CN101448949A - Method for the enzymatic production of 2-hydroxy-2-methyl carboxylic acids - Google Patents

Abstract

The present invention relates to a process for the enzymatic preparation of 2-hydroxy-2-methylcarboxylic acids from 3-hydroxycarboxylic acids, wherein the 3-hydroxycarboxylic acids are prepared in an aqueous reaction solution and/or are added to the reaction solution and incubated . The aqueous reaction solution contains units having 3-hydroxycarboxylic acid-CoA-mutase-activity, which not only has the activity of generating 3-hydroxycarbonyl-CoA-esters but also isomerizes 3-hydroxycarbonyl-CoA-esters activity and causes the conversion of 3-hydroxycarboxylic acids to the corresponding 2-hydroxy-2-methylcarboxylic acids (which are available as acids or in the form of their salts). In a preferred embodiment, the unit having 3-hydroxycarboxylic acid-CoA-mutase-activity is one comprising an isolated cobalamin-dependent mutase and optionally producing a 3-hydroxycarbonyl-CoA-ester Enzymes or enzyme systems or units of microorganisms containing them. The present invention preferably relates to a biotechnological process for the production of 2-hydroxy-2-methylcarboxylic acids, wherein microorganisms having the desired activity are cultured in an aqueous system with the aid of simple natural substances, and the 3- The hydroxycarbonyl-CoA-ester is converted to the corresponding 2-hydroxy-2-methylcarboxylic acid. The invention also includes the preparation of unsaturated 2-methylcarboxylic acids, wherein the 2-hydroxy-2-methylcarboxylic acids obtained are converted by dehydration to the corresponding unsaturated 2-methylcarboxylic acids (methacrylic acid and more advanced homologues).

Description

Method for enzymatically preparing 2-hydroxy-2-methylcarboxylic acid

technical field

The present invention relates to a process for the enzymatic preparation of 2-hydroxy-2-methylcarboxylic acid from 3-hydroxycarboxylic acid, wherein the 3-hydroxycarboxylic acid is prepared in an aqueous reaction solution and/or added to the reaction solution and incubated . The aqueous reaction solution contains units having 3-hydroxycarboxylic acid-CoA-mutase-activity, which not only have the activity of generating 3-hydroxycarbonyl-CoA-esters but also have the ability to isomerize 3-hydroxycarbonyl-CoA-esters The activity of oxidization and causes the conversion of 3-hydroxycarboxylic acid to the corresponding 2-hydroxy-2-methylcarboxylic acid, which is obtained as acid or in the form of its salt. In a preferred embodiment, the unit having 3-hydroxycarboxylic acid-CoA-mutase-activity comprises an isolated cobalamin-dependent mutase and optionally comprises an enzyme producing 3-hydroxycarbonyl-CoA- The enzyme or enzyme system of the ester or the microorganism containing them. The present invention preferably relates to a biotechnological method for the production of 2-hydroxy-2-methylcarboxylic acids, wherein microorganisms having the desired mutase-activity are cultivated in an aqueous system with the aid of simple natural substances and intracellularly produced The 3-hydroxycarbonyl-CoA-ester is converted to the corresponding 2-hydroxy-2-methylcarboxylic acid. The invention also includes the preparation of unsaturated 2-methylcarboxylic acids, wherein the 2-hydroxy-2-methylcarboxylic acids obtained are converted by dehydration to the corresponding unsaturated 2-methylcarboxylic acids (methacrylic acid and more advanced homologues).

In a preferred embodiment of the invention, the microbial strain HCM-10 (DSM 18028) is used as the microorganism producing and isomerizing 3-hydroxycarbonyl-CoA-thioesters.

Background technique

Methacrylic acid and its homologous unsaturated 2-methylcarboxylic acids are widely used in the preparation of acrylic glass sheets, die-cast molded products, coatings and many other products.

A number of methods for the preparation of methacrylic acid and its homologues are known. Although the vast majority of commercially available products worldwide are based on the amide-sulfate hydrolysis of methacrylic acid and its homologs (which are prepared from the corresponding 2-hydroxynitriles) (W. Bauer, "Methacrylic acid and derivatives", in Ullmann's Encyclopedia of Industrial Chemistry, 5th edition, editors: B.Elvers, S.Hawkins, G.Schulz, VCH, New York, 1990, Bd.A16, pp. 441-452; A.W.Gross, J.C. Dobson, "Methacrylic acid and derivatives", in Kirk-Othmer Encyclopedia of Chemical Technology, 4th ed., eds. J.I. Kroschwitz, M. Howe-Grant, John Wiley & Sons, New York, 1995, Bd.16, 474- 506 pages). In this process, for example, about 1.6 kg of sulfuric acid are required for the production of 1 kg of methacrylic acid. For such reasons, an alternative process for the commercial production of methacrylic acid which does not require the recovery of sulfuric acid (and the high energy consumption associated therewith) is advantageous.

