purification can be accomplished in fewer steps without the need for refrigeration equipment
  • the effect of tiiermophilic biocatalysts is to lower catalyst cost by simplifying purification and increase catalyst half life to reduce the frequency of catalyst replacement
  • TT medium This medium consists of (per liter): BBL Polypeptone (8 gm), Difco Yeast Extract (4 gm), and NaCl (2 gm). Small scale cultures for screening are grown at 55-65°C at 250-300 rpm with 1 liter of medium in a 2 liter flask.
  • Samples ( ⁇ 1 gm) are resuspended in 2 ml of TT broth and 50-100 ⁇ of these samples were plated onto TT agar plates containing twice the usual amount of agar (3%). The increased agar concentration in the initial screening helped keep highly motile isolates from covering the entire plate.
  • agar was usually added to a final concentration of 1.5% for solid media Plates are incubated at 55°C or 65° C for one to two days and isolates then purified by numerous restreaks onto fresh plates for single colony isolation. The initial basis for differentiation is color, colony morphology, microscopic examination, temperature of growth. Several hundred strains were initially isolated. Over 100 different microorganisms were chosen for further study.
  • ⁇ pMYFl is a derivative of high capacity phagemid vector ⁇ pSL5, which was successfully applied for cloning and expression of a number of prokaryotic and eukaryotic genes in E. co/f.(Nick K.
  • ⁇ pSLS vector is a hybrid of phage ⁇ vectors ⁇ gtWEC and 1 L47.1 and plasmid pUC19.
  • ⁇ pSL5 DNA contains all the genes required for lytic ⁇ development, and provides for effective amplification of the primary libraries in phage form.
  • the vector itself cannot be effectively packaged into die ⁇ capsid in monomeric or oligomeric forms due to its size of 35 kb.
  • recombinant molecules of appropriate length have a selective advantage, which provides for very low background of non-recombinant molecules ( ⁇ 1% for ⁇ pSL5 vector).
  • Recombinant libraries collected as phage particles can be transduced into host strain containing a helper phage and screened for dehydrogenase activity its bacterial colonies.
  • the therm ⁇ sensitive ⁇ repressor cI857 from resident ⁇ prophage facilitates stability of ⁇ pSL5 based clones at 30°C.
  • E. coli LE392 (supE supF hsdR) was chosen as a host strain. LE392 is a standard host for phage and plasmid vectors bearing amber-mutations (such as ⁇ pSL5 and ⁇ pMYFl).
  • ⁇ pMYFl A map of ⁇ pMYFl is shown in Drawing 1.
  • ⁇ pMYFl was constructed by elimination of one of two BamHI sites on ⁇ pSL5 (Fonstein, unpublished). This vector allows cloning of Sau3A generated fragments into its remaining BamHI site.
  • ⁇ pMYFl was chosen as cloning vector because it provides d e highest level of foreign genes expression, presumably from P_ and PR promoters. Also, by unclear mechanism, copy number of ⁇ pMYFl is regulated dependent on toxicity of expressed foreign protein, i.e. ⁇ pMYFl allows cloning of unbearable for standard plasmid vectors proteins, such as proteases.
  • thermostable microorganisms from ThermoGen collection. Strains were re-streaked from frozen stocks on TT agar plates, and 50 mi TT liquid cultures were inoculated from single colonies. Cultures grew overnight at 55 C C, and were washed twice in 1 x TE buffer. Pellets were resuspended in 2 ml of fresh buffer SI (50 mM Glucose, 50 mM Tris HC1 pH 8.0, 50 mM EDTA pH 8.0, 10 mg/ml lysozyme) and incubated at room temperature (RT) for 5 minutes.
  • SI 50 mM Glucose, 50 mM Tris HC1 pH 8.0, 50 mM EDTA pH 8.0, 10 mg/ml lysozyme
  • DNA was extracted from water phase by precipitation with 95% ethyl alcohol, and resuspended in 500 ⁇ l of 10 mM Tris HC1 pH 7.5 solution. Construction of recombinant DNA libraries on pMYFl.
  • For the clone bank preparation we used the following microorganisms from die collection: # 2, 4, 7, 14, 16, 17, 19, 20, 22, 23, 24, 26, 27, 30, 31, 39, 45, 49, 51, 55, 57, 67, 69, 71, 75, 77, 83, 90, 98, 99, 116, 118, 122, 136, 146. In total, there were 55 new libraries constructed from 37 different strains (see Table 1 below).
  • Genomic DNA of each strain was partially cleaved with serial dilutions of which gave a gradient of restriction fragments lengths. The reactions continued for lh at 37°C followed by agarose electrophoresis to determine the sample widi optimal an average fragment size of 10-20 kb.
