ES2803273T3 - Alcaligenes faecalis mutant 3-hydroxybutyrate dehydrogenase, as well as procedures and uses involving the same - Google Patents

Abstract

A mutant 3-hydroxybutyrate dehydrogenase (3-HBDH) from Alcaligenes faecalis with improved performance relative to wild-type 3-HBDH, in which the mutant enzyme comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 (3-HBDH from Alcaligenes faecalis; wild-type 3-HBDH) or the amino acid sequence of SEQ ID NO: 2 (extended core sequence of wild-type 3-HBDH) and in which the mutant enzyme has at least three amino acid substitutions relative to natural 3-HBDH, in which the amino acid at position corresponding to - position 253 of SEQ ID NO: 1 is substituted with Ile (253Ile), Ala (253Ala) or Cys (253Cys) , - position 233 of SEQ ID NO: 1 is replaced with Ser (233Ser), Ile (233Ile), Ala (233Ala), Pro (233Pro), Arg (233Arg), Thr (233Thr), Lys (233Lys) or Cys (233Cys), and - position 234 of SEQ ID NO: 1 is replaced with Thr (234Thr).

Description

DESCRIPTION

Alcaligenes faecalis mutant 3-hydroxybutyrate dehydrogenase , as well as procedures and uses involving the same

The present invention relates to a mutant 3-hydroxybutyrate dehydrogenase (3-HBDH) with improved performance relative to natural 3-HBDH, a nucleic acid encoding mutant 3-HBDH, a cell comprising mutant 3-HBDH or nucleic acid, a method of determining the amount or concentration of 3-hydroxybutyrate in a sample, the use of the mutant 3-HBDH to determine the amount or concentration of 3-hydroxybutyrate in a sample, and a device for determining the amount or concentration of 3-hydroxybutyrate in a sample.

Ketone bodies are produced in the liver, mainly from the oxidation of fatty acids, and are exported to peripheral tissues for use as an energy source. They are particularly important for the brain, which has no other substantial energy source not derived from glucose. The two main ketone bodies are 3-hydroxybutyrate and acetoacetate. Biochemically, abnormalities of the body's ketone metabolism can be subdivided into three categories: ketosis, hypoceotic hypoglycemia, and 3-hydroxybutyrate / acetoacetate ratio abnormalities.

An abnormal elevation of the 3-hydroxybutyrate / acetoacetate ratio usually implies an unoxidized state of the mitochondrial matrix of hepatocytes as a result of hypoxia-ischemia or other causes.

Typically, the presence of ketosis implies that lipid energy metabolism has been activated and that the entire lipid breakdown pathway is intact. In rare cases, ketosis reflects an inability to use ketone bodies. Ketosis is normal during fasting, after prolonged exercise, and when consuming a high-fat diet. During the neonatal period, infancy and pregnancy, times when lipid energy metabolism is particularly active, ketosis develops easily.

Pathologic causes of ketosis include diabetes, childhood ketotic hypoglycemia, corticosteroid or growth hormone deficiency, alcohol or salicylate intoxication, and various inborn errors of metabolism.

The formation of ketone bodies is increased when lipolysis is increased, for example, in insulin deficiency (diabetes mellitus; in particular, type I diabetics), when the glucagon concentration is increased and in the fasting state. In such cases, the normal physiological concentration of less than 7 mg / dl can increase by more than a factor 10. In recent years, 3-hydroxybutyrate has proven to be an extremely reliable parameter for monitoring insulin treatment.

The absence of ketosis in a patient with hypoglycemia is abnormal and suggests the diagnosis of hyperinsulinemia or an inborn error of fat energy metabolism.

Consequently, ketone bodies are an interesting diagnostic target, particularly for diabetes. Therefore, there is a continuing need for robust and sensitive test systems for ketone bodies, especially 3-hydroxybutyrate. The ratio of 3-hydroxybutyrate and acetone or acetoacetic acid is normally 3: 1. In ketoacidosis, the ratio increases to 6: 1 to 12: 1. Adequate 3-HBDH should allow the development of a quantitative test for 3-hydroxybutyrate.

Surprisingly, a variety of Alcaligenes faecalis 3-hydroxybutyrate dehydrogenase mutant enzymes have been found to show improved performance, in particular increased thermal stability and / or affinity for substrate and / or cofactor. As shown in the examples, there are many sites on the natural enzyme that allow mutations, which increase the thermal stability and / or the affinity for the substrate and / or cofactor of the mutant enzyme relative to the natural enzyme.

One site that is currently considered particularly suitable for increasing yield is the amino acid at position corresponding to position 253 of SEQ ID NO: 1 (see below). In particular, the amino acid can be substituted for Ile (253Ile), Ala (253Ala) or Cys (253Cys) to increase the affinity for the substrate and / or cofactor (for 3-hydroxybutyrate and / or carba-NAD) and optionally the stability thermal (see tables 1A, 1B and 4).

In addition, it was discovered that mutations in one or more of the positions corresponding to positions 18, 19, 33, 38, 39, 62, 125, 143, 148, 170, 175, 187, 216, 233 and / or 234 of SEQ ID NO: 1, in particular at least positions 62, 143, 148, 233 and / or 234 of SEQ ID NO: 1 were also suitable (see Tables 1B, 2A-C and 4). The yield of the mutant 3-HBDH could be further increased by combining mutations at the above sites (see Tables 2A-D and 3). Particularly suitable examples of those mutant enzymes include those with the mutations (for definitions of abbreviations, see below)

- 253Ile, 233Lys, 234Thr; OR

- 253Ile, 62Val, 233Lys, 234Thr; OR

- 253Ile, 62Val, 147Arg, 233Lys, 234Thr; OR

- 253Ile, 39Gly, 62Val, 143Val, 148Gly, 233Lys, 234Thr; OR

- 253Ile, 62Val, 143Val, 148Gly, 233Lys, 234Thr; OR

- 62Val, 111Leu, 140Met, 148Gly

(see Tables 2A-D).

Surprisingly, the mutant enzyme having 253Ile, 233Lys and 234Thr mutations named AFDH3 showed a dramatic increase in thermostability, compared to a mutant enzyme with 253Ile mutation, alone (named AFDH2) (see Table 2D), in which AFDH2 already it had increased its thermostability against the natural enzyme (called AFDH1). Additional mutations, namely 18Arg, 18Lys, 18Glu, 18Pro, 19Gly, 33Ala, 33Leu, 33Thr, 38Lys, 39Gly, 62Phe, 62Met, 62Lys, 62Arg, 62Leu, 62Val, 125Gly, 143Val, 147Arg, 148Gly, have been shown to 170Gly, 175Thr, 175Val, 175Ile, 187Phe, and 216Val further enhance the performance of mutant enzymes relative to wild type 3-HBDH or AFDH2 (see Tables 2D, 3 and 4).