The chemical conversion of 2-hydroxyisobutyric acid to methacrylic acid is disclosed by patents US 3,666,805 and US 5,225,594. 2-Hydroxyisobutyric acid is dehydrated here by using metal oxides, metal hydroxides, ion exchange resins, alumina, silica, amines, phosphine, alkali metal alcoholates and alkali metal carboxylates. Typical reaction temperatures are between 160°C and 250°C. Methacrylic acid yields of up to 96% can be achieved with this method.

An alternative method for the production of methacrylic acid and its homologues is produced by the hydrolysis of 2-hydroxynitriles to the corresponding 2-hydroxy-2-methylcarboxylic acids with the use of nitril-hydrolases. Nitrilase or a combination consisting of nitrile hydratase and amidase is involved here (A. Banerjee, R. Sharma, U.C. Banerjee, 2002, "The nitrole-degrading enzymes: current status and future prospects", Appl. Microbiol. Biotechnol ., 60:33-44). This method is protected by multiple patents (US6582943B1). A serious disadvantage of this method is the instability of the nitrile in the neutral pH range necessary for efficient nitrilytic enzymatic activity. Decomposition of nitriles in the reaction mixture leads to the accumulation of ketones and cyanides, both of which inhibit nitril-hydrolase activity.

A general disadvantage of these two methods (ie the amide-sulfate based method and the enzymatic nitrile-hydrolysis based method popular today) is the requirement for 2-hydroxynitriles. It must first be prepared from environmentally damaging reactants, namely ketones and cyanides.

Therefore, a process for the production of methacrylic acid and its homologues based on simple, non-environmentally damaging reactants would be advantageous.

It was therefore the object of the present invention to find alternative possibilities for the production of 2-hydroxy-2-methylcarboxylic acids and to provide means and methods which are based as far as possible on simple, non-environmentally disruptive reactants, consume little amounts of energy and produce a small amount of waste.

The object is solved by an enzymatic process for the preparation of 2-hydroxy-2-methylcarboxylic acids from 3-hydroxycarboxylic acids. According to the invention, 3-hydroxycarboxylic acids are prepared in aqueous reaction solutions containing units having 3-hydroxycarboxylic acid-CoA-mutase activity and/or are added to such reaction solutions. A unit having 3-hydroxycarboxylic acid-CoA-mutase-activity means in the sense of the present invention comprising a cobalamin-dependent mutase and optionally an enzyme producing a 3-hydroxycarbonyl-CoA-ester or Enzyme systems or units of biological systems containing or producing them which have 3-hydroxycarboxylic acid-CoA-mutase-activity and which exhibit not only the activity of producing 3-hydroxycarbonyl-CoA-esters but also the production of 3-hydroxycarbonyl-CoA-esters - Activity for the isomerization of hydroxycarbonyl-CoA-esters. Immediately after incubation, the correspondingly converted 2-hydroxy-2-methylcarboxylic acid is obtained as acid or in the form of its salt.

Preferably the present invention relates to a biotechnological process for the production of 2-hydroxy-2-methylcarboxylic acid under the utilization of microorganisms. Said microorganisms generally have the activity of synthesizing 3-hydroxycarbonyl-CoA-esters and are able to produce such cobalamin-dependent mutases or contain such mutases and pass 3-hydroxycarboxylic acid-CoA-mutases - activity capable of intracellularly converting 3-hydroxycarbonyl-CoA-esters formed from simple natural substances (from reactants such as sugars and/or alcohols and/or organic acids and their derivatives) to the corresponding 2-hydroxy-2 -Methylcarbonyl-CoA-ester.

The method of the present invention is characterized in that microorganisms (which produce or contain a cobalamin-dependent mutase and have 3-hydroxycarboxylic acid-CoA-mutase-activity) are used in an aqueous system for deriving from 3- Conversion of hydroxycarboxylic acids to the corresponding 2-hydroxy-2-methylcarboxylic acids.

In a preferred method variant, a microorganism (which contains 3-hydroxycarboxylic acid-CoA-mutase-activity and has not only the activity of producing 3-hydroxycarbonyl-CoA-thioester but also the activity of making 3-hydroxycarbonyl-CoA - Activity for thioester isomerization) is cultivated in an aqueous system with renewable raw materials or wastes from the utilization of renewable raw materials as carbon and energy sources. Here, the 3-hydroxycarboxylic acid-CoA-thioester formed in the cell is converted into the corresponding 2-hydroxy-2-methylcarboxylic acid. Preferably the reaction is carried out with the addition of exogenous 3-hydroxycarboxylic acid. The corresponding 2-hydroxy-2-methylcarboxylic acid is then isolated as the acid or in the form of its salt.