  • ⁇ pMYFl DNA (0.5 ⁇ J ⁇ ) was digested widi Bam HI to completion. Both genomic Sau 3A1 fragments and linearized ⁇ pMYFl DNA were precipitated wid etiiyl alcohol and resuspended in sterile distilled water at concentration 0.5 ⁇ / ⁇ and 0.8 g/ l.
  • 2 ⁇ l of ⁇ pMYFl was ttien ligated with 3 ⁇ of genomic Sau 3A1 fragments overnight at 16°C using 1 U of T4 DNA ligase (Stratagene) in a ligation mixture volume of 10 ⁇ .
  • 2 ⁇ l of die ligation mixture was incubated widi 12 ⁇ of 1 packaging extract (Promega) for 90 minutes at room temperature. Extracts were plated on LB and covered widi top agar containing fresh E. coli LE392 cells. Plates were incubated for approximately 16 hours, and recombinant phages were collected. Libraries containing 10 ⁇ - 10 ⁇ independent plaques, were stored at 4 ⁇ C in SM buffer.
  • thermophilic organisms were screened for new dehydrogenase activities using a colorimetric para-rosaniline test (described below) since some enzymes can be produced more easily from die host organism, and others can be produced more easily in a cloned format since the expression control signals which may inhibit production of the gene in the native host are removed in the clone.
  • a colorimetric para-rosaniline test described below
  • botii native tiiermophilic organisms as well as clone banks from these organisms so we would have an optimal chance of finding new and unique enzyme activities.
  • clone banks were plated out at about 1,000 colonies per plate and native organisms were screened individually.
  • die first step a para-rosaniline screen was used widi edianol as a substrate to identify colonies with alcohol dehydrogenase activity.
  • This broad range of enzymes was then screened in a second step with a set of different alcohol substrates which allowed us to identify the key enzymes of interest for the project Once a set of potential candidates were identified, the enzymes were more carefully analyzed quantitatively.
  • Transduction and activity tests 50 ⁇ l of ⁇ pMYFl libraries were mixed widi 200 ⁇ l of fresh overnight LE392 (1) cells at room temperature. After 20 minutes, 600 ⁇ l of LB were added, and incubation continued for 60 minutes at 30°C. Upon transduction, cells were plated on LB agar, and incubated at 30° C for 24-36 hours. Grown colonies were screening for dehydrogenase activity according the following protocol. Clone banks were transduced into l_E392 ⁇ ( ⁇ lysogen) for MYF derivatives or XLOLR for pBK derivatives.
  • LE392 ⁇ and XLOLR transduction were identical, however die growth temperature was 30°C for LE392 ⁇ derivatives which contain a temperature inducible lambda instead of 37°C for XLOLR derivatives. Plates were grown with a colony density of approximately 500 colonies per plate.
  • a number of different primary and secondary alcohols such as ethanol, hexanol, isopropanol and cyclohexanol were included in agar containing LB media, 50 mg/ml p-rosaniline and 250 mg/ml sodium bisulfite (Conway, et al., 1987).
  • Ethanol or another substrate diffuses into the bacterial cells to produce die acetaldehyde (or the appropriate product) by alcohol dehydrogenase.
  • the leuco dye serves as a sink, reacting with the acetaldehyde to form a Schiff base which is intensely red. Utilizing this metiiod, we screened dirough several hundred new strain isolates as well as all of the clone banks described above.
  • Plasmid pBPP containing d e cloned Horse Liver Alcohol Dehydrogenase - HLADH
  • plasmid pBPP containing d e cloned Horse Liver Alcohol Dehydrogenase - HLADH
  • Strains LE392 ⁇ harboring MYF derivatives widi cloned adh genes were grown in a 17 liter fermenter (LH Fermentation, Model 2000 series 1) in 15 liters of LB medium widi 100 g/ml Ampicilin at 30°C overnight Strains XLOLR/49.12 and ADOl were grown in the LB medium widi 40 g/ml Kan at 37°C. Native strains were grown in 15 liters of TT brotii at 55-65°C. All cells were grown with approximate stirring at 250 rpm and 03 to 0.5 wm (volumes air/volume media per minute).
  • Quantitative Assay A standard method for die quantification of alcohol dehydrogenase based on NAD(P) utilization was used. Overnight cultures of cells to be assayed are grown in rich media. The cells are washed, resuspended in 600 ⁇ l of assay buffer (83 mM KH2PO4 [pH 7.3], 40 mM KCl, 0.25 mM EDTA), sonicated, and centrifuged. The assay mixture typically contained 50 ⁇ l of cell extract, 100 ⁇ l EtOH, 20 ⁇ l 100 mM NAD and/or NADP, 830 ⁇ l buffer and is carried out at room temperature.