Consequently, in a first aspect, the present invention relates to a mutant 3-hydroxybutyrate dehydrogenase (3-HBDH) of Alcaligenes faecalis with an improved performance relative to natural 3-HBDH.

3-Hydroxybutyrate dehydrogenase (3-HBDH) (EC 1.1.1.30) belongs to the family of oxidoreductases, specifically it is an enzyme that catalyzes the stereospecific oxidation of 3-hydroxybutanoate / 3-hydroxybutyrate / (Rj-3-hydroxybutyrate / 3-hydroxybutyric acid (3-HB) to acetoacetate with NAD or derivatives as cofactor:

(R) - 3-hydroxybutyrate NAD + ^ acetoacetate NADH H +

The systematic name of this class of enzymes is (R) -3-hydroxybutanoate: NAD + oxidoreductase. Other commonly used names include NAD + -6epha-hydroxybutyrate dehydrogenase, hydroxybutyrate oxidoreductase, oepha-hydroxybutyrate dehydrogenase, D-bepha-hydroxybutyrate dehydrogenase, D-3-hydroxybutyrate dehydrogenase, D - (-) - 3-hydroxybutyrate dehydrogenase, D - (-) - 3-hydroxybutyrate dehydrogenase, hydrogenase dehydrogenase dehydrogenase, 3-D-hydroxybutyrate dehydrogenase and oepha-hydroxybutyric dehydrogenase. This enzyme participates in the synthesis and degradation of ketone bodies and the metabolism of butanoate and can be used to determine ketone bodies in samples, such as a blood sample.

The term "natural 3-HBDH" refers to a 3-HBDH as it typically occurs in nature. A natural 3-HBDH from the gram negative bacterium Alcaligenes faecalis was cloned in E. coli and was further described in 1996 (Jpn. Kokai Tokkyo Koho (1996), JP 08070856 A 19960319). However, the enzyme is characterized by a rather low stability (resulting in a short shelf life), which is disadvantageous in biotechnical, biomedical and diagnostic applications. In particular, the short shelf life has a negative impact on the applicability of the enzyme, if the use of a previously prepared enzyme is intended. In particular, this includes commercial products such as enzyme preparations, kits, test strips, etc., which are normally prepared on a large scale, stored and then marketed, for example, in smaller batches. Additionally, it is obviously desirable to increase the affinity of the enzyme for substrates and / or cofactors. Increasing the affinity and / or stability will increase the performance of the enzyme in biotechnical applications and devices. The commonly accepted amino acid sequence of the typical wild-type 3-HBDH from Alcaligenes faecalis is provided below as SEQ ID NO: 1.

The term " Alcaligenes faecalis mutant 3-hydroxybutyrate dehydrogenase (3-HBDH) " refers to a 3-HBDH enzyme with an amino acid sequence that differs from that of the native Alcaligenes faecalis 3-HBDH , in particular the amino acid sequence of SEQ ID NO: 1, in at least one mutation, that is, one or more substitutions, additions, amino acid deletions or combinations thereof, in particular in at least one substitution.

The expression "with improved performance relative to natural 3-HBDH" means that the performance of the mutant enzyme has been improved relative to natural 3-HBDH from Alcaligenes faecalis. Performance is improved if the mutant enzyme has a higher yield, for example, higher activity in the conversion of 3-HB to acetoacetate, under any condition (for example, after storage, at a particular pH, with a specific buffer, at a chosen temperature, with a particular cofactor (eg carba-NAD), at a specific concentration of substrate or cofactor, etc.). A higher yield can especially be increased stability (such as thermal stability) and / or affinity for one or more substrates / cofactors (eg, carba-NAD and / or 3-HB). Preferably, performance is improved if the mutant enzyme, for example, has a higher relative remaining activity in converting 3-HB to acetoacetate when subjected to more extreme conditions (for example, storage, buffer conditions, temperature, cofactor concentration or substrate) compared to activity without being subjected to these more extreme conditions and / or if the mutant enzyme, for example, has a higher relative activity (i.e. activity at a subsaturation concentration / activity at a saturation concentration).

The sequence of the natural 3-HBDH from Alcaligenes faecalis is shown as SEQ ID NO: 1:

SEQ ID NO: 1 (3-HBDH from Alcaligenes faecalis)

MLKGKKAWT GSTSGiGLAMATELAKAGAD W iWGFGQL; 2 DIERERSTLE SKFGVKAYYL 60

NADLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH 12C

GTAAALPIMQ KQGWGRIINI ASAHGLVASV NKSAYVAAKH GWGLTKVTA LENAGKGITC 18C

NAICPGWVRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGGAAVFLSS 240

AAADQMTGTT LSLDGGWTAR 260

Figure 1 shows the various domains (catalytic center and binding sites) of the natural enzyme. Based on these domains, the core sequence of the enzyme can be determined. As detailed above, mutant 3-HBDH with mutations at position corresponding to position 253 of SEQ ID NO: 1 were found to be particularly useful. Consequently, an extended core sequence of 3-HBDH from Alcaligenes faecalis spanning this position was established and is shown as SEQ ID NO: 2:

SEQ ID NO: 2 (3-HBDH extended core sequence from Alcaligenes faecalis)

ADLSDAQATR DFIAKAAEAL GGLDILVNNAGIQHTAPIEEFPVDKWNAIIALNLSAVFHG 60

TAAALPIMQK QGWGRIINIA SAHGLVASVNKSAYVAAKHGWGLTKVTALENAGKGITCN 12C

AICPGWVRTP LVEKQIEAIS QQKGIDIEAAARELLAEKQP SLQFVTPEQLGGAAVFLSSA 180

AADQMTGTTL SL 192

Accordingly, a preferred example of a natural 3-HBDH is given above, in Figure 1 and as SEQ ID NO: 1.

If one or more mutations are introduced in the natural sequence, especially the sequence of SEQ ID NO: 1, a mutant 3-HBDH is obtained. With regard to the mutant 3-HBDH of the present invention, it should be noted that the mutant enzyme is functionally active and shows improved performance relative to natural 3-HBDH. This means that the mutant enzyme has maintained its enzymatic function of converting 3-HB into acetoacetate. Additionally, preferably at least one of the stability and affinity of the enzyme for the substrate and / or cofactors is increased. The mutant enzyme differs from natural 3-HBDH in one or more amino acid substitutions, additions and / or deletions, especially at least one or more substitutions.

