CA2308095A1 - Thermostable alcohol dehydrogenases - Google Patents

Abstract

The present invention provides proteins having alcohol dehydrogenase activity. In a particular embodiment, proteins having chiral alcohol selective alcohol dehydrogenase activity. In another embodiment, proteins having thermostable alcohol dehydrogenase activity are provided.

Description

- - WO 99121971 - . PCTIUS98I22607 TFIERMOSTABLE ALCOHOL DEHYDROGENASES
Relation To Previous Applications This application is a continuation of U.S. Provisional Application 60/063,517 filed October 2?. 1998 (Allen, et al., THERMOSTABLE ALCOHOL DEHYDROGENASES).
Statement of Government Rights Some of the work in this specification has been performed under NSF Grant No.

61842, therefore the United States Government may have certain rights in the invention.
Field of the Invention The present invention relates to the filed of catalytic reagents for use in chemical synthesis. In particular, the present invention relates to novel thermostable enzyme proteins with alcohol dehydrogenase activity.
Background of the Invention There is great need in the chemical industry to develop catalytic methods capable of efficiently converting molecules to products without generating excessive waste streams or emissions. Molecular conservation methods based on biocatalyt reagents, such as dehydrogenase enzymes, offer opportunities to solve the challenges found in attempting industrial scale synthesis. Current dehydrogenase based technology has been limited by the lack of available enzymes. The few enzymes which are available have narrow substrate specificities, poor stabilities or yield only a single type of stereocenter. New dehydrogenase enzymes are needed in order to increase the chemo- and stereoselectivity for different substrates, thereby increasing the spectrum of useful chemical synthetic reactions over that which is currently available.
Dehydrogenase biocatalysi reagents efficiently reduce ketones to chiral alcohols. The reduction of 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). Although many methods exist for operation on carbonyl groups, few combine the selectivity, mildness, cost effectiveness and minimal waste streams offered by enzyme catalyzed reactions. Alcohol dehydrogenases are one of the principal enzymes that effectively operate on the carbonyl group. In spite of industrial interest, very 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 acre increasingly being nrcognized 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 aro user friendly, economical and produce high yields of chiral chemicals are crucial to the future of industrial chemistry. Over 5096 of existing therapeutic agents are chiral molecules and of those that are synthetic (528), 7596 are propared 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 rocently c~edeveloped antiasthma agent (R)-albuterol, and the nonsteroidal andinflammatory 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). In many such cases, biocatalysts offer the potential to dramatically szrearnline synthetic processes by minimizing the total number of I S steps andlor complex purification schemes (Grout 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. Qne emerging technology involves the use of dehydrogenase enzymes to effectively roduce carbonyl groups to the corresponding alcohols. Figure I highlights a few ketone substrates stemoselectively reduced to chiral alcohols that have been used as intermediates in chemical synthesis.
Commercially available dehydrogenases are limited in chemo- and stereoselectivity. Alcohol dehydrogenases are a family of enzymes capable of formal reversible two electron chemistry in which alcohols are oxidized to the con~esponding ketones (Table I (Faber.
1992)).
Table I. Characteristics of commonly studied alcohol dehydrogenases.
Dehydrogenase Specificity Commercial yeast-ADH Prelog +

horse liver-ADH Prelog +

T. brockii-ADH Prelog +

Hydoxysteroid-DH Pt~elog +

Curvularia falcata-ADHPrelo ~ -lactobacillus kefir-ADH Anti-Prelog -Pseudamorcas sp.-ADH Anti-Prelog -.._._,_ .-.
a. The specificity is reversed for small ketone substrates.

- - WO 99/21971 - - PCT/US98~2607 Depending on the conditions (Lemiere, 1986), ketones can be reduced to the respective alcohols via a stereospecific delivery of a hydride equivalent catalyzed by the enzyme coupled to a bound cofactor (NADH or NADPH). 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 generating a variety of compounds with capacity for industrial scale-up (See Drawing below) (Bradshaw, et al. 1992; Hummel, 1990; Seebach, et al., 1984}.
O
O ~ CF3 CI~
Ketone substrates reduced by T. brockii alcohol dehydrogenase O O

\I -Brief Summary of the Invention 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
(ADO 1 ). In a particular embodiment, the present invention provides for a protein with chiral alcohol selective alcohol dehydrogenase activity selected from the group consisting of AD19, AD30. AD39, AD49.4, AD49.12, ADSS, AD69, AD71, AD98, and XY (ADO1).
The present invention also provides for a protein with thermostable alcohol dehydrogenase activin~ selected ti-om the group consisting of AD7.1, AD14.1. AD31.3, AD39.4, AD49.-1.
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. Such 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. Thus 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 - - WO 99/21971 - . PCT/US98I22607 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.
Thus 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.
Brief Description of the Drawings The enzymes of the present invention will be better understood in conjunction with the following figures in which:
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), are shown. The data is presented as percent of original activity.