  • assay buffer 83 mM KH2PO4 [pH 7.3], 40 mM KCl, 0.25 mM EDTA
  • the assay mixture typically contained 50 ⁇ l of cell extract, 100 ⁇ l EtOH, 20 ⁇ l 100 mM NAD and/or NADP, 830 ⁇ l buffer and is carried out at room
  • the assay measures a substrate dependent reduction of NAD(P) + monitored by the change in absorbence at 340 nm in order to confirm dehydrogenase activity. Since crude lysates were expected to have background activity towards NAD(P) + , control experiments were performed to correct for spontaneous reaction with botii reduced and oxidized cofactors in the absence of added substrate. The reactions were run for approximately 3 minutes while continually measuring absorbence at 340 nM. This mediod produced a reliable quantitative determination of ADH activity present in the cell. Units of activity were calculated as ⁇ m A per minute of product formed. Specific activity is calculated as units per mg of protein used.
  • Isolated plasmid clones which express the desired proteins of die present invention can be sequenced to determine die nucleic acid sequence for the expressed protein.
  • By direct analysis of the DNA sequence it is possible to determine the start-codon for die initiation of transcription of die full-length transcript from which the amino acid sequence of die protein can be determined. Where tiiere may be more titan one possible start-codon, performing N-terminal amino acid sequence determination on isolated protein will allow for the identification of the proper start-codon.
  • Methods for nucleic acid sequnencing, amino acid sequencing, protein isolation and die procedures for growing and/or manipulating cells, proteins and/or nucleic acids can be found in die general literature, for example see Sambrook et al., Molecular Cloning 2nd edition, 1989, Cold Spring Harbor Press.
  • the turbid cultures were spun down in 50 ml conical tubes at 3,000 rpm for 10 minutes. The clear supernatant was discarded and die cell pellets were washed by resuspending in ⁇ 1 ml of TE buffer and transferring die volume into 1.5 ml eppendorf tubes. The cells were pelleted by centrifuging at high speed for 2-3 minutes. The supernatant was tiien aspirated and die cell pellets were resuspended in 0.5-1.0 ml of ADH buffer (80 mM KH 2 PO 4 , 40 mM KC1, 0.25 mM EDTA, pH 7.3) and put on ice.
  • ADH buffer 80 mM KH 2 PO 4 , 40 mM KC1, 0.25 mM EDTA, pH 7.3
  • the cell solution was sonicated twice for 20-30 seconds (output control at 4, percent duty cycle at 50, with pulsing). After sonication, die cell debris was spun down for 5 minutes. The clear supernatant was transferred to fresh eppendorf tubes and kept on ice.
  • Figure 3 depicts analysis of crude extracts from the ADH candidates. Crude extracts from ADH positive colonies were run on SDS gels. Fifteen microliters of each extract was mixed widi 15 ⁇ l of 2X SDS loading dye (100 mM Tris pH 6.8, 200 mM DTT, 4% SDS, 0.2% Bromophenol Blue, 20% glycerol). The mixtures were heated at 100°C for 5 minutes.
  • 2X SDS loading dye 100 mM Tris pH 6.8, 200 mM DTT, 4% SDS, 0.2% Bromophenol Blue, 20% glycerol.
  • the gel was run for ⁇ 2 hours at 80-100 V and tiien stained with Coomassie Brilliant Blue (0.25g Coomassie Brilliant Blue R250 in 90 ml methanol.water (1: 1 v/v) and 10 ml glacial acetic acid) for 30-45 minutes and destained widi destaining solution (90 ml metiianol: water (1:1 v/v) and 10 ml glacial acetic acid).
  • Labels 5, 7, 14, 19, 30, 39, 49.4, 49.12, 55, 69, 71, 98, 136, XY, CA, CB, CC refer to extracts made from strains AD5, AD7, AD 14, AD 19, AD30, AD39, AD49.4, AD49.12, AD55, AD69, AD71, AD98, AD136, XY (ADOl), CA, CB, and CC respectively. As can be seen, they did not show any outstanding protein bands which corresponded to the ADH gene. In order to further characterize the ADH content of die cells, Native protein gels and semipurified extracts were tested as described in die following section.
  • the molecular size of the active form of die enzymes was determined by separation by size on a gel filtration column containing Sephadex S-200 (Pharmacia ) agarose gel.
  • the column was equilibrated by the dehydrogenase enzyme assay buffer (83 mM potassium phosphate buffer, 43 mM Potassium chloride, 1 mM EDTA).