The sequence of the mutant 3-HBDH according to the present invention comprises (optionally, in addition to the substitutions specified herein) one or more amino acid substitutions, deletions or additions, especially one or more substitutions, in particular one or more substitutions conservative amino acids.

In one embodiment of the present invention, the mutant 3-HBDH according to the present invention may comprise one or more amino acid substitutions, in particular a limited number of substitutions (for example, substitutions of up to 50, 40, 30, 20 , especially 10 amino acids). Suitable substitutions are given in the examples, in particular in the tables. All the substitutions identified in the examples as suitable for increasing the yield of 3-HBDH can be used in the invention, either alone or in combination with each other and / or with additional substitutions, eg conservative substitutions. "Conservative amino acid substitution" refers to a substitution of one residue with a different residue that has a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same defined amino acid class or from a similar class. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid, eg, alanine, valine, leucine, and isoleucine; an amino acid with a hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain, eg, serine and threonine; an amino acid having aromatic side chains is substituted with another amino acid having an aromatic side chain, eg, phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with a basic side chain is substituted with another amino acid with a basic side chain, eg, lysine and arginine; an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain, for example, aspartic acid or glutamic acid; and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively. Examples of conservative amino acid substitutions include those listed below:

Original residue Conservative substitutions

Ala, Leu, Val, Ile Other aliphatics (Ala, Leu, Val, Ile)

Other non-polar (Ala, Leu, Val, Ile, Gly, Met)

Gly, Met Other non-polar (Ala, Leu, Val, Ile, Gly, Met)

Asp, Glu Other acids (Asp, Glu)

Lys, Arg Other basic (Lys, Arg)

Asn, Gln, Ser, Thr Other poles (Asn, Gln, Ser, Thr)

His, Tyr, Trp, Phe Other aromatics (His, Tyr, Trp, Phe)

Cys, Pro None

In an embodiment of the present invention, the mutant 3-HBDH according to the present invention may comprise one or more amino acid additions, in particular small internal amino acid additions (eg up to 50, 40, 30, 20, especially 10 amino acids). Alternatively, additions can be achieved by combining the mutant 3-HBDH into a fusion protein comprising the mutant 3-HBDH of the present invention.

Fusion proteins are proteins created by joining two or more originally separate proteins or peptides. This procedure results in a polypeptide with functional properties derived from each of the parent proteins. Accordingly, depending on the intended use of 3-HBDH, it can be combined with an additional peptide or protein in a fusion protein. Proteins can be fused via a linker or spacer, which increases the likelihood that the proteins will fold independently and behave as expected. Especially in the case where linkers allow purification of proteins, linkers in protein or peptide fusions are sometimes genomanipulated with cleavage sites for proteases or chemical agents that allow the release of the two separate proteins. Dimeric or multimeric fusion proteins can be made through genomanipulation by fusion with the parent proteins of peptide domains that induce dimerization or multimerization of artificial proteins (eg, leucine or streptavidin zippers). Fusion proteins can also be made with toxins or antibodies attached to them. Other fusions include the addition of signal sequences, such as a lipidation signal sequence, a secretion signal sequence, a glycosylation signal sequence, a translocation signal peptide, etc.

Preferably, the fusion protein of the present invention comprises a label. Labels are attached to proteins for various purposes, for example, to facilitate purification, to aid in proper folding of proteins, to prevent protein precipitation, to alter chromatographic properties, to modify the protein, or to label the protein. Examples of tags include the Arg tag, the His tag, the Strep tag, the Flag tag, the T7 tag, the V5 peptide tag, the GST tag, and the c-Myc tag.

In an embodiment of the present invention, the mutant 3-HBDH according to the present invention may comprise one or more amino acid deletions, in particular N and / or C terminal deletions, especially in the absent portions of SEQ ID NO: 1 in SEQ ID NO: 2. Deletions can be small (eg, up to 50, 40, 30, 20, especially 10 amino acids).

In another embodiment, the sequence of the mutant 3-HBDH according to the present invention may comprise, preferably in addition to the substitutions specified herein, a combination of one or more deletions, substitutions or additions as defined above.

In a preferred embodiment of the present invention, the mutant 3-HBDH comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 (3-HBDH from Alcaligenes faecalis; 3- Wild type HBDH) or the amino acid sequence of SEQ ID NO: 2 (wild type 3-HBDH extended core sequence).

The term "at least 80% identical" or "sequence identity of at least 80%", as used herein, means that the sequence of the mutant 3-HBDH according to the present invention has a sequence of amino acids characterized in that, within a stretch of 100 amino acids, at least 80 amino acid residues are identical to those of the corresponding natural sequence. The sequence identities of other percentages are defined accordingly.

Sequence identity according to the present invention can be determined, for example, by sequence alignment procedures in the form of sequence comparison. Sequence alignment procedures are well known in the art and include various alignment programs and algorithms that have been described, for example, in Pearson and Lipman (1988). Additionally, the NCBI Basic Local Alignment Search Tool (BLAST) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with blastp sequence analysis programs, blastn, blastx, tblastn, and tblastx. The percent identity of mutant enzymes according to the present invention in relation to the amino acid sequence of, for example, SEQ ID NO: 1 is typically characterized using Blast blastp from NCBI with standard settings. Alternatively, sequence identity can be determined using GENEious software with standard settings. The alignment results can be derived, for example, from the GENEious software (version R8), using the global alignment protocol with free-end gaps as the alignment type and Blosum62 as the cost matrix.

As detailed above, the mutant 3-HBDH of the present invention in one embodiment comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 or 2. In one embodiment, A preferred embodiment, the mutant 3-HBDH comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences of I KNOW THAT ID NO: 1 or 2. Sequence identity can be determined as described above.

In another preferred embodiment, the mutant enzyme of the present invention has at least one amino acid substitution in relation to natural 3-HBDH, in which the amino acid at the position corresponding to position 253 of SEQ ID NO: 1 is substitute Ile (253Ile), Ala (253Ala) or Cys (253Cys), in particular Ile (253Ile). It has been shown in the examples that the substitution of the amino acid in the position corresponding to position 253 of SEQ ID NO: 1 increases the stability and / or affinity for substrates and / or cofactors (see, for example, Tables 1A, 1B and 4).