Detailed Description of the Invention Dehydrogenases have been iden'ed 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. The 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 WO 99/21971 - . PCT/US98l22607 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 nactions 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. The broad range of molecules which serve as substrates has advanced baker's yeast as an important synthetic organic reagent and its use has been documented in several reviews (Csuk and Glanzer, 1991;
Neuberg.1949; Servi, 1990). In spite of its apparent general utility there are some limitations to intact Baker's yeast including the risk of secondary reactions resulting from other active proteins within the yeast Other challenges associated with. whole cell biocatalytic systems include limited substrate access to intracellular proteins and contaminating products of normal metabolic processes. Whole cells are often sensitive to immobilization and intolerant to temperature and inclusion of organic solvents.
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 (Cosy, et ai..1992) and resolution of racemic alcohols (Jones and Jakovic, 1982).
Until recently, application of purified dehydrogenases has been limited by both the cost of cofactors (NAD(P)I-17 and the intolerance of cell free dehydrogenases to extreme environments often found in synthetic organic transformations. Biocatalysts developed from thermophilic organisms could increase environmental tolerance although thermophiles have yet to be widely investigated for useful dehydrogenase catalysts. The alcohol dehydrogenase isolared from Thermoanaerobium brockii is one of the few successfully isolated dehydrogenases from - - WO 99/21971 - . PtyT/US98I22607 thermophilic organisms which show promise for industrial applications (Keinan, et al.. 1986;
Lamed and Ziekus. 1981 ).
NAD(P)H Cofactors am 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,OOQImole) and are used stoichiometrically during the reaction. Cofactor cost currently limits the use of cofactor requiting enzymes to high value applications such as the preparation of pharmaceuticals. Generally, cofactors can not be simply disposed of at the conclusion of the reaction but rather need to be recycled. Ideally, cofaewrs should function at catalytic concentrations, necessitating in situ t~egeneration of the active species.
Cutreztt cofactor recycling techniques allow dehydrogenase biocatalysts to be used as valuable synthetic tools. the question of cofactor tECycling has received considerable attention and several elegant means exist to regenerate reduced nicotinamide analogs for use in dehydrogenase catalyzed reactions (Jones. 1986). Promising methods for cofactor recycling include multiple dehydrogenase reactions in which the reduction by the dehydrogenase of interest is coupled to a second dehydrogenase such as formate dehydrogenase (Hummel, et al., 1987). Other methods involve using simple alcohols like isopropanol which have more favorable equilibria to regenerate cofactors without reoxidizing newly formed products (van Eys, 1961). 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. Recently NAD+ molecules have been treated with vanadate to produce a less expensive analog to the highly cosily 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 ozidatian state. For the reduction of carbonyl groups, analysis of t~eaction 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. Several general observations have been made mgarding the stereoselectivity of dehydrogenases which has led to some empirical rules for dehydrogenase mediated catalysis. In an early attempt to explain dehydrogenase stereoselectivity, 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).
b WO 99/21971 - - PCf/US98I22607 c ~r~ ~~ ~ The Prelog Rule for reduction of x R s ,, M carbonyl groups.
g M NAD(Plli NAD(PJ
A more recent reinvestigation of dehydmgenase selectivity was carried out by Jones and coworkers who developed the cubic surface model for predicting steteoselecdvity of dehydrogenase ructions shown in the drawing below (Dolls and Jones. 1988; Jacovac, et al.. 1982;
Jones and Jakovac, 1982).
'Ibe Cubic Space modtl fa alcohol debydtogenase catalyzed reactiocu (Jones and Jaltavac,1982). Cubic surfact model of the active site of horse liver aloo6ol debydtogenase. Cubes with solid lines are focbiddea spars occupied by active site aimino acid residues. Dotted lines are cubes with disfavored spaces originating fmm potential interactions with charged groups on active site residues. Open spaces are available for substrate access to the active site, w6icb allows positioning of the substrate in such a way that the alive carbonyl functionality is located just above the hydride equivalent of tbc cofactor at the Cl-D1 boundary.
This model mote accurately describes the structure activity relationship between the active site binding surface and the structural geometry imposed by the substrate. The cubic surface model remains an effective steneoselectivity model since it evaluates catalysis based on both a library of substrates with differing structure and high resolution X-ray crystal data instead of addttrssing specific interactions between substrate and protein. These investigations allow an un~rstanding of dehydrogenase function that may be extended to newly developed catalysts. A
newly discovered dehydrogenase can be assayed with a group of molecules to define the potential applications of the isolated catalyst.
Thet~e is significant commercial utility for alcohol dehydmgenas~es.
Development of reliable catalytic systems will provide an efficient means of generating chiral compounds without the need for alternative often tedious, costly resolution and purification techniques.
Dehydrogenase catalysts will be synthetic reagents that ate both mild and selective even in the presence of sensitive functionality to produce chiral building blocks for assembly of mote complex compounds.
Dehydrogettase catalyzed reactions ate expected to be applied to the synthesis of complex therapeutic compounds such as antibiotics and antiviral agents.

Few options ane 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). These methods represent relatively low cost biocatalytic options for the reduction of ketones to directly insert chiral centers into molecules that have demonstrated the potential of dehydrogenases for industrial use.
Unfortunately, these two catalysts are limited in their substrate range and the HLADH enzyme in particular, is sensitive to inactivation. The structural specificity of these two enzymes has, however, laid the groundwork for development of other dehydrogenases with different chemo- and stereoselectivities in addition to providing opportunities for dehydrogenase catalysts that are more tolerant to conditions found in industrial applications. One technology which has been developed address the lack of stable enzymes is Altus Biologics' Cross-Linked Enyzme Crystal (CLEC) technology. Although expensive, this technology is useful to stabilize proteins for some applications and could possibly be used to stabilize thenmophific proteins even fiu~ther if needed.
Thermophilic organisms provide stable biocatalyst reagents for organic synthesis. One of the greatest challenges still facing synthetic chemists is to develop inexpensive, efficient ways of producing compounds. In the field of biocatalysis, the problem is not only to find a system to effect a given transformation but also to implement the reaction on a large scale. Proueins derived from thermophilic sources provide an opportunity to satisfy the demands of the industrial process chemist Thermophile derived biocatalysts maintain activity under relatively harsh conditions (Daniel, et al., 1990; Fontana, 1984; Pham and Phillips, 1990) including: non-refrigerated storage, presence of organic cosolvents, and resistance to temperature inactivation.
Thermophile derived biocatalysts can reduce production and catalyst cost. The ability to function at elevated temperatures should not be confused with a need to operate under such conditions.
Tolerance to temperature reflects the inherent ability to survive as a functioning catalyst long after mesophile derived proteins have been inactivated, regardless of tine means by which deactivation occurs. In addition, the costs associated with cloned thermophilic enzymes (resulting from longer catalyst lifetime) should be lower than the corresponding mesophilic proteins, particularly for systems requiring purified catalyst preparations. Purification can be accomplished in fewer steps without the need for refrigeration equipment. The effect of thermophiIic biocatalysts is to lower catalyst cost by simplifying purification and increase catalyst half life to reduce the frequency of catalyst r~eplacemen~