The novel biotechnological approach, which utilizes the production of 3-hydroxycarboxylic acids from simple starting materials and isomerization to 2-hydroxy-2-methylcarboxylic acids, is able to solve the above-mentioned problems.

In a preferred embodiment of the present invention, the method comprises the following steps:

a) Production of 3-hydroxycarboxylic acids from simple natural substances and subsequent conversion to 2-hydroxy-2-methylcarboxylates in suitable biological systems with activity for the synthesis of 3-hydroxycarbonyl-CoA-esters and mutase activity sour, and

b) Isolation of 2-hydroxy-2-methylcarboxylic acid as free acid or as its corresponding salt.

The 2-hydroxy-2-methylcarboxylic acid thus obtained can advantageously be used for the preparation of C2-C3-unsaturated isoalkenic acids (methacrylic acid and its homologues), which can be obtained by Dehydration of the acid prepared in or its corresponding salt is carried out. The reaction is illustrated below:

- simple natural substances (e.g. regenerative raw materials or wastes resulting from the utilization of regenerative raw materials, e.g. sugars, organic acids or alcohols) → 3-hydroxycarboxylic acids → 2-hydroxy-2-methylcarboxylic acids (e.g. by strain HCM-10)

- 2-Hydroxy-2-methylcarboxylic acid → methacrylic acid and homologues (eg in the presence of NaOH and at a temperature of 185°C)

Here, of course, the reaction conditions (pH-value, ion concentration, oxygen/carbon dioxide requirement, trace elements, temperature and the like) are selected in such a way that the microorganisms can optimally react the 3-hydroxycarboxylic acid to form 2 -Hydroxy-2-methylcarboxylic acid. Under such reaction conditions, the cobalamin-dependent mutase has higher stability and efficiency than the isolated enzyme in the natural microenvironment (ie, inside the cell). Furthermore, under suitable conditions, cell propagation and thus an increase in the concentration of the mutase is possible. Thus, the enzymatic reaction by means of microorganisms optionally represents significant advantages with regard to reliability, autonomy and simplicity as well as the quality and yield of the end product of the process.

It is also possible for the enzymatic reaction of the present invention from 3-hydroxycarboxylic acids to 2-hydroxy-2-methylcarboxylic acids to combine units with 3-hydroxycarboxylic acid-CoA-mutase-activity (i.e. preferably with The active bound cobalamin-dependent mutase for the synthesis of CoA-esters) is introduced into the reaction solution in purified, enriched and/or isolated form, wherein said enzyme may be, for example, of natural origin. Of course, the enzyme may be recombinantly produced by genetically modified organisms.

In the method according to the invention, the enzyme is used as catalyst not only in the form of intact microbial cells but also in the form of permeated microbial cells. Further use possibilities exist in the form of component(s) of microbial cell extracts, but also in partially purified or purified form. Other CoA-ester-synthesizing enzymes, such as CoA-transferases or CoA-synthetases, are optionally used according to the invention. The enzymatic catalysts may be immobilized or attached to dissolved or undissolved support materials.

In preferred embodiment variants, specific cellular compartments or parts thereof are separated from each other or combined, i.e. carbohydrate structures, lipids or proteins and/or peptides and nucleic acids (which can positively or negatively affect A unit with mutase-activity) binds or separates them. In order to make use of such influences consciously, crude extracts are prepared from microorganisms, eg according to the field of expertise, which are optionally centrifuged so that the precipitate or supernatant can be used for carrying out the reactions according to the invention.

3-Hydroxycarboxylic acids (eg 3-hydroxybutyric acid) or more precisely their intracellular CoA-thioester 3-hydroxycarbonyl-CoA are readily produced from simple natural substances by a large variety of bacterial strains. These acids are the basis substances (Grundbausteines)/monomers for the carbon and energy storage substances poly-3-hydroxyalkanoates of ubiquitous bacteria. Translocation of carbons within the carboxylic acid backbone is ubiquitous in bacteria as well as in other biological systems. However, to date no biological system can be demonstrated for the conversion of 3-hydroxycarbonyl-CoA-esters to the corresponding 2-hydroxy-2-methylcarboxylic acid-CoA-esters. The present invention is based on the surprising realization that systems with cobalamin-dependent mutase-activity possess both properties.

Microorganisms containing cobalamin-dependent mutases are, for example, Methylibiumpetroleiphilum PM1, Methylibium sp.R8 (Stammsammlung UFZ, Leipzig), β-protein strain HCM-10, Xanthobacter autotrophicus Py2, Rhodobacter sphaeroides) (ATCC17029) or Nocardioides sp. JS614.