Additionally, it could be shown that the substitution at position corresponding to position 253 of SEQ ID NO: 1 can be combined with a variety of other substitutions to further optimize the enzyme. The substitution of Leu at position 253 with Ile (253Ile) is particularly suitable for increasing thermal stability, as well as affinity for the substrate, as shown in Tables 1A-C and Tables 2 to 4 for simple mutations and multiple relative to natural, respectively. Performance could be shown to improve after previous incubations at various temperatures and under different conditions (buffers, pH). The positive effect of this substitution is particularly surprising since the amino acid in natural (leucine) is replaced with a fairly similar amino acid (isoleucine). Both amino acids are constitutional isomers. Both are considered nonpolar and hydrophobic and have the same molecular weight.

Substitution of leucine at a position corresponding to position 253 of SEQ ID NO: 1 with the non-polar amino acid alanine or the non-acid, non-polar, non-charged hydrophilic amino acid cysteine significantly increased the affinities for substrate and cofactor (see Table 1B). Due to the chemical differences between the introduced amino acids Ala and Cys, this result is also unexpected and surprising.

A highly preferred embodiment of the present invention relates to a mutant 3-hydroxybutyrate dehydrogenase (3-HBDH) of Alcaligenes faecalis with an improved yield relative to natural 3-HBDH, in which the mutant enzyme comprises a sequence of amino acids that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 (3-HBDH from Alcaligenes faecalis; wild-type 3-HBd H) or the amino acid sequence of SEQ ID NO: 2 (extended core sequence of 3 -HBDH natural) and

wherein the mutant enzyme has at least three amino acid substitutions relative to natural 3-HBDH, wherein the amino acid at the position corresponding to

- position 253 of SEQ ID NO: 1 is replaced with Ile (253Ile), Ala (253Ala) or Cys (253Cys),

- position 233 of SEQ ID NO: 1 is replaced with Ser (233Ser), Ile (233Ile), Ala (233Ala), Pro (233Pro), Arg (233Arg), Thr (233Thr), Lys (233Lys) or Cys ( 233Cys), and

- position 234 of SEQ ID NO: 1 is replaced with Thr (234Thr).

More preferably, the amino acid at position corresponding to position 253 of SEQ ID NO: 1 is substituted with Ile (253Ile) and / or the amino acid at position corresponding to position 233 of SEQ ID NO: 1 is substituted with Lys (233Lys) in this highly preferred mutant enzyme and optionally the mutant enzyme has at least one additional amino acid substitution at one or more of the positions corresponding to positions 18, 19, 33, 38, 39, 62, 125, 143, 148, 170, 175, 187 and / or 216 of SEQ ID NO: 1, in particular at least positions 62, 143 and / or 148 of SEQ ID NO: 1.

As already detailed above, it could be shown that amino acids in one or more additional positions of SEQ ID NO: 1 can be substituted alternatively or additionally, to improve the performance of the enzyme. Consequently, in another embodiment of the present invention, the mutant enzyme of the present invention has at least one amino acid substitution in one or more of the positions corresponding to positions 18, 19, 33, 38, 39, 62, 125, 143, 148, 170, 175, 187, 216, 233 and / or 234 of SEQ ID NO: 1, in particular at least positions 62, 143, 148, 233 and / or 234 of SEQ ID NO: 1, especially at least positions 62, 143, 148, 233 and 234 of SEQ ID NO: 1. Substitutions at these positions can be combined with each other, as well as with a substitution at position 253, especially 253Ile, 253Ala, 253Cys, in particular 253Ile (see also examples).

Particularly suitable examples of positions for substitutions, as well as combinations thereof for mutant 3-HBDH of the present invention are given below:

- position 253 of SEQ ID NO: 1 is substituted with Ile (253Ile), Ala (253Ala) or Cys (253Cys), especially Ile (253Ile);

sustituye con Arg (18Arg), Lys (18Lys), Glu (18Glu) o Pro (18Pro); - position 18 of SEQ ID NO:

substitute Arg (18Arg), Lys (18Lys), Glu (18Glu) or Pro (18Pro);

sustituye con Gly (19Gly);- position 19 of SEQ ID NO:

replace with Gly (19Gly);

- position 33 of SEQ ID NO: 1 is replaced with Ala (33Ala), Leu (33Leu) or Thr (33Thr);

- position 38 of SEQ ID NO: 1 is replaced with Lys (38 Lys);

- position 39 of SEQ ID NO: 1 is replaced with Gly (39Gly);

- position 62 of SEQ ID NO: 1 is replaced with Phe (62Phe), Met (62Met), Lys (62Lys), Arg (62Arg), Leu (62Leu) or Val (62Val), especially Val (62Val);

e sustituye con Gly (125Gly);- position 125 of

e substitute Gly (125Gly);

e sustituye con Val (143Val);- position 143 of

e substitute Val (143Val);

e sustituye con Gly (148Gly);- position 148 of

e substitute Gly (148Gly);

e sustituye con Gly (170Gly);- position 170 of

e substitute Gly (170Gly);

e sustituye con Thr (175Thr), Val (175Val) o Ile (175Ile);- position 175 of

e substitute Thr (175Thr), Val (175Val) or Ile (175Ile);

- position 187 of SEQ ID NO: 1 is replaced with Phe (187Phe);

- position 216 of SEQ ID NO: 1 is replaced with Val (216Val);

- position 233 of SEQ ID NO: 1 is replaced with Ser (233Ser), Ile (233Ile), Ala (233Ala), Pro (233Pro), Arg (233Arg), Thr (233Thr), Lys (233Lys) or Cys ( 233Cys), especially Lys (233Lys); I

- position 234 of SEQ ID NO: 1 is replaced with Thr (234Thr).

In a preferred embodiment of the present invention, the mutant 3-HBDH has at least or only the following mutations:

- 253Ile;

- 253Ile, 233Lys, 234Thr;

- 253Ile, 62Val, 233Lys, 234Thr;

- 253Ile, 62Val, 147Arg, 233Lys, 234Thr;

- 253Ile, 39Gly, 62Val, 143Val, 148Gly, 233Lys, 234Thr;

- 253Ile, 62Val, 143Val, 148Gly, 233Lys, 234Thr; or

- 62Val, 111 Leu, 140Met, 148Gly,

in particular where the mutant 3-HBDH has at least or only the mutations 253Ile, 62Val, 143Val, 143Val, 148Gly, 233Lys, 234Thr, optionally in combination with

- 19Gly;

- 170Gly;

- 125Gly, 187Phe;

- 18Glu, 33Leu;

- 33Leu, 125Gly, 187Phe;

- 33Leu, 125Gly, 187Phe, 216Val;

- 18Glu, 187Phe;

- 33Leu, 125Gly, 175Val, 187Phe;

- 175Val, 216Val;

- 175Val, 187Phe, 216Val;

- 19Gly, 125Gly, 187Phe;

- 125Gly, 175Thr, 187Phe;

- 19Gly, 33Leu;

- 19Gly, 175Ile;

- 19Gly, 175Thr;

- 170Gly, 175Thr;

- 18Glu, 125Gly, 187Phe;

- 33Leu, 125Gly, 175Thr, 187Phe;

- 33Leu, 125Gly, 175Ile, 187Phe;

- 33Leu, 125Gly, 170Gly, 187Phe;

- 18Glu, 175Ile, 187Phe;

- 33Leu, 125Gly, 175Val, 187Phe;

- 33Leu, 125Gly, 175Val, 187Phe, 216Val;

- 33Leu, 125Gly, 175Thr, 187Phe; or

- 33Leu, 38Lys, 39Gly, 125Gly, 175Val, 187Phe.