- - WO 99121971 - . PCT/US98/22607 The pn"sent invention and its various embodiments will be better understood in view of the following examples. 'l7ie examples are presented only by way of illustration, and not 1'smitation, of certain embodiments of the pt~esent invention. One of ordinary skill in the art would be able to understand and use the teachings of the p~sent invention to develop additionat equivalent emodimnents within the scope of the teaching and spirit of the disclosure.

Example 1. Isolation of Strains Strains. Most isolated strains and cultures ore grown un TT medium. This medium c:wns~sV of (per liter): BBL Polypeptone {8 gm), Difco Yeast Extract (4 gm), and NaCI (2 gm). Small scale rultums for screening; are ~,~rown at 5S-65°C at ''S0-300 rpm with 1 liter of medium in a 2 liter flask.
Enrichment Procedures for Newly Isolated Therniophiles. Multiple stream sediments, composting organic materials, and soil samples are used to isolate new strains. These samples were collected from numerous geographic sites ranging from the Midwest to the Southeast United States.
Samples (~I gm) are resuspended in 2 ml of TT broth and 50-100 ~Cl of these samples were plated onto TT agar plates containing twice the usual amount of agar (3~1e). The increased agar concentration in the initial screening helped keep highly motile isolates from covering the entire plate. After initial purif c.:atiun, agar was usually added to a Coral cvnc;entration of l.S~c fur solid media Plates are incubated at 55°C or 65°C for ane to two days and isolates then purified by numerous restr~ks onto fresh plates for sinble cwlony isolation. The initial basis for differentiation is color, colony morphology, microscopic examination, temperature of growth.
Several hundred strains were initially isolated. Over lUU different microorganisms were chosen for further study.
Example 2. Construction of 7~pMYF
pMYFl is a derivative of high eapac,~ity phal;emid vector 7~pSL5, which was successfully applied 2S for cloning and expression of a number of prokaryotic and eukaryotic genes in E. coli.(Nick K.
Yankovsky, et al. 1989. Phasmids as effective tools for cunswction and analysis of gene libraries.
Gene, 81, ?.03-210). The origiaal hpSLS vector is a hybrid of phage ~ vectors ~gtWEC and 1 L47.1 and plasmid pUCl9. 7~pSL5 DNA contains all the genes required Cur lyvc 7~ development, and provides for effective amplification of the primary libraries in phage form. The vector itself exurncx be effectively p~ackabed into the ~. c;apsld in munumeric or oligomeric forms due t~~ its size of 35 kb. Thus, recombinant molecules of appropriate length have a selective advantage, which provides fur very low background of non-recombinant molecules (<I~Ic for xpSLS
vector).
Recombinant libraries collected as phage particles can be transdt>ced into host strain containing a helper phube and sc,Teened fur dehydrugenase activity :is bacterial wlunies.
The thermusensztive ~
repressor cI857 from resident 7~ prophage facilitates stability of ~pSLS based clones at 30°C.
AuW nc~mc~us replic;alicm of phagemids in lysagens is mainldined by a pUC
replic:atiun origin. The - - WO 99/21971 - . PCT/US98/22607 average size of cloned fragments is 12-15 kb, with maximum at about 20 kb, which is determined by the capacity of 7~ capsid (4$ kb). E. roll 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 7~pSL5 and 7~pMYF1).
A map of 7~pMYF1 is shown in Drawing 1. 7~pMYF1 was oonstnrcted by elimination of one of two BamHI sites on kpSLS (Fonstein, unpublished). This vector allows cloning of Sau3A
generated fragments into iu remaining BamHI site. ~pMYFI was chosen as cloning vector because it provides the highest Level of foreign genes express~iun, presumably from Pl" and PR promoters.
Also, by unclear mechanism, copy number of ~.pMYFl is regulated dependent on toxicity of expressed foreign protein, i.e. 7~pMYF1 allows cloning of unbearable for standard plasmid va;tors proteins, such as prot~eases.
Example 3. Preparation of Clone Banks Choice of the vectors and E. roll host strains. Several cloning systems were used to clone new dehydrogenases. The first was the ~ZAPIXLOLR system from Promega which we had previously used to clone many other types oC enzymes. This system led to the cloning of several new dehydrogenases but was not as effective as statistical analysis suggested it could be for the cloning of new genes. As a result, a second system, ~.pIviYFl, which we have also used regularly in the ~t was employed (and led to most of the new clones desv,,ribed in this work).
Preparation of genomic DNA of thermostablc microorganisms from ThermoGen collection.
Strains were re-streaked from frozen shocks on TT agar plates, and 50 m1 TT
Liquid cultures were inoculated Crom single colonies. Cultures grew overnight at 55°C, and were washed tvvce in 1 x TE buffer. Pellets were resuspended in 2 ml of fresh buffer S1 (50 mM Glucose, 50 mM Tris HCl pH 8.0, 50 mM EDTA pH 8.0, 10 mglml lysozyme) and incubated at room temperature (RT) for 5 minutes. 5 ml of buffer S2 (50 mM Tris HCI pH 8.0, 50 mM Tris HCl pH 8.0, 1 %
SDS, 10 mglml Proteinase K)was added in each tubes, and samples were iysed at 65°C until solution a ant. Normally, this procedure required 3-4 hours, but time varied depending on strain. After completion of lysis several extractions with organic solvents were applied to get rid off call proteins. The first two extractions were done with pure phenol saturated with Tris HCI, pH
7.5, followed by phenollchloraform 1:1 and chloroCarmlisoamyl afwhol 24:1 extrac;tions. DNA
was extracted from water phase by precipitation with 95°k ethyl alcohol, and resuspended in 500 Pl of 10 mM Tris HCl pH 7.5 solution.