  • the column conditions were, flow rate, 14.4 ml per hour. Sample volume, 200 ⁇ L and die column size 50 cm by 1 cm (BioRad Labs.), and bed volume,
  • Labels 5, 7, 14, 19, 30, 39, 49.4, 49.12, 55, 69, 71, 98, 136, XY, CA, CB, CC refer to extracts made from strains AD5, AD7, ADM, AD19, AD30, AD39, AD49.4, AD49.12, AD55, AD69, AD71, AD98, AD136, XY (ADOl), CA, CB, and CC respectively.
  • the activity stain was made either in 0.4-0.7% agarose or in liquid form (1 mg/ml nitro blue tetrazolium, 0.1 mg/ml phenazine metiiylsulfale (dissolved in 1.0-1.2 ml ethanol before adding to mixture), 0.25- 0.35 mM NAD, 0.25-0.35 mM NADP) and added to the top of native PAGE gels as shown in Figure 4. Approximately 30 ml was adequate for staining each gel. The activity staining reaction was run at 37°C in the dark. In just a few minutes dark bands began to appear for highly active enzymes.
  • Lyophilized ADH samples were dissolved in ADH buffer (80 mM KJL-PO ⁇ 40 mM KC1, 0.25 mM EDTA, pH 7.3) at 20 mg/ml and put on ice.
  • the reaction mixture was made up of an appropriate dilution of die extract 30 l 0.1 M NAD or NADP, 100 ⁇ l ethanol, and measured up to 1 ml widi ADH buffer.
  • the activities were measured on die spectrophotometer at 340 nm for 80 seconds at room temperature.
  • the enzymes were assayed for activity at 30°C, 40°C, 50°C, 60°C, and 70°C.
  • d e spectrophotometer chamber was heated by a circulating water temperature bath.
  • the ADH buffer is also heated to die respective temperature, but the NAD or NADP and ethanol were at room temperature.
  • the amount of tiiose reagents in the total volume is not enough to lower die temperature significantly.
  • the 80 second activity reading is run 2-3 times consecutively to get an average. At 40°C, 50°C, and 60 ⁇ C, activity increases over several minutes. At 70°C, the activity is die highest for nearly all the enzymes, but die duration of the activity lasts only up to 5-7 minutes before die enzymes denature.
  • the optimal temperature charts for enzymes produced from strains AD19, AD30, AD39, AD49.4, AD49.12, AD55, AD69, AD71, AD98, and strain XY are depicted in Drawing 6. The data is presented as specific activity. Reactions were run for 80 seconds at the various temperature, and generally reflect die initial rates of the reaction. Most enzymes begin to denature at around 60°C and have very short half lives at 70°C.
  • Lyophilized samples were dissolved in ADH buffer (80 mM KH,PO 4 , 40 mM KC1, 0.25 mM EDTA, pH 7.3) at 20 mg/ml.
  • the enzymes were initially assayed at room temperature as a standard measurement.
  • the samples were tiien incubated at 40°C, 50°C, and 60 ⁇ C for varying lengths of time.
  • Plots for many of the enzymes we analyzed during die course of this work are depicted in Figure 7. As can be seen, most enzymes retain a significant amount of tiieir activity with very little loss in activity at 40°C.
  • Example 13 Specific Activity and Optimal Cofactor Analysis.
  • ⁇ Specific Activity is calculated from one run in the 15 Uter fermenter using ethanol as a substrate. Reactions were run at room temperature. Extracts from the clones were heat purified crude extracts, nd - not determined We tested die substrate specificity of the newly discovered catalysts on a variety of alcohols which could easily be analyzed spectrophotometrically. Table 5 lists relative activities of the ten enzymes we chose from die previous section of the work to study against a series of alcohols. The activities presented are relative to tiieir activity on edianol . The data which is presented has been compiled from several independent runs of enzyme in order to verify repeatability of the ratios which have been presented here.
  • die enzymes prefer alternative alcohols compared to edianol. Very few have a high degree of activity on cyclohexanol relative to edianol, but enough activity is present to detect in the short reaction.
  • AD49-12 is not highly selective on at least one of die two compounds. This underscores die fact tiiat we can find selective catalysts using die methods employed during tiiis project Another interesting note is that AD49-4 and AD49-12 both were isolated from the same clone bank (created from strain #49) yet are clearly different dehydrogenase activities.
  • AD55-1 3.0 5.0 4.8 0.0 0.0 100% 167% 160% 0% 0%