In a preferred embodiment of the present invention, the mutant 3-HBDH comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. to the amino acid sequence of any of SEQ ID NO: 1 to 16, in particular wherein the mutant 3-HBDH comprises or consists of the sequence selected from the group consisting of SEQ ID NO: 3 to 16.

As detailed above, the sequences of SEQ ID NO: 1 and 2 refer to natural 3-HBDH and a fragment thereof, respectively. The sequences of SEQ ID NO: 3 to 16 are the highly preferred mutant enzyme sequences. The sequences of the mutant enzymes and the substitutions made are shown in the "Sequences" section. Sequence identity can be determined as described above.

In a preferred embodiment of the present invention, the improved yield relative to natural 3-HBDH is

- increased stability, especially thermal stability, relative to natural 3-HBDH; I

- an increased affinity for substrate, especially for 3-hydroxybutyrate, relative to natural 3-HBDH; and / or - an increased affinity for cofactor, especially for NAD or a derivative thereof, in particular where the derivative is carba-NAD, relative to natural 3-HBDH.

The expression "increased stability, especially thermal stability, relative to natural 3-HBDH" means that mutant 3-HBDH is less prone to loss of (enzymatic) activity under certain conditions, especially at elevated temperatures. Stabilizing enzymes, including bypassing denaturation mechanisms to carry out their full potential as catalysts, is an important goal in biotechnology. Stabilization of enzymes is of remarkable importance due to the increasing number of applications of enzymes. Increased stability allows for sustained usability (eg, longer storage, longer usability, etc.). The increase in the stability of the mutant enzyme in relation to the natural one can be determined by comparing the remaining activity of both enzymes (natural and mutant), for example, after storage or exposure to particular conditions (for example, elevated temperature, dried, buffer or salt) (absolute remaining activity). Alternatively, the stability is improved compared to the natural enzyme, if the mutant enzyme, for example, has a higher relative remaining activity in the conversion of 3-HB to acetoacetate. The relative remaining activity can be determined by comparing the remaining or residual acidity after storage under given conditions (eg, storage time, temperature) with the initial activity before storage.

The term "enzymatic activity" and its determination are well known to those skilled in the art. Activity Enzymatic is generally defined as the conversion of an amount of substrate over time. The SI unit for enzyme activity is the katal (1 katal = 1 m ols-1). A more practical and commonly used value is the enzyme unit (U) = 1 pmolmim1. 1 U corresponds to 16.67 nanokatals and is defined as the amount of enzyme that catalyzes the conversion of 1 micromole of substrate per minute. The specific activity of an enzyme is the activity of an enzyme per milligram of total protein (expressed in pmol min-1 mg-1).

Enzyme activity can be determined in an assay by measuring substrate or cofactor consumption or product formation over time. There are a large number of different procedures for measuring the concentrations of substrates and products, and many enzymes can be assayed in a number of different ways, which are known to those of skill in the art. In the present invention, the subject 3-HBDH is incubated, for example, with 3-hydroxybutyrate and cofactor (for example, NAD or a derivative thereof such as carba-NAD) and the conversion of the cofactor (NAD to NADH) is monitored or carba-NAD to carba-NADH). Monitoring can be done, for example, by measuring the absorbance of light at 340 nm. The change in absorption obtained per minute (dE / min) represents the enzymatic activity. For more details, see example 2.

In a preferred embodiment of the present invention, the increased stability of the mutant 3-HBDH relative to the respective non-mutated 3-HBDH can be expressed as an increase in the half-life of the mutant enzyme (t1 / 2 (mutant) ) relative to the half-life of the respective wild type 3-HBDh (t1 / 2 (wild type)). The half-life (t1 / 2) of the enzyme indicates the amount of time it takes for 50% of the original activity to be lost (activity at t = 0) and after which the remaining activity represents 50%. Consequently, increased stability results in an increased half-life of the mutant enzyme relative to the respective natural enzyme. The increase in stability can be determined as t1 / 2 (mutant) / t1 / 2 (wild type). The percentage increase in half-life can be determined as [t1 / 2 (mutant) / t1 / 2 (wild-type) - 1] * 100.

In a highly preferred embodiment of the present invention, the increased stability of the mutant 3-HBDH relative to the respective non-mutated 3-HBDH can be determined and expressed as remaining activity after incubation under extreme conditions (eg, 30 min at, for example, 64 ° C or any other condition given in the examples) relative to the initial activity before incubation / storage under extreme conditions (see the examples). For this, the enzymatic reaction can be monitored (for example, at room temperature at 340 nm for 5 minutes) and the change in absorption per time (for example, dE / min) can be calculated for each sample. The values obtained for the samples subjected to extreme conditions can be compared with those of the respective sample not subjected to extreme conditions (value established as 100% activity) and calculated in percentage of activity ((dE / min (sample in extreme conditions) ) / dE / min (sample in non-extreme conditions) * 100). Consequently, a mutant enzyme value greater than the value obtained with the natural enzyme represents an improvement in thermal stability. Stability increases if [% of remaining activity of the mutant enzyme] - [% remaining activity of the natural enzyme]> 0. Alternatively, the remaining activity of the mutant enzyme can also be expressed as a percentage activity and can be calculated as follows: [% of remaining activity of the mutant enzyme] / [% of remaining activity of the natural enzyme] * 100%. The stability of the mutant enzyme relative to the natural enzyme is increased if the resulting value is> 100%. A suitable test p joint to determine stability is described in detail in Example 2. Accordingly, stability can be expressed as remaining activity and calculated as [dE / min (sample in cond. extreme)] / [dE / min (sample in non-extreme cond.)] * 100. Other details are given in Example 2. A value obtained with a mutant enzyme that is greater than the value obtained with the natural enzyme represents a stability increased mutant enzyme.