Construction of recombinant DNA libraries on pMYFJ. For the clone bank preparation we used the following microorganisms from the 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 Ih at 37°C
followed by agardse electrophoresis to determine the sample with optimal an average fragment size of 10-20 kb.
xpMYFl DNA (0.5 y~gl~l ) was digested with Bam HI to completion. Both genotnic Sau 3A 1 fragments and linearized apMYFI DNA were precipitated with ethyl alcohol and resuspended in sterile distilled water at concentration 0.5 yegl~l and 0.8 y~glui. 2 yd of 7~pMYF1 was then ligated with 3 ~1 of genomic Sau 3A1 fragments overnight at 16°C using 1 U of T4 DNA ligase (Stratagene) in a ligation mixture volume of 10 yd. Upon of the completion of the reaction, 2 yel of the ligation mixture was incubated with 12 ~I of 1 packaging extract (Promega) for 90 minutes at room temperature. Fxtiacxs were plated on LB and covered with top agar containing fresh E. toll LE392 cells. Plates were incubated for approximately 16 hours, and recombinant phages were uuilected. Libraries wntaining 103 - 105 independent plaques, were stored at 4'C in SM buffer.
Table 2. Organisms and Clone Banks Screened in this Work Strains used from ThermoGen collection Organisms Scxeened Nearly 200 strains from the ThermoGen uollec;tion.
pSL.S based clone banks 2;10; 14; 16; 17; 19; 20; 23; 27; 30; 36; 51; 67; 69;
71; 75; 77; 83; 90;
98; 99; 116; 118; 122;136; 146; xy (26total) pMYFl based clone banks 2; 4; 7; I4; 17;19; 20; 22; 23; ?A; 26b; 30; 31; 39;
45; 49; 51; 55; 57;
69; ?1; 77; 83; 90; 98; 122; 136; xy (28 total) pZAP based clone banks 11; 13; 14; 15; 18; 21; 26b; 35; 36; 37; 49; 51; 59;
65; 83 Example 4. Screening for Novel Alcohol Dehydragenases (ADH) The following approach for the identification of new dehydrogenase catalysts from thermophilic organisms was used. Both native thermophilic organisms and clone banks from thermophilic organisms were screened for new dehydrogenase activities using a oolorimetric para-rosaniline test (desc:ribed below) since same enzymes v;an be produced more easily frcxn the host urganiscn, and 12 ', WO 99121971 - . PCT/US98/22607 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. As a result, we screened both native thetmophilic organisms as well as clone banks from these organisms so we would have an optimal chance of finding new and unique enzyme activities. In general, clone banks were plated out at about 1,000 colonies per plate and native organisms were screened individually. In the Cirst step, a para-rosaniline screen was used with ethanol as a substrate to identify cdonies 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. For transduction, 50 ~l of kpMYFl libraries were mixed with 200 ~1 of fresh overnight LF392 (1) cells at room temperature. After 20 minutes, 600 gel of LB
were added, and incubation continued for 60 minutes at 30°C. Upon transduction, cells were plated IS 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 LE3927~ (7~ lysogen) for MYF derivatives or XLOLR for pBK derivatives. The conditions for LF392~ and XLOLR transduction were identical, however the growth temperature was 30°C for LF3927. derivatives which contain a temperature inducible lambda instead of 37°C f~ XLOLR
derivatives. Plates were grown with a colony density of approximately 500 colonies per plate.
plate Assay. Both native strains and clone banks were screened for the presence of ADH activity using para-rosaniline method (modified from T. Conway et al., 198'1, J.
Bacteriol., 169:2591-2597) as follows: Indicator plates were prepared by adding 8 ml of para-rosaniline (2.5 mglml in 96°k ethanol) and 100 mg of sodium bisulfate to 400 ml batches of preoooled (45°C) Lucia agar.
Most of the dye was immediately converted to the leuco form by reaction with bisulfate to produce a rose~olor~ed medium. Plates were stored away from fumes which contain aldehydes in the dark.
A number of different primary and secondary aloohols such as ethanol, hexanol, isopropanol and cyclohexanol were included in agar containing LB media, 50 mglml p-rosaniline and 250 mg/ml sodium bisulfate (Conway, et al., 198'7). Ethanol (or another substrate) diffuses into the bacterial cells to produce the acetaldehyde (or the appropriate product) by alcohol dehydrogenase. The leuoo dye serves as a sink, reacting with the acetaldehyde t~o form a Schiff base which is intensely red.