A preferred suitable biological system was found with strain HCM-10. Said strain was deposited with DeutschenSammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany under the deposit number DSM 18028 on March 13, 2006 under the Budapest Treaty on the Deposit of Microorganisms for the Purposes of Patent Processes.

Particularly good yields of 2-hydroxy-2-methylcarboxylic acids, especially 2-hydroxyisobutyric acid, can be achieved with the use of the preferred microbial systems. However, said enzymatic reactions by microorganisms are by no means limited to said strains. According to the invention, all organisms capable of converting 3-hydroxycarboxylic acids to 2-hydroxy-2-methylcarboxylic acids can be used.

These are microorganisms which, on the one hand, have the same genes or gene products, or, on the other hand, similar genes which lead to gene products with similar or similar activity). Thus, 3-hydroxycarbonyl-CoA-mutase activity from other sources is also revealed by the present invention. The invention also includes transformed systems having one or a similar 3-hydroxycarbonyl-CoA-mutase-activity as strain HCM-10 or other sources.

Belonging to mutants (variants of genetically modified and isolated microorganisms) may be, for example, organisms which, due to the introduction of a mutase-encoding nucleotide sequence, have the desired cobalamin-dependent mutase-activity .

The biological system preferably used (strain HCM-10-DSM 18028) produces 3-hydroxycarbonyl-CoA-esters as thioesters from simple natural substances such as sugars and/or organic acids and/or alcohols and their derivatives. In the preferred system used here, 3-hydroxycarbonyl-CoA-esters are converted to 2-hydroxy-2-methylcarbonyl-CoA-esters by a cobalamin-dependent carbon backbone-engineered mutase, such as As shown in Equation 1 exemplarily for the case of (R)-3-hydroxybutyryl-CoA. The CoA-thioesters are hydrolyzed in the system and excrete the acid into the culture medium.

(R)-3-Hydroxybutyryl-CoA 2-Hydroxyisobutyryl-CoA

For the method of the present invention, the microbial strain HCM-10 (DSM 18028), the autotrophic Xanthobacterium Py2, the Rhodobacter sphaeroides (ATCC17029) or the sphaeroides containing cobalamin-dependent mutase are used as preferred enzyme-catalysts. Karstella sp. JS614, crude extracts or parts thereof. The bacterial strain used in the present invention produces and preferably has the protein of sequence SEQ ID NO:2 and/or SEQ IDNO:4 or comprises nucleic acid sequence SEQ ID NO:1 and/or SEQ ID NO:3 (HCM-10), has sequence SEQ ID NO: 5 and/or the protein of SEQ ID NO: 6 or comprise nucleic acid sequence SEQ ID NO: 7 and/or SEQ ID NO: 8 (autotrophic yellow bacillus Py2), have sequence SEQ ID NO: 9 and/or SEQ ID NO: the protein of 10 or comprise nucleic acid sequence SEQ ID NO: 11 and/or SEQ ID NO: 12 (Rhodobacterium sphaeroides ATCC17029) or have the protein of sequence SEQ ID NO: 13 and/or SEQ ID NO: 14 or comprise Nucleic acid sequence SEQ ID NO: 15 and/or SEQ ID NO: 16 (Nocardia-like genus JS614). Within the meaning of the present invention, the proteins can be used in enriched, isolated or synthetically prepared form.

In a further preferred embodiment variant of the invention, the enzyme catalyst (in particular the microorganism, its crude extract, its fraction and/or the enriched or isolated enzyme) is used immobilized. Immobilization places enzymes, organelles, and cells into an insoluble and space-restricted state for reaction. In this way, the enzyme can be immobilized, for example, in a polymer matrix (eg alginate-, polyvinyl alcohol- or polyacrylamide-gel). Immobilization can also be performed on dissolved or undissolved support materials (eg diatomaceous earth) to simplify catalyst-recovery and-reuse. Methods for cell-immobilization in polymer matrices or on soluble or undissolved supports are known to those skilled in the art and have been described in detail. The enzymatic activity can likewise be isolated from microbial cells. The enzymatic activity can then be used directly as a catalyst or immobilized in a polymer matrix or on a dissolved or undissolved support. The methods necessary for this are known to the person skilled in the art and are described, for example, in Methods in Biotechnology, Volume 1: Immobilization of enzymes and cells, editor: G.F. Bickerstaff, Humana Press, Totowa, New Jersey, 1997.

The conversion of 3-hydroxycarboxylic acids to 2-hydroxy-2-methylcarboxylic acids is preferably carried out in the context of a continuous process, which can be carried out in a through-flow reactor in which microorganisms are grown and produced by This product was formed. However, a continuous process is also to be understood as any system of growing cells and catalytic enzymes into which, on the one hand, a nutrient solution is delivered and from which, on the other hand, a culture solution (including enzymatically formed 2-hydroxy-2-methyl carboxylic acid) extracted. The process according to the invention can also be carried out in a semi-continuous process or in a batch process.