Preferably, the stability is increased by at least 10%, 20%, 30% or 40%, preferably by at least 50%, 75% or 100%, still more preferably by at least 125%, 150%, 175 % or 200%, especially 250% and most preferably 300%.

In addition, there are commercial advantages to performing enzymatic reactions at higher temperatures. Consequently, the thermal stability of the mutant enzyme is preferentially increased. This means that the resistance of the mutant enzyme relative to the natural enzyme at increased temperatures is higher. The increase in thermal stability can be determined in particular as shown in the examples, for example in example 3 (tables 2A-D). In general, the mutant enzyme and the natural enzyme can be pre-incubated at an increased temperature (for example, above 50 ° C, such as 64 ° C or 68 ° C or 75 ° C, etc.) for a period of time. defined (such as 10 minutes or 30 minutes), after which the remaining activity in the conversion of 3-HB to acetoacetate of the mutant enzyme is compared to the remaining activity of the natural enzyme. If the remaining activity of the mutant enzyme is greater than that of the natural enzyme, the mutant enzyme has increased thermal stability.

A suitable procedure for the determination of increased thermal stability is detailed in the examples. Exemplary extreme conditions can be pre-incubation at 60-90 ° C (eg 64 ° C or 68 ° C or 75 ° C) for 30 minutes and then testing with 62.22 mM 3-hydroxybutyrate; 4.15 mM cNAD; 0.1% Triton X-100; 200 mM Hepes pH 9.0 or 150 mM 3-hydroxybutyrate; 5 mM cNAD; 0.1% Triton X-100; Mops 70 mM at pH 7.5.

The expression "increased affinity for substrate, especially for 3-hydroxybutyrate, relative to natural 3-HBDH" means that the affinity of the mutant enzyme for the substrate 3-hydroxybutyric acid / 3-hydroxybutyrate / 3-hydroxybutanoate (3-HB) that is converted to acetoacetate is increased. For determination, the enzymatic reaction can be monitored (eg at room temperature at 340 nm for 5 minutes) and the dE / min can be calculated for each sample. The affinity of the mutant enzyme is increased compared to the natural enzyme if the mutant enzyme, for example, has a higher absolute or relative affinity for the substrate, in particular 3-HB. The affinity of the mutant enzyme compared to the natural enzyme can be determined by comparing the absolute affinities of both enzymes (natural and mutant) (absolute comparison) and can be calculated in percentage of activity ((dE / min (mutant) / dE ( natural)) * 100) (%). Alternatively, the affinity of the mutant enzyme compared to the wild type enzyme can be determined by comparing the relative affinities of both enzymes (wild type and mutant) (relative comparison). The relative affinity of the wild-type or mutant enzyme can be determined by adjusting the affinity at the subsaturation substrate concentration relative to the affinity at the saturation substrate concentration. As detailed in Examples 2-4, the affinity for 3-hydroxybutyrate can be determined in an activity assay with a reduced amount of substrate (i.e., at a subsaturation concentration), for example, with 3-hydroxybutyrate 1, 94 mM (other exemplary conditions: 4.15 mM cNAD; 0.1% Triton X-100; 200 mM Hepes at pH 9.0) or 5mM 3-hydroxybutyrate (other exemplary conditions: 5 mM cNAD; Triton X-100 0.1%; Mops 70 mM at pH 7.5). A particular suitable test to determine the affinity is described in detail in Example 2. Accordingly, the affinity can be expressed as relative activity and calculated as [dE / min (subsaturation substrate concentration)] / [dE / min (saturation substrate concentration)] * 100. Other details are given in Example 2. A value obtained with a mutant enzyme that is greater than the value obtained with the natural enzyme represents an increase in the affinity for the mutant enzyme.

An increased affinity is correlated with a lower Km value. The Michaelis constant Km is the concentration of the substrate at which the reaction rate of the enzyme is half the maximum and is an inverse measure of the affinity of the substrate for the enzyme.

The expression "increased affinity for cofactor, especially for NAD or a derivative thereof, in particular where the derivative is carba-NAD, relative to natural 3-HBDH" means that the affinity of the mutant enzyme for the cofactor ( NAD or a derivative thereof, especially carba-NAD) required to convert 3-HB to acetoacetate is increased. As detailed in Examples 2-4, affinity for the cofactor can be determined in an activity assay with a reduced amount of cofactor (i.e. below the saturation level), for example with 0.032 mM cNAD (other Exemplary conditions: 62.22 mM 3-hydroxybutyrate; 0.1% Triton X-100; 200 mM Hepes at pH 9.0) or 0.5 mM cNAD (other exemplary conditions: 150 mM 3-Hydroxybutyrate; Triton X- 0.1% 100; Mops 70 mM at pH 7.5). The above details given regarding the affinity for the substrate are likewise applicable to the affinity for the cofactor. A particular suitable test for determining affinity is described in detail in Example 2.

In particular, the mutant 3-HBDH of the present invention is characterized in that the mutant 3-HBDH has a stability increased by at least a factor 2 relative to the natural 3-HBDH, preferably a stability increased by at least a factor 3 , preferably a stability increased by at least a factor 4, more preferably a stability increased by at least a factor 5; and / or is characterized in that the mutant 3-HBDH has an increased affinity for the substrate and / or cofactor in relation to the natural 3-HBDH, in particular an increased affinity for (i) 3-hydroxybutyrate and / or (ii) nicotinamide adenine dinucleotide (NAD) or a functionally active derivative thereof and / or in particular in which the affinity for the substrate and / or cofactor is increased by at least 5%, more particularly by at least 10% , even more particularly by at least 15% or 20%.

In another aspect, the present invention relates to a nucleic acid encoding the mutant 3-HBDH of the present invention as described above.

The term "nucleic acid" as used herein refers generally to any nucleotide molecule that encodes the mutant 3-HBDH of the invention and that can be of variable length. Examples of a nucleic acid of the invention include, but are not limited to, plasmids, vectors, or any type of DNA and / or RNA fragment (s) that can be isolated by standard molecular biology procedures, including, for example , ion exchange chromatography. A nucleic acid of the invention can be used for the transfection or transduction of a particular cell or organism.

The nucleic acid molecule of the present invention may be in the form of RNA, such as mRNA or cRNA, or in the form of DNA, including, for example, cDNA and genomic DNA, for example, obtained by cloning or produced by synthetic techniques. chemistry or by combination thereof. DNA can be tri-stranded, double-stranded, or single-stranded. Single-stranded DNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also known as the antisense strand. Nucleic acid molecule, as used herein, also refers to, among others, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded RNA and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or triple-stranded, or a mixture of single-stranded and double-stranded regions. Furthermore, the nucleic acid molecule, as used herein, refers to triplex regions comprising RNA or DNA or both RNA and DNA.