- - WO 99121971 - . PCTIUS98/22607 Utilizing this method, we screened through several hundszd new strain isolates as well as all of the clone banks described above. A number of different candidates was identified for further study.
By far, the clone banks gave us the most colonies with the best overall enzyme production and yield. In fact, many native strains which did not show dehydrogenase activity on the screening plates, were actually good sources for cloned dehydrogenases from gene banks made from their genome. About twenty positive clones per bank were picked on the para-rosaniline plates.
l~asmid DNA was isolated from all the positive clones, and transductattts containing the unique plasmids were used for the further analysis that included ADH activity assay with ethanol as a substrate. Strains harboring plasmid pBPP (containing the cloned Home Liver Alcohol Dehydrogenase - HLADH) were used as a control. Upon restreaking, and testing for dehydrogenase activity, the most active isolates were identified for further study.
Easmple 5. Production of Novel ADH Candidates Psoduetion of ADH enrymes in shake~lasks for analysis. Strains LF~92 harboring MYF-ADH
plasmids were grown as 1 l cultures in 21 shake flasks for 2 days in LB medium with 100 Pglml ampicillin (Amp) at 30°C. Strains XLOLRl49.12 and ADOl were grown in LB-Kan4p medium at 37~C overnight. Cetls were harvested by centrifugation, lysed using a Sonics &
Materials homogenizes, incubated for 5 min at 65~C in the 50 ml tubes (without temperature control inside of the solution). After centrigugation to remove the denatured protein debris the supernatant containing the purified protein was lyophilized.
Production of ADH enzymes in fermenters. Strains LE392k harboring MYF
derivatives with cloned adh genes were grown in a 17 liter fermenter (LH Fermentation, Model 2000 series 1) in 15 liters of LB medium with 100 ug/ml Ampicilin at 30'°C overnight.
Strains XLOLRI49.12 and AD01 were grown in the LB medium with 40 ~glml Kan at 37°C. Native strains were grown in 15 liters of TT broth at 55-65°C. All cells were grown with approximate stirring ai 250 tpm and 03 to 0.5 vvm (volumes airlvolume media per minute). Temperature is maintained by circulating water from a 28 liter water reservoir through hollow baffles within the stirred bars. The cells were disrupted by using two passes of an Avestin Emulsiflex CS homogenizes between 10,000-15,000 PSI. The cells were then spun down and purified by heat treatment at 65~C for 5 min. with temperature control inside of the solution. The sample was then spun down again in a centrifuge to remove the cell debris. The protein was lyophilized and characterized by substrate specificity assay.

- - WO 99/21971 . . PCT/US98/22607 Example 6. Assay for ADH In cell extracts Quareaitative Assay. A standard method for the 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 b00 pl of assay buffer (83 mM KH2P04 [pH
7.3], 40 mM
KCI, 0.25 mM EDTA), sonicaied, and centrifuged. The assay mixture typically contained 50 y~l of cell extract, 100 ~1 EtOH, 20 ~! 100 mM NAD andlor NADP, 830 ~C! buffer and is carded out at room temperature. 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)+, coatroi experiments were performed to correct for spontaneous reaction with both reduced and oxidized oofacLars in the absence of added substrate. The reactions were run for approximately 3 minutes while continually measuring absorbence at 340 nM. This method producer! a reliable quantitative determination of ADH activity present in the cell. Units of activity were calculated as yCmol pa minute of product formed.
Specific activity is calculated as units per mg of protein used Example 7. DNA Analysts.
For the cloned candidate dehydrogenases, the DNA insert size was analyzed for comparison to each other. To analyze plasmids which were cloned using the pMYF system, as EooRV digestion was used and to analyze plasmids which were cloned from the XLOLRIaZAP system, an EcoRI +
Pstl restriction digestion was used. Insert sizes for the key new dehydrogenase activities we have discovered during the course of this work are compiled in Table 4 below. It is interesting to note that done numbers 19 and 39 show nearly identical restriction patterns, yet they were obtained from colonies with significantly different morphologies, and their substrate specificities are different (see substrate characterization data below). This may indicate that they are highly related, but not identical enzymes.
Isolated plasmid clones which express the desired proteins of the present invention can be sequenced to determine the nucleic acid sequence for the expressed protein. By direct analysis of the DNA sequence it is possible to determine the start-colon for the initiation of transcription of the full-length izanscript from which the amino acid sequence of the protein can be determined. Where .there may be more than one possible start-colon, performing N-terminal amino acid sequence determination on isolated protein will allow for the identification of the proper start-colon.