As already specified, the 3-hydroxycarboxylic acids, which are the starting materials for the 2-hydroxy-2-methylcarboxylic acids, are preferably prepared by enzymatic reactions of carbohydrates and/or organic acids and/or alcohols or derivatives thereof. In connection with the present invention, enzymes for the synthesis of CoA esters, which are present in the microorganisms or which are added, are optionally used in addition to the cobalamin-dependent mutases. Here, the conversion of hydrocarbons and/or carbohydrates and/or organic acids and/or alcohols or derivatives thereof to 3-hydroxycarboxylic acids and from 3-hydroxycarboxylic acids to 2-hydroxy-2-methylcarboxylic acids occurs in a single The process step is carried out, that is, the conversion of the initial substrate to the 3-hydroxycarboxylic acid and the enzymatic conversion of the 3-hydroxycarboxylic acid to the corresponding 2-hydroxy-2-methylcarboxylic acid are carried out simultaneously or slightly time-delayed in a And run in the same reaction solution.

In a particularly particular embodiment of the invention for the cultivation a substrate with tert-butyl residues is used as carbon and energy source, preferably tert-butanol is used as the sole carbon and energy source in the basal medium.

The process according to the invention can preferably be used for the preparation of 2-hydroxy-2-methylpropionic acid (2-hydroxyisobutyric acid). The preferred preparation of 2-hydroxyisobutyric acid is also characterized by the addition of exogenous 3-hydroxybutyric acid.

The method can be carried out aerobically, preferably when using whole cells, or also anaerobically, for example under nitrogen gassing, preferably when using extracts or purified enzymes.

The invention also relates to a nucleic acid molecule encoding an enzyme having cobalamin-dependent mutase activity selected from the group consisting of

A) a nucleic acid molecule, which encodes a protein comprising the amino acid sequence shown under Seq.No.2 and/or Seq.No.4;

b) A nucleic acid molecule comprising the nucleotide sequence shown under Seq. No. 1 and/or Seq. No. 3.

It has been shown that the enzymes of the invention are preferably heterodimeric proteins comprising the subunits described under Seq. No. 2 and Seq. No. 4 and thus possessing outstanding enzymatic activity.

A nucleic acid molecule may be a DNA molecule, preferably a cDNA molecule or a genomic DNA and/or RNA molecule. Both nucleic acids and proteins can be isolated from natural sources (preferably by DSM 18028, but, for example, also by Methylibium petroliphilum PM1, Methylibium sp. R8 (Stammsammlung UFZ Leipzig), autotrophic Xanthobacterium Py2, Rhodobacter sphaeroides (ATCC 17029) or Nocardioid Bacteria JS614) or synthesized according to known methods.

Mutations can be produced in the nucleic acid molecules used according to the invention by means of molecular biology techniques known per se, which allow the synthesis of other enzymes with similar or similar properties, which are likewise used in the method according to the invention. The mutation may be a deletion mutation, which results in a truncated enzyme. Modified enzymes with similar or analogous properties can likewise be produced by other molecular mechanisms such as insertion, replication, translocation, gene fusion, nucleotide exchange or also gene transformation between various microbial strains.

Identification and isolation of such nucleic acid molecules can be performed using nucleic acid molecules or parts thereof. Molecules that hybridize to nucleic acid molecules also include fragments, derivatives and allelic variants of the aforementioned nucleic acid molecules, which encode enzymes that can be used according to the invention. Here, a fragment refers to a part of a nucleic acid molecule which is sufficiently long to encode the enzyme in question. A derivative refers to a sequence of a molecule which differs in one or more positions from the sequence of the aforementioned nucleic acid molecule, but which has a high degree of homology to this sequence. Homology here means a sequence identity at the nucleic acid level of at least 40%, in particular at least 60%, preferably more than 80% and particularly preferably more than 90%, 95%, 97% or 99%. Here, the encoded enzyme has a sequence identity at the amino acid level of at least 60%, preferably at least 80%, particularly preferably at least 95%, very particularly preferably at least 99%, to the indicated amino acid sequence. Deviations here can be produced by deletions, substitutions, insertions or recombinations. This can be a naturally occurring variation, for example to a sequence from another organism, or to a mutation, wherein the mutation can occur naturally or by targeted mutagenesis (UV-rays, X-rays, chemical agents or other) Appear. Furthermore, said variants may relate to synthetically prepared sequences. The variants have certain common characteristics, such as enzyme activity, active enzyme concentration, subunits, functional groups, immunoreactivity, conformation and/or physical properties, such as migration behavior in gel electrophoresis, chromatographic behavior , solubility, precipitation coefficient, optimum pH, optimum temperature, spectral properties, stability and/or others.