In addition, the nucleic acid can contain one or more modified bases. Such nucleic acids may also contain modifications, for example, in the ribose-phosphate backbone to increase the stability and half-life of such molecules in physiological environments. Therefore, DNAs or RNAs with backbones modified by stability or for other reasons are "nucleic acid molecules" as that characteristic feature is intended herein. Furthermore, DNAs or RNAs that comprise unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are nucleic acid molecules within the context of the present invention. It will be appreciated that a wide variety of modifications to DNA and RNA have been prepared that serve many useful purposes known to those of skill in the art. The term nucleic acid molecule, as used herein, encompasses such chemically, enzymatically or metabolically modified forms of the nucleic acid molecule, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including single cells. and complex, among others.

Furthermore, the nucleic acid molecule encoding the mutant 3-HBDH of the invention can be functionally linked, using standard techniques such as standard cloning techniques, to any desired sequence, such as a regulatory sequence, leader sequence, marker sequence. heterologous or a heterologous coding sequence to create a fusion protein.

The nucleic acid of the invention can be formed originally in vitro or in a cultured cell, in general, by manipulation of nucleic acids by endonucleases and / or exonucleases and / or polymerases and / or ligases and / or recombinases or other known procedures. by one of skill in the art to produce the nucleic acids.

The nucleic acid of the invention can be comprised of an expression vector, wherein the nucleic acid is operably linked to a promoter sequence that can promote the expression of the nucleic acid in a host cell.

As used herein, the term "expression vector" refers, in general, to any type of nucleic acid molecule that can be used to express a protein of interest in a cell (see also above details on acids nucleic acids of the present invention). In particular, the expression vector of the invention can be any plasmid or vector known to those skilled in the art that is suitable for expressing a protein in a particular host cell, including, but not limited to, mammalian cells, bacterial cells, and yeast cells. An expression construct of the present invention can also be a nucleic acid encoding a 3-HBDH of the invention, and which is used for subsequent cloning into a respective vector to ensure expression. Plasmids and vectors for protein expression are well known in the art, and are commercially available from a variety of vendors, including, for example, Promega (Madison, Wl, USA), Qiagen (Hilden, Germany), Invitrogen (Carlsbad, CA, USA) or MoBiTec (Germany). Protein expression procedures are well known to those of skill in the art and are described, for example, in Sambrook et al., 2000 (Molecular Cloning: A laboratory manual, Third Edition).

The vector may further include nucleic acid sequences that allow it to replicate in the host cell, such as an origin of replication, one or more therapeutic genes and / or selectable marker genes, and other genetic elements known in the art, such as regulatory elements that direct the transcription, translation, and / or secretion of the encoded protein. The vector can be used to transduce, transform, or infect a cell, thereby causing the cell to express nucleic acids and / or proteins other than natural to the cell. The vector optionally includes materials to help achieve entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coat, or the like. Numerous types of expression vectors appropriate for the expression of proteins are known in the art, by standard techniques of molecular biology. Such vectors are selected from conventional vector types including insects, eg, baculovirus expression, or yeast, fungal, bacterial, or viral expression systems. Other appropriate expression vectors, of which numerous types are known in the art, can also be used for this purpose. Procedures for obtaining such expression vectors are well known (see, for example, Sambrook et al., Supra).

As detailed above, the nucleic acid encoding a mutant 3-HBDH of the invention is operably linked to a sequence that is suitable for directing the expression of a protein in a host cell, to ensure expression of the protein. However, it is encompassed within the present invention that the claimed expression construct may represent an intermediate product, which is subsequently cloned into a suitable expression vector to ensure expression of the protein. The expression vector of the present invention may further comprise all types of nucleic acid sequences, including, but not limited to, polyadenylation signals, splice donor and splice acceptor signals, intermediate sequences, transcription-enhancing sequences, translation enhancers, drug resistance genes or the like. Optionally, the drug resistance gene can be functionally linked to an internal ribosome entry site (IRES), which could be cell cycle specific or cell cycle independent.

The term "operably linked" as used herein generally means that the gene elements are arranged so that they function in concert for their intended purposes, for example, where transcription is initiated by the promoter and proceeds through the DNA sequence encoding the protein of the present invention. That is, RNA polymerase transcribes the sequence encoding the fusion protein into mRNA, which is then spliced and translated into a protein.

The term "promoter sequence" as used in the context of the present invention refers, in general, to any a type of DNA regulatory sequence operably linked to a coding sequence downstream, in which said promoter can bind RNA polymerase and initiate transcription of the encoded open reading frame in a cell, thereby directing expression of said coding sequence in the 3 'direction. The promoter sequence of the present invention can be any type of promoter sequence known to those skilled in the art, including, but not limited to, constitutive promoters, inducible promoters, cell cycle specific promoters, and cell type specific promoters.

Another aspect of the present invention relates to a cell comprising the mutant 3-HBDH of the present invention or the nucleic acid of the present invention. In one embodiment of the present invention, a cell comprising the mutant enzyme is used in the context of the present invention. The cell is preferably a host cell. A "host cell" of the present invention can be any type of organism suitable for application in recombinant DNA technology, and includes, but is not limited to, all types of bacterial and yeast strains that are suitable for expressing one or more recombinant proteins. Examples of host cells include, for example, various strains of Bacillus subtilis or E. coli. A variety of E. coli bacterial host cells is known to one of skill in the art and includes, but is not limited to, strains such as DH5-alpha, HB101, MV1190, JM109, JM101, or XL-1 blue that are commercially available from various vendors, including, for example, Stratagene (CA, USA), Promega (Wl, USA) or Qiagen (Hilden, Germany). A particularly suitable host cell is also described in the examples, namely E. coli XL-1 Blue cells . Bacillus subtilis strains that can be used as a host cell include, for example, wild type 1012: leuA8 metB5 trpC2 hsdRM1 and 168 Marburg: trpC2 (Trp-), which are commercially available from MoBiTec (Germany).