Methods for nucleic acid sequnencing, amino acid sequencing, protein isolation and the procedures for growing andior manipulating cells, proteins andlor nucleic acids can be found in the general literature, for example see Sambrook et al., Molecular Clc~'ng_2nd edition, 1989, Cold Spring Harbor Press.
Example 8. SDS-PAGE Gel Analysis.
In order to prepare samples for protein gel analysis, 50 ml of LB containing I00 pUml ampicillin were inoculated with different E. coil LE3921 cells containing ADH plasmids derived from pSLS
vector. The cultures were grown overnight at 30°C. The strains were named according to the native organism's DNA library number. 5, 7, 14, 19, 30, 39, 55, 69, 71, 98, 136 and 49.4 AD49.12 were clones derived from the pBK vector and transformed into E. coil XLOLR cells instead of LE3927~. XY, CA, CB, and CC are native cells that were found as new organisms on I5 para-rosaniline selection plates (which may be mesophiles). The turbid cultures were spun down in 50 ml conical tubes at 3,000 rpm for 10 minutes. The clear supernatant was discarded and the cell pellets were washed by resuspending in ~l ml of TE buffer and transferring the volume into i.5 ml eppendorf tubes. The cells were peileted by centrifuging at high speed for 2-3 minutes.
The supernatant was then aspirated and the cell pellets were resuspended in 0.5~ 1.0 ml of ADH
buffer (80 mM KH2P04, 40 mM KCI, 0.25 mM EDTA, pH 7.3) and put on ice. The cell solution was sonicated twice for 20-30 seconds (output control at 4, percent duty cycle at 50, with pulsing).
After sonication, the ce~1 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. FiCt~een microliters of each extract was mixed with 15 ~l of 2X SDS loading dye (104 mM Tris pH 6.8, 200 mM DTT, 4% SDS, 0.2%.Bromophenol Blue, ZO~Yo glycerol). The mixtures were heated at 100°C for 5 minutes.
Thirty microliters oC each mixture was loaded onto an 8°k SDS Gel (8~o acrylamide, 375 mM Tris pH
8.8, O.I°6 SDS, 0.1°k ammonium persulfate, 10-12 ~Cl TEMEDI20 ml gel solution) with 10-15 ~l of molecular weight standard (Kaleidoscope Prestained Standards, Catalog number 16I-0324, Bio-Rad). The gel was run for -2 hours at 80-100 V and then stained with Coomassie Brilliant Blue (0.25g Coomassie Brilliant Blue 8250 in 90 ml methanol:water (1:1 vlv) and 10 ml glacial acetic acid) for 30-45 minutes and destained with destaining solution (90 ml methanol:water (1:1 vlv) and 10 ml glacial acetic acid). Labels 5, 7, 14, 19, 30, 39, 49.4, 49: I2, 55, 69, 7I, 98, 13b, XY, CA, CB, CC

refer to extracts made from strains ADS. AD7, AD14, AD19, AD30, AD39, AD49.4, AD49.1?, AD55, AD69, AD71, AD98, AD136, XY (ADO1), CA, CH, 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 the cells, Native protein gels and semiputifted extracts were tested as described in the following section.
Example 9. Determination of Molecular Size of the Proteins by Gel Filtration Chromatography The molecular size of the active form of the enzymes was determined by separation by size on a gel filtration column containing Sephadex S-200 (Pharmaaa ) agarose gel. The column was equilibrated by the dehydrogenase enzyme assay buffer (83 mM potassium phosphate buffo, 43 mM Potassium chloride, 1 mM EDTA). The column conditions were, flow rate, 14.4 ml per hour.
Sample volume, 200 ~L and the column size 50 cm by 1 cm (HioRad Labs.), and bed volume, -37.5 ml. The proteins were detected by absorption at 280 nm and for the enzymes the activity was monitored and, the fraction with highest activity was taken as the elution volume. Molecular sizes were calculated from the plot of Log of the known protein standards against the ratio of elution volume (Ve) w void volume (Vo) of the column. The ratio of Ve/Vo values obtained for the unknown proteins (dehydrogenases) was used to determine the molecular size from the standard plot developed for the proteins of known molecular size. The standard proteins used were, yeast alcohol dehydrogenase ( 150 kDa), bovine serum albumin (67 kDa), ovalbumin (49 kDa) and blue dextran (2'_00 kDa). When possible, subunit composition was determined by comparing active protein sizes of the fractions from the chromatography with SDS Gel electrophoresis results of the semipurified protein. The results of this analysis is presented in the Summary Table 4 below.
Example 10. Method of Protein CharacteHzation by migration on Native PAGE.
We were able to identify the active protein band in a crude extract by running the sample on a native (nondenaturing) protein gel and staining with a phenazine-based method.
The cell e!ctracts were run on an 8% Native PAGE, which (which were identical to our 8% SDS PAGE
gels described above without the SDS). Fifteen microliters of cell extract was mixed with 15 Pl of 2X
Native gel loading dye (100 mM Tris pH 6.8, 200 mM DTT, 0.29 Bromophenol Blue, ?09c glycerol). The mixture was loaded onto the gel without boiling. The gels were run at 80-100 V
for 2-3 hours. 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 ADS, AD7, AD14, AD19, AD30, AD39, AD49.4, AD49.12, 17 ' - - WO 99/21971 - . PCTIUS98/22607 AD55, AD69, AD71, AD98, AD136, XY (ADO1), CA. CB, and CC respectively. The activity stain was made either in 0.4-0.7% agarose or in liquid form ( 1 mglml vitro blue tetrazolium, 0.1 mglml phenazine methylsulfate (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.
Example 11. Optimum Temperature Analysis.
Lyophilized ADH samples were dissolved in ADH buffer (80 mM KH~P4~, 40 mM KCI, 0.25 ~ EDTA, pH 7.3) at 20 mglml and put on ice. The reaction mixture was made up of an appropriase dilution of the extract, 30 y~l 0.1 M NAD or NADP, 100 ~l ethanol, and measured up to 1 ml with ADH buffer. The activities were measured on the 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. At each temperature, the spectrophotometer chamber was heated by a circulating water temperature bath. The ADH buffer is also heated to the respective temperature, but the NAD or NADP and ethanol were at room temperature. The amount of those reagents in the total volume is not enough to lower the 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 the highest for nearly all the enzymes, but the duration of the activity lasts only up to 5-7 minutes before the enzymes denature. The optimal temperature charts for enzymes produced from strains AD19, AD30, AD39, AD49.4, AD49.I2, 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 the initial rates of the reaction.
Most enzymes begin to denadue at around 60°C and have very short half lives at 70°C.
Example 12. Residual Activity Analysis.
Lyophilized samples were dissolved in ADH buffer (80 mM KHZPO~, 40 mM KCI, 0.25 mM
~TA, pH 7.3) at 20 mglml. The enzymes were initially assayed at room temperature as a standard measurement. The samples were then incubated at 40°C, 50°C, and 60°C for varying lengths of time. Plots for many of the enzymes we analyzed during the course of this work are depicted in Figure 7. As can be seen, most enzymes retain a significant amount of their activity with very little loss in activity at 40'°C.
18 i.