Furthermore, a subject of the present invention is also novel proteins having the sequences No. 2 and 4 and proteins of heterodimers comprising Seq. No. 2 and Seq. No. 4 and at least 99% of their homologues.

SEQ ID NO: 1 shows a nucleotide sequence comprising 1644 bp encoding the large subunit of the cobalamin-dependent mutase derived from DSM 18028.

SEQ ID NO: 2 shows the amino acid sequence comprising 548 AS of the large subunit of the cobalamin-dependent mutase derived from DSM 18028.

SEQ ID NO: 3 shows the 369bp partial nucleotide sequence encoding the small subunit of the cobalamin-dependent mutase derived from DSM 18028.

SEQ ID NO: 4 shows the 123AS-containing partial sequence derived from the cobalamin-dependent mutase subunit of DSM 18028.

SEQ ID NO: 5 and 6 show the amino acid sequence comprising 562 or 135 AS of the cobalamin-dependent mutase derived from Flavobacterium autotropha Py2.

SEQ ID NO: 7 and 8 show the 1689 or 408 bp nucleotide sequence encoding the cobalamin-dependent mutase derived from Flavus autotrophicum Py2.

SEQ ID NO: 9 and 10 show the amino acid sequence comprising 563 or 135 AS of the cobalamin-dependent mutase derived from Rhodobacter sphaeroides ATCC 17029.

SEQ ID NO: 11 and 12 show a 1692 or 408 bp nucleotide sequence encoding a cobalamin-dependent mutase derived from Rhodobacter sphaeroides ATCC 17029.

SEQ ID NO: 13 and 14 show the amino acid sequence comprising 569 or 164 AS of the cobalamin-dependent mutase derived from Nocardioides sp. JS614.

SEQ ID NO: 15 and 16 show the 1710 or 495 bp nucleotide sequence encoding the cobalamin-dependent mutase derived from Nocardioid sp. JS614.

The 2-hydroxy-2-methylcarboxylic acid prepared according to the invention can be isolated by known methods by treatment of the culture medium (after removal of undissolved components such as microbial cells). Such methods are, for example, concentration, ion exchange, distillation, electrodialysis, extraction and crystallization, among others. The product can be isolated as a salt or (after acidification) as protonated 2-hydroxy-2-methylcarboxylic acid.

2-Hydroxy-2-methylcarboxylic acids (or their corresponding salts) can be dehydrated to the corresponding unsaturated 2-methylcarboxylic acids by a number of methods. For the preparation of the C2-C3 unsaturated isoalkenic acid, the 2-hydroxy-2-methylcarboxylic acid obtained is dehydrated according to methods known from the prior art. The dehydration of the 2-hydroxy-2-methylcarboxylic acid can be achieved by using metal oxides, metal hydroxides, ion exchange resins, alumina, silica, amines, phosphine, alkali metal alcoholates and alkali metal Carboxylate proceeds. Typical reaction temperatures are between 160°C and 250°C. The preparation of methacrylic acid is thus carried out at a temperature of about 185° C., for example by dehydration of 2-hydroxyisobutyric acid in the presence of NaOH.

The methacrylic acid and its homologues produced by this process are suitable for use in a range of industrial branches, for example as additives and in coatings. In contrast to the hitherto known methods, the method combines the advantages of a low-temperature process, the use of non-environmentally damaging reactants and a small amount of waste generation.

The invention is subsequently described in more detail in the examples, without however restricting the invention thereto.

Detailed ways

Materials and methods

Microbial Enzyme Catalyst

Protein subunits with sequences No. 2 and No. 4 derived from or isolated from microbial cells of strain HCM-10 (DSM 18028) characterized by the activity of producing 3-hydroxycarbonyl-CoA-esters and activity to isomerize 3-hydroxycarbonyl-CoA-esters.

Growth of microbial enzyme-catalysts

The microbial strains used for the production of 2-hydroxy-2-methylcarboxylic acid were isolated as described below. Strain cultures in 20% glycerol-solution were stored in liquid nitrogen.

Strain HCM-10 derived from groundwater was enriched on basal medium (Table 1) with tert-butanol as the sole carbon and energy source.

The strains are phylogenetically in the Rubrivivax-Leptothrix genus.

Table 1

Strain HCM-10 was grown aerobically under the following conditions (Table 2) for testing 3-hydroxycarbonyl-CoA-mutase activity.

Table 2

Cells were used directly after harvesting. Whole cells can be used without further pretreatment such as permeabilization. In addition, the cells may be used permeabilized (eg, by treatment with toluene, washes, or by freeze-thaw cycles) in order to improve the rate of diffusion of substances into and out of the cells.

