Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1999-12-17
2001-05-01
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S190000, C536S023200, C536S023700
Reexamination Certificate
active
06225099
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns new enantioselective alcohol dehydrogenases (ADH) from microorganisms such as Lactobacillus species especially from
Lactobacillus brevis
. The new enzymes are particularly advantageous for the reduction of organic keto compounds to form the corresponding hydroxy compounds, these reductions leading enantioselectively to the corresponding R compounds. The enantiomeric excess in this process which is calculated as
ee
(%)=(
R
)-product−(
S
)-product/(
R
)product+(
S
)-product×100
is usually more than 95%. S hydroxy compounds were not detectable when using the ADHS according to the invention. Due to their broad substrate spectrum the enzymes according to the invention can be used, for example, to produce chiral alcohols, chiral hydroxy esters (for example &agr;- and &bgr;-hydroxy esters) and also hydroxy acids.
Optically active hydroxy compounds are valuable chiral building blocks which are difficult to prepare with classical chemical processes. Hence biotechnological processes are usually considered for the production of chiral compounds either using whole cells of microorganisms or by means of isolated enzymes. As is shown by, e.g. F. Aragozzini et al. (Appl. Microbiol. Biotechnol. (1986) 24, 175-177), processes using whole cells often result in low yields, only a small enantiomeric excess (i.e. low ee values) and long reaction periods so that enzymes which can be used in a purified and concentrated form are more advantageous. In the case of chiral hydroxy compounds such as alcohols, alcohol dehydrogenases which reduce the prochiral compound with the aid of a coenzyme (often NADH or NADPH) may be used. As a rule these reactions are highly enantioselective. The previously available alcohol dehydrogenases (ADH) all lead to S-alcohols; the substrate spectrum for some of these enzymes is relatively narrow (yeast ADH, horse liver ADH). An NADP-dependent alcohol dehydrogenase from
Lactobacillus kefir
is described in DE 40 14 573 C1. The enzyme produces R-alcohols; however, it has turned out this enzyme is relatively unstable; thus purification to form a homogeneous enzyme could only be accomplished with substantial losses (>98%).
SUMMARY OF THE INVENTION
Surprisingly, an R-specific alcohol dehydrogenase with considerably higher stability was found by intensive screening. If one determines the thermal deactivation of this enzyme compared to the most similar previously known ADH enzyme, it turns out that the previously known enzyme is already inactivated by 50% at 45° C. whereas the enzyme according to the invention is only inactivated by 50% at temperatures of about 65° C. In addition the higher thermostability means a higher storage stability and a higher stability under particular reaction conditions.
A R-ADH enzyme of corresponding stability was found in particular in microorganisms of the genus Lactobacillus such as
L. brevis
and the subgroup Betabacterium (group A). Previously no microorganism or organism was known that had a stable R-specific alcohol dehydrogenase. With the aid of an antibody against the
L. kefir
ADH protein it has now been shown that all Lactobacilli of the subgroup Betabacterium (group A) produce a protein that reacts with the antibody. Particularly good enzymatic activity is detectable in
Lactobacillus brevis
. Other strains of this group also produce an enzyme which, in principle, is suitable for such reactions even if they exhibit a somewhat lower activity under the given culture and test conditions. In contrast, Lactobacilli of other subgroups (Thermobacterium IA, Streptobacterium IB, Betabacterium group B) exhibit neither a reaction in the antibody test nor do they have an appropriate enzymatic activity.
It is possible to purify the stable enzyme obtainable from
L. brevis
to homogeneity. Data for the protein sequence were determined using the purified enzyme.
The biochemical characterizations that were carried out showed further differences the enzymes of the invention and previously known enzymes. These differences include relative activities towards ketones. The stability of the ADHS according to the invention shown maximum stability at a pH value of about 9.0. In addition the enzymes show good stability at about pH 5.5. This is true example, in a 50 mM MES buffer (MES=2-morpholinoethane sulfonic acid). In the same buffer system the enzyme according to the invention still retains activity of over 95% after about 30 minutes at a temperature between 25° C. and 60° C. The enzyme has a stability maximum at ca. 40° C. In addition the enzyme has an activity maximum at ca. 50° C.
Furthermore the enzyme according to the invention ADH enzymes. For example the N-terminal end has five amino acid substitutions over a total length of 38 amino acids (AA), i.e., an AA difference of over 12%. Hence all comparisons demonstrate that it is advantageous to use the alcohol dehydrogenases according to the invention especially those for
Lactobacillus brevis
for corresponding applications.
A further object of the invention involves isolation of the enzyme according to the invention from suitable microorganisms. Good enzyme yields were achieved with a strain of
Lactobacillus brevis
which was deposited on the 06.06.1972 under the number DSM 20054 at the “Deutsche Sammlung von Mikroorganismen und Zellkulturen”, Braunschweig and is freely available.
The process for the isolation and purification of the ADH enzyme proceeds essentially via the following process steps: disruption of the cultured cells, such is by glass beads, carrying out hydrophobic chromatography or interaction chromatography and by anion exchange and affinity chromatography. In particular it has proven to be advantageous when all homogenization and elution buffers contain from about 0.5 to about 5 mM magnesium. In this connection a Mg
2+
concentration of about 1 mM has proven to be particularly advantageous. In this manner an enzyme is obtained which has a specific activity of at least 400 U/mg and in many cases up to 500 U/mg.
In addition the enzyme according to the invention can be produced by remombinant processes i.e. by expressing an appropriate DNA molecule coding for the enzyme in a suitable prokaryotic or eukaryotic strain. In particular a system which is based on an ADH structural gene from
Lactobacillus brevis
transformed into
Escherichia coli
is suitable.
A further subject matter of the invention is a process for the enantioselective reduction of organic keto compounds according to the general formula (I)
in which R
1
and R
2
are different or identical, and may be hydrogen, straight-chained or branched alkyl or alkenyl group, an aryl or arylenyl group each group containing of 1 to 20 C atoms which maybe substituted by one or several halogen atoms, nitro, hydroxyl or alkoxy residues, the alkoxy residues having 1 to 20 C atoms, an optionally substituted nitrogen, oxygen or sulphur heterocycle moiety, or may be an optionally substituted polycondensed saturated and/or unsaturated aromatic residue said reaction comprising a keto compound or an appropriate mixture with a microbial alcohol dehydrogenase according to the invention. In this case the enzyme may be used, as may a culture of microorganisms which produce this enzyme or cells containing the enzyme. The reaction is preferably carried out in an aqueous buffer such as potassium phosphate, Tris/HCl or triethanolamine (TEA) buffer, in the presence of magnesium ions, at a pH value of 5 to 10 preferably pH 6 to 9 and at a temperature of 10° C. to 70° C. preferably of 30° C. to 60° C. In addition, an appropriate coenzyme such as NAD(P)H and a suitable agent for the regeneration of oxidized coenzyme such as, isopropanol may be present in the reaction mixture. When an adequate conversion such as a 50% conversion has been achieved, the reaction is terminated, preferably by direct extraction preferably in chloroform. However, the reaction can also be terminated by lowering the pH value, by heating or by adding a suitable enzyme inhibitor. Subsequentl
Hummel Werner
Riebel Bettina
Forschungszentrum Julich GmbH
Fronda Christian L.
Fulbright & Jaworski LLP
Nashed Nashaat T.
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