Example 13. Specific Activity and Optimal Cofactor Analysis.
The specific activity of each enzyme identified in a crude extract from shake flasks or from fermentets was analyzed using ethanol as the main substrate to determine relative reaction rates. In addition, both NAD and NADP were tested as cofactors in order to determine the ideal cofactor.
For all of the enzymes discussed in this report, NAD was the optimal cofactor . Other enzymes preferred NADP, however activities were generally low and made it difficult to develop these enzymes further. Since NAD is a more cost effective cofactor than NADP, we decided to preferentially focus on the NAD utilizing enzymes during the initial characterization. The optimized results from cultures which were grown in 15 L fermenters are listed in Table 4 at the end of this section. In many instances these number are several orders of magnitudc higher than the original specific activities identified in the crude extracts before growth optimization.
Example 14. General Substrate Specificity Analysis Further characterisation was done to assess the initial structurelfunction parameters of the active site of the newly discovered dehydrogenases and to investigate the stereochemical preference of the catalysts. This would help develop a reasonable initial set of enzymes with a different range substrate specificities. The compounds and methods we have chosen to study in the feasibility portion of this project were chosen for their ease of analysis. The general type of alcohol resolution reaction we tested is shown in the scheme below.
Ho _ r~to~,~
RWR2 ~ Rr R2 + R~ Rz Scheme 1. Resolution of alcohols by dehydrogenase During the course of this work we screened nearly Z00 native organisms and 57 clone banks (approximately 5-10,000 individual clones from each bank on plate assay). Of these, we detected about 100 positive dehydrogenase activities from the native organisms and clone banks combined.
After performing preliminary activity analysis similar to that which was described above for these positive activities, about 16 were chosen for a more complete study based on their overall performance and production level consistency. These enzymes and their origin are fisted in Table 3. Of these about a dozen showed unique dehydrogenase activity and were followed up in more detail - and a summary of their physical properties are shown below in Table 4.

WO 99/21971 - . PCT/US98l22607 Table 3. Kcy to Main Strains and Initially Chosen for Follow-up Strain Name Source Vector Resistance Comments Strains*
#1-#246 Strains in the'I'ticrmoGen collection. Used for screening sad consUvction of clone banks AA-XY Strains in the ThermoGea collection. Used primarily for screening Native ADH
crmdidares XY (ADOl) native - ~, ppr ThetznoGcn strain coDection CA native - Kmr. Apr T~~ strain a~llection ~ native - Kmr. Apr 'IhermoGea strain collection CC native - Kmr, ppr ThermoGen strain colloction Cbnad ADH s candidate Apr/ clone from pTGADI4 ppr ~pMYF clone strain olr7 AD14 clone from pTGADI4 ppr 7~pMYF clue strain #14 AD19 clone fmm pTGADI9 Apr kpMYF clone strain X19 p close from pTGAD3 t Apr ~.pMYF clone strain X30 pp31 clone from pTGAD14 ,~pr ~pMY'F clone strain X31 pp39 clone from pTGADI4 ppr ~pMYF clone Basin #39 AD49.4 clone from pTGADI4 A~ ~pMYF clone strain #49 AD49.12 clone from pTGADI4 ~ pBK clone strain #49 clone fmm pTGADi4 Apt' ~pM7c'F clone strain #55 pp69 clone from pTGADI4 ppr ~pMYF clone strain #69 AD71 clone from pTGADI4 Apr 7~pMYF clone strain *'Il pp9g clone from pTGADI4 ppr 7~pMYF clone strain #98 *approximately 200 ThermoGen strains total were xrcened Table 4. ADH Physical Properties Summary Table Fmzyme mw DNA useful Sp. Aci*.1112 Temp (1~1~ laSCfIRange ~ll~mg~ 4~C ~hrS~

AD14 ~ 16.8 RT.40C 28 m AD19 105 6 RTfiO'C nd 20 AD30 121 16.4 RT-00C 4 24 AD31 nd 11.8 RTriOC 1..15 ai pp39 73.6 6 RT-40C 60 15 AD49.4 78.2 8.4 RT~OC 16 15-20 AD49.12 121.3 3.3 RT-50C 306 >50 AD55 97.3 9.3 RT~OC 30 20 AD69 121.3 12.8 RT-40C 30 20 pp71 78.1 7.4 RT-40C 136 12 ppgg 78.1 12 RT~OC 80 28 XY (AD01)78-131 NA RT-00C 460 >SO
_ *Spe~c Activity is calculated Iron one run in the 15 liter fermeater using ethanol as a substrate. Reactions were run at room temperature. R7CtrdCtS from the clones were heat puriCted crude eztracts. nd = not detccmined WO 99/21971 - . PCTNS98/22607 We tested the substrate specificity of the newly discovered catalysts on a variety of aloohols which could easily be analyzed specuophotometrirally. Table S lists relative activities of the ten enzymes we chose from the previous section of the work to study against a series of aloohols. The activities presented are relative to their activity on ethanol . 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.
'~' -"- r ~~ ~_~~at s ~....Lr.l Preference As can be seen, several of the enzymes prefer alternative alcohols compared to ethanol. Very few have a high degree of activity on cyclohexanol relative w ethanol, but enough activity is present to detect in the short reaction.
Example 15. Enantioselectivity of the Dehydrogenases To obtain data on chital compounds, we utilized chiral alcohois and measured relative rates on the ?0 two different enantiomecs of substrates of interest This type of data is presented in Table 6.
Again, for comparison, the data is presented as activity relative to using ethanol as the substrate.
Two pairs of chiral alcohols were tested: R- and S-butanol; and R- and S-pentanol.
The data clearly indicates different preferences for the enzymes. Most of the enzymes prefer the S-alcohols, however the XY dehydrogenase prefers R-pentanol over S-pentanol nearly six times -. - WO 99/21971 . . _ PCT/US98/22607 more. Interestingly enough, it does not have the same degree of selectivity for the butanol compound. Several of the other enzymes prefer the S~nantiomer up to twenty times higher than the R-enantiomer. Most enzymes are either as selective or more selective using pentanol as a substrate versus butanol, however, the AD49.4 is more selective with the butanol. It is comforting to see that only one enzyme in this test, AD49-12, is not highly selective on at least one of the two compounds. This underscrares the fact that we can Cmd selective catalysts using the methods employed during this project Another interesting note is that AD49-4 and AD49-12 both were isolated from the same clone bank (cue from strain #49) yet are clearly different dehydrogenase activities.
to 1 W /p I %O a.7V m v. a i ~ v .v v.w Xy ~ lpofo ~'7~10 ~ 85% I 1.14 I62dn I 11'0 15.91 Summary. After having screened nearly 200 new organisms and over 50 clone banks we have isolated and performed characterization of about a dozen new dehydrogenases.
The enzymes of the present invention have demonstrated s~~nthetic utility as dehydrogenases by identifying R- and S-selective catalysts.