The concentrations of 2-hydroxyisobutyric acid and 3-hydroxybutyric acid in the culture broth or in the reaction batch were determined by gas chromatography after alcoholysis with acidic methanol using an FFAP column and an FID detector.

Embodiment 1 :

Conversion of 3-hydroxybutyrate to 2-hydroxyisobutyrate by strain HCM-10.

A suspension of 1 g (dry weight) of cells of strain HCM-10 in 100 mL of basal medium was filled into a 120 mL serum bottle. 50 mg of 3-hydroxybutyric acid were added to the suspension and the suspension was incubated at 30° C. on a rotary shaker (Schüttler). After 0.3 h of aerobic incubation, the suspension was gassed with nitrogen and incubated for a further 4.4 h at 30° C. with shaking. Samples were taken at different time points and the content of 2-hydroxyisobutyric acid and 3-hydroxybutyric acid in the cell-free supernatant after centrifugation of the suspension was determined. 2-Hydroxyisobutyric acid was determined to be the only product released in the anaerobic phase. In contrast, in the aerobic initial phase, 3-hydroxybutyric acid was apparently completely decomposed ( FIG. 1 ). The yield of 2-hydroxyisobutyric acid was 5.1% in this case, and about 80% of 3-hydroxybutyric acid remained in the reaction liquid.

Embodiment 2 :

Conversion of 3-hydroxybutyric acid to 2-hydroxyisobutyric acid by crude extract of strain HCM-10.

Cell-free crude extracts of strain HCM-10 were prepared by deaggregation of cells in a ball mill, followed by separation of cell debris by centrifugation. The cell-free crude extract at a concentration of 10 mg protein in 5 mL of 50-mM-potassium phosphate buffer (containing 1 mM MgCl ₂ at pH 7.2) was added to a sealable 10 mL-glass container. Then 0.01 mM of coenzyme B12, 1 mM of coenzyme A, 1 mM of ATP and 4.25 mg of 3-hydroxybutyric acid were added to the extract. The reaction solution was gassed with nitrogen, the reaction vessel was tightly closed and incubated at 30° C. for 2 h with shaking. The reaction products were analyzed as indicated above. The yield of 2-hydroxyisobutyric acid was 9% in this case, about 88% of 3-hydroxybutyric acid remained in the reaction liquid ( FIG. 2 ).

Embodiment 3 :

Dehydration of 2-hydroxyisobutyric acid produces methacrylate.

A solution of 2-hydroxyisobutyric acid (1 mg/5 mL) (prepared corresponding to the procedure carried out in Example 2) was admixed with NaOH (0.06 mg) with stirring. The solution was incubated at 185-195° C. under vacuum (300 Torr) with stirring and reflux cooling. An aliquot of 0.5 mg per 5 ml of 2-hydroxyisobutyric acid, additionally containing 0.4% by weight of p-methoxyphenol, was added every hour over a period of 5 h in order to avoid polymerization of methacrylate . The reaction was terminated after 24 h of incubation. The conversion of 2-hydroxyisobutyric acid to methacrylate amounts to 97%. Methacrylic acid is separated from the reaction mass by distillation.

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<400>16

Claims (29)