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Abstract

La présente invention se rapporte à des protéines possédant une activité d'alcool déshydrogénase. Dans une réalisation particulière, l'invention concerne des protéines possédant une activité d'alcool déshydrogénase produisant sélectivement des alcools chiraux. Dans une autre réalisation, l'invention concerne des protéines possédant une activité d'alcool déshydrogénase thermostable.
PCT/US1998/022607 1997-10-27 1998-10-26 Alcool deshydrogenases thermostables WO1999021971A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP98957372A EP1027427A2 (fr) 1997-10-27 1998-10-26 Alcool deshydrogenases thermostables
CA002308095A CA2308095A1 (fr) 1997-10-27 1998-10-26 Alcool deshydrogenases thermostables
AU13648/99A AU1364899A (en) 1997-10-27 1998-10-26 Thermostable alcohol dehydrogenases

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US6351797P 1997-10-27 1997-10-27
US60/063,517 1997-10-27

Publications (3)

Publication Number Publication Date
WO1999021971A2 true WO1999021971A2 (fr) 1999-05-06
WO1999021971A9 WO1999021971A9 (fr) 1999-08-19
WO1999021971A3 WO1999021971A3 (fr) 1999-10-28

Family

ID=22049741

Family Applications (1)

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PCT/US1998/022607 WO1999021971A2 (fr) 1997-10-27 1998-10-26 Alcool deshydrogenases thermostables

Country Status (4)

Country Link
EP (1) EP1027427A2 (fr)
AU (1) AU1364899A (fr)
CA (1) CA2308095A1 (fr)
WO (1) WO1999021971A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8476051B2 (en) 2008-09-26 2013-07-02 Kesen MA Thermostable alcohol dehydrogenase derived from Thermococcus guaymasensis

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4352885A (en) * 1980-05-09 1982-10-05 Wisconsin Alumni Research Foundation Preparation of a novel NADP linked alcohol-aldehyde/ketone oxidoreductase from thermophilic anaerobic bacteria for analytical and commercial use
FR2706906A1 (en) * 1993-06-21 1994-12-30 Ifremer Alcohol dehydrogenase, microorganism producing it and its uses
KR19990087330A (ko) * 1996-02-27 1999-12-27 로저 윌킨슨 써모언에어로박터 에탄올리쿠스 39e의 2차 알콜 탈수소 효소를코딩하는 유전자의 클로닝 및 발현과 효소의 생화학적 특성화
DE19610984A1 (de) * 1996-03-21 1997-09-25 Boehringer Mannheim Gmbh Alkohol-Dehydrogenase und deren Verwendung zur enzymatischen Herstellung chiraler Hydroxyverbindungen

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8476051B2 (en) 2008-09-26 2013-07-02 Kesen MA Thermostable alcohol dehydrogenase derived from Thermococcus guaymasensis

Also Published As

Publication number Publication date
AU1364899A (en) 1999-05-17
WO1999021971A9 (fr) 1999-08-19
WO1999021971A3 (fr) 1999-10-28
CA2308095A1 (fr) 1999-05-06
EP1027427A2 (fr) 2000-08-16

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