WO 99/21971 . . PCT/US98/22607 Example 16. Heat Denaturation Purification for Production of Enzyme Tables 7A and 7B below list the results for characterizations of the Alcohol dehydrogenase specific activities (u=~mollmg of the total protein) in the ADH producing strains after heat treatment at SSoC.
Table 7 Heat purified ADH activity Sin SSC
heat inactivation 0 15 30min.60 90 0 min.t5 30 60 90 min. min. min. min. min. min. min. mia.

AI77-19.0 3.5 2.0 6.8 0.0 100dc 39% 22b 76~ 0~fo AD14-114.0 60.0 60.0 0.0 0.0 100k 429k 429k 0do 0~0 AI731-34.7 14.0 10.5 0.0 0.0 1006 298k 223do0do 06 AD39-467.0 149.0 127.039.0 24.0 100k 222.6 190b 58k 366 AD49.436.5 22.0 15.0 8.1 0.0 100!a 60k 41k 22k 0do AD49-12 1830.01060.0597.0450.0 100k 84k 49~ 28.6 21.b 2170.0 AD55-13.0 5.0 4.8 0.0 0.0 100~do167dv 1606 0i6 0b Al>63-521.0 28.0 23.0 6.5 0.0 100% 133!o 1106 31% 0do J(y 1680.04450:07822.01048.0822.0 100b 265~O 466 62k 49k ADS-1 35 75 50 180 100k 214.6 1436 514k 096 Strains were grown in 1 liter of the LBpmp (or LBKan for the 49-12 and XY) at 30~C overnight, washed in TE buffer, concentrated x20 times in assay buffer and homogenized in EMulsiFlex homogenizer. Activities were assayed with ethanol as a substrate; cofactors that have been used were: NAD for the strains #14-1, 39-4, 49-4, 49-12 and XY, and NADP for the #7-1, 7-2, 31-3, 55-1, 83-5; Protein concentrations are given in mglml.
?3 - WO 99121971 - . PCTIUS98/22607 Tabte 8. Total ADH activities (u/l liter) in the grown cultures without and with heat treatment.
Total ADH
activities (u=~mol;1 liter) with and without heat tteatmcat strain SSoC
heat inactivation 0 15 30 60 90 0 min.15 30 60 90 min. min. min. min. min. min. mia. mia. min.

AD7-1 ?30 14i 72 170 0 100k 196 10k 23do 0.b ADi4-i3.4001935 1250 850 100 57b 37% 25Xo 0b AL2il-31050 504 314 0 0 100b 48k 30k 0k O~C

AD39-45700 4500 3200 770 360 100k 7996 56b 14.6 66 pp49~42400 968 532 242 0 100b 40~O 22k 106 06 AD49-12 110000580003000020000 100k 22~O 12k 6.6 4b AD55-1300 101 72 0 0 100~b 34k 24b 0b 06 AD63-52100 706 580 97 0 100do 34k 286 5~fo 0b XY 28600011130078200524000 100k 39b 27b 18k 0.6 ADS-i 3500 1130 900 850 100k 326 26% 246 06 - - WO 99/21971 . . PCT/US98/22607 Literature Cited Reagent cost estimated from prices found in the Sigma catalog.
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Claims (8)

We Claim:

1. 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 (ADO1).

2. A protein with thermostable alcohol dehydrogenase activity selected from the group consisting of AD7.1, AD14.1, AD31.3, AD39.4, AD49.4, AD49.12, AD55.1, AD83.5, XY, and AD5.1.

3. A protein with chiral alcohol selective alcohol dehydrogenase activity selected from the group consisting of AD19, AD30, AD39, AD49.4, AD49.12, AD55, AD69, AD71, AD98, and XY(AD01).

4. An expressible recombinant DNA construct which contains a DNA sequence which when expressed encodes for a protein of claim 1.

5. A recombinant DNA which hybridizes with a recombinant DNA of claim 4.

6. A recombinant DNA which encodes for the amino acid sequence of the protein of claim 1.

7. A DNA sequence which hybridizes with the DNA of claim 6 under high stringency conditions.

8. A cell transformed with the construct of claim 4.