1. A process for the enzymatic preparation of 2-hydroxy-2-methylcarboxylic acids from 3-hydroxycarboxylic acids, characterized in that the 3-hydroxycarboxylic acids are prepared in an aqueous reaction solution and/or added to the reaction solution, Incubating and subsequently obtaining the correspondingly converted 2-hydroxy-2-methylcarboxylic acid as an acid or as a salt thereof, the reaction solution contains units having 3-hydroxy-carboxylic acid-CoA-mutase-activity, the Said unit not only has the activity of generating 3-hydroxycarbonyl-CoA-ester but also has the activity of isomerizing 3-hydroxycarbonyl-CoA-ester. 2. The method according to claim 1, wherein the unit having 3-hydroxycarboxylic acid-CoA-mutase-activity comprises an isolated cobalamin-dependent mutase. 3. The method according to claim 1 or 2, characterized in that the unit having 3-hydroxycarboxylic acid-CoA-mutase-activity comprises an enzyme or enzyme system producing 3-hydroxycarbonyl-CoA-ester . 4. The method according to any one of claims 1-3, characterized in that the unit having 3-hydroxycarboxylic acid-CoA-mutase-activity is a microorganism or its crude extract. 5. The method according to claim 1 or 4, characterized in that the use of a cobalamin-dependent mutase that produces or contains a cobalamin-dependent mutase that has 3-hydroxycarboxylic acid-CoA-mutase-activity and not only has the ability to produce 3- The activity of hydroxycarbonyl-CoA-esters and the activity of isomerizing 3-hydroxycarbonyl-CoA-esters, which are used in aqueous systems to generate the corresponding 2-hydroxy-2-methylcarboxylates from 3-hydroxycarboxylic acids acid conversion. 6. The method according to any one of claims 1-5, characterized in that, a) A microorganism having 3-hydroxycarboxylic acid-CoA-mutase-activity is cultivated in an aqueous system using regenerative raw materials or waste products resulting from the utilization of regenerative raw materials as carbon and energy sources, through which intracellular Synthesis of 3-hydroxycarboxylic acid-CoA-thioester and conversion to the corresponding 2-hydroxyl-2-methylcarboxylic acid, the microorganism not only has the activity of producing 3-hydroxycarbonyl-CoA-thioester but also has the ability to make 3-hydroxyl carbonyl-CoA-thioester isomerization activity, and b) Isolating the corresponding 2-hydroxy-2-methylcarboxylic acid as the acid or in the form of its salt. 7. The method according to any one of claims 1-6, characterized in that the reaction is carried out with the addition of exogenous 3-hydroxycarboxylic acid. 8. according to the method described in any one of claim 1-7, it is characterized in that, described microorganism is selected from bacterial strain HCM-10DSM18028, autotrophic yellow bacillus (Xanthobacterautotrophicus) Py2, Rhodobacter tersphaeroides (Rhodobacter tersphaeroides) (ATCC17029 ) or Nocardioides sp. JS614. 9. The method according to any one of claims 1-8, characterized in that whole cells of the microorganism are used unchanged, osmotically or carrier-immobilized. 10. The method according to any one of claims 1-8, characterized in that cell extracts and/or cobalamin-dependent mutases, and optionally other enzymes, such as enzymes for the synthesis of CoA-esters , optionally used in purified form after partial or complete isolation from the microorganism. 11. The method according to claim 10, characterized in that a cell-free crude extract of the microorganism is used. 12. according to the described method of any one of claim 1-11, it is characterized in that, use has sequence SEQ ID NO:2 and/or SEQ ID NO:4, has sequence SEQ ID NO:5 and/or SEQ ID NO : 6, a protein having the sequence SEQ ID NO: 9 and/or SEQ ID NO: 10 or a protein having the sequence SEQ ID NO: 13 and/or SEQ ID NO: 14, and at least 60% of its homologues. 13. The method according to any one of claims 1-12, characterized in that sugars and/or alcohols and/or organic acids and/or hydrocarbons or derivatives thereof are used as carbon sources for enzymatic reactions during the cultivation and energy source. 14. The method according to any one of claims 1-13, characterized in that a substrate with tert-butyl residues is used as carbon and energy source for the cultivation. 15. The method according to any one of claims 1-14, characterized in that tert-butanol is used as the only carbon source and energy source in the basal medium for culturing. 16. The method according to any one of claims 1-15, characterized in that 3-hydroxycarboxylic acid is generated from sugar and/or alcohol and/or organic acid and/or hydrocarbon or derivatives thereof and from 3-hydroxycarboxylic acid The enzymatic reaction of acid to 2-hydroxy-2-methylcarboxylic acid is carried out in a single process step. 17. The method according to any one of claims 1-16, characterized in that the preparation of 2-hydroxyisobutyric acid is carried out with the addition of exogenous 3-hydroxybutyric acid. 18. Process for the preparation of C2-C3-unsaturated isoalkenic acids, characterized in that 2-hydroxy-2-methylcarboxylic acids prepared according to any one of claims 1-17 are dehydrated. 19. Process for the preparation of methacrylic acid, characterized in that 2-hydroxyisobutyric acid prepared according to any one of claims 1-17 is dehydrated. 20. Microbial strain HCM-10-DSM 18028. 21. A nucleic acid molecule encoding an enzyme having cobalamin-dependent mutase activity selected from the group consisting of A) a nucleic acid molecule, which encodes a protein comprising the amino acid sequence shown under Seq.No.2 and/or Seq.No.4; b) A nucleic acid molecule comprising the nucleotide sequence shown under Seq. No. 1 and/or Seq. No. 3. 22. The nucleic acid molecule according to claim 21, characterized in that, the nucleic acid molecule encodes a protein which is composed of two different subunits as an oligomerase, which is contained in Seq.No.2 and Seq. Amino acid sequence shown under No.4. 23. The nucleic acid molecule of claim 21 or 22, which is a DNA molecule. 24. The nucleic acid molecule of claim 23, which is cDNA or genomic DNA. 25. The nucleic acid molecule of claim 21 or 22, which is an RNA molecule. 26. A protein having cobalamin-dependent mutase activity encoded by the nucleic acid molecule of claims 21-22. 27. The protein having sequence No. 2 and at least 99% homologues thereof. 28. The protein having sequence No. 4 and at least 99% homologues thereof. 29. A protein as a heterodimeric enzyme comprising Sequence No.2 and Sequence No.4.

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