Host cell culture according to the invention is a routine procedure known to those skilled in the art. That is, a nucleic acid encoding a mutant 3-HBDH of the invention can be introduced into a suitable host cell or cells to produce the respective protein by recombinant means. These host cells can be any suitable cell type, preferably bacterial cells such as E. coli, which can be easily cultured. In a first step, this approach may include the cloning of the respective gene into a suitable plasmid vector. Plasmid vectors are widely used for gene cloning and can be easily introduced, ie transformed, into bacterial cells that have become competent. After the protein has been expressed in the respective host cell, the cells can be disrupted by means of chemical or mechanical cell lysis which is well known to the person skilled in the art and includes, but is not limited to, for example, solution treatment. hypotonic saline, detergent treatment, homogenization or ultrasound.

In another aspect, the present invention relates to a method for determining the amount or concentration of 3-hydroxybutyrate in a sample, the method comprising

a) contacting the sample with the mutant 3-HBDH of the present invention under conditions conducive to 3-HBDH activity;

b) reacting the 3-hydroxybutyrate with nicotinamide adenine dinucleotide (NAD) or a functionally active derivative thereof; Y

c) determining the change in the redox state of NAD or the derivative thereof, thus determining the amount or concentration of 3-hydroxybutyrate in the sample.

The above procedure is based on the fact that 3-HBDH can be used to catalyze the conversion of 3-HB to acetoacetate according to the following scheme:

(R) -3-hydroxybutyrate NAD + ^ acetoacetate NADH H +

In a first step of the process of the present invention, a sample is contacted with the 3-HBDH of the present invention. Contact of the sample with the mutant 3-HBDH can be direct (for example, in liquid assays) or indirect (for example, in sensor systems where only a fraction of the sample (containing the analyte) is in contact with 3-HBDH).

It is clear that the contact must be carried out under conditions conducive to the activity of 3-HBDH, that is to say, that allow the enzyme to convert the 3-HB into acetoacetate. The incubation step can range from about 5 seconds to several hours, preferably from about 10 seconds to about 10 minutes. However, the incubation time will depend on the assay format, the volume of the solution, the concentrations, and the like. Typically, the test will be carried out at room temperature or at the temperature required for other test formats carried out concomitantly (for example, 25 ° C to 38 ° C; such as 30 ° C or 37 ° C), although it can be performed in a range of temperatures, such as 10 ° C to 40 ° C.

Optionally, the enzyme can be attached to or immobilized on a support layer prior to contact with the sample to facilitate the assay. Examples of support layers include glass or plastic in the form of, for example, a microtiter plate, a glass slide or coverslip, a rod, a microsphere or a microbead, membranes (for example, used in test strips), and layers of biosensors.

The sample can be any sample suspected of containing 3-HB, in particular a sample from a subject. The term "sample from a subject" includes all biological fluids, excretions and tissues isolated from any given subject, particularly a human. In the context of the present invention, such samples include, but are not limited to, samples of blood, blood serum, blood plasma, nipple aspirate, urine, semen, seminal fluid, seminal plasma, prostatic fluid, excretions, tears, saliva , sweat, biopsy specimen, ascites fluid, cerebrospinal fluid, milk, lymph, samples of bronchial lavage and other washes, or samples of a tissue extract. Preferably the subject is an animal (including a human), more preferably a mammal, still more preferably a human. Preferably, the sample is a body fluid, in particular a blood sample or a urine sample.

Typically, blood samples are preferred test samples for use in the context of the present invention. For this, blood can be drawn from a vein, usually from the inside of the elbow or the back of the hand or fingertip. In particular, in infants or young children, a sharp tool called a scalpel can be used to pierce the skin and cause it to bleed. Blood can be collected, for example, into a pipette or cannula, or onto a slide or test strip.

After contact and conversion of 3-HB, if present, the change in the redox state of NAD or derivative mediated by 3-HBDH is determined, thereby determining 3-HB in the sample. Obviously, the amount of NADH or derivative thereof produced and the amount of NAD or derivative thereof consumed correlate with the amount of 3-HB present in the sample. Consequently, the change of the NAD redox state includes determining the amount or concentration of NAD and / or NADH, as well as the ratio of the two. The same applies to derivatives of NAD.

A variety of procedures for determining NADH / NAD or derivative thereof are known in the art and any of them can be used.

Exemplary procedures for determining NADH / NAD or derivative thereof include electrochemical procedures (for example, as described in US 6,541,216) or optical procedures (for example, measuring the conversion of NAD / NADH by absorbance of light to eg 340 nm or 365 nm or by assays based on a reductase to form luciferin, which is then optically quantified). If electrochemical procedures are used, the NADH / NAD or derivative thereof can a) react directly on a measuring electrode or b) the NADH / NAD or derivative thereof reacts in a first step with an additional oxidoreductive mediating substance that changes its state of redox in a defined relationship with the redox state of the NADH / NAD or derivative thereof and this redox mediator reacts at a later stage at the measurement electrode.

Both NAD and NADP are base-labile molecules whose degradation pathways are described in the literature (see, for example, NJ Oppenheimer in The Pyridine Nucleotide Coenzymes Academic Press, New York, London 1982, J. Everese, B. Anderson , K. You, authors of the edition, chapter 3, pages 56-65). Therefore, NADH / NAD derivatives have been developed and are commercially available. Some derivatives are described in The Pyridine Nucleotide Coenzymes, Academic Press, New York, London 1982, editors J. Everese, B. Anderson, K. You, chapter 4, WO 01/94370, WO 98/33936 and US 5,801,006.

Preferably, carba-NAD (cNAD) is used as a derivative of NAD. In carba-NAD, ribose is replaced by a carbacyclic sugar unit. Carba-NAD has the following structure (I):

The compound, its production and use are described in detail in WO 2007/012494, WO 2011/012270 and WO 2014/195363. The cofactor in the present invention is preferably carba-NAD. In an embodiment of the present invention, the cofactor is a functionally active derivative of NAD as disclosed in formula III of WO 2011/012270 to which reference is explicitly made. In one embodiment of the present invention, NADP is used instead of NAD.

The method of the present invention can be carried out in a so-called liquid or wet test, for example, in a cuvette, or as a so-called dry test in an appropriate reagent holder, thus the necessary test reagents being present in or on a solid support, which is preferably an absorbent or swellable material.

Alternatively or additionally, the 3-HBDH can be part of a sensor, a test strip, a test element, a test strip device, or a liquid test.

A sensor is an entity that measures a physical / chemical quantity and converts it into a signal that can be read by an observer or an instrument. In the present invention, 3-HBDH can be part of a sensor. The sensor converts 3-HB and NAD or a derivative thereof into acetoacetate and NADH or a derivative thereof, which is then converted into a signal such as a color change or a value displayed, for example, on a screen or monitor.

In one embodiment, the sensor may comprise 3-HBDH and an amperometric device for determining 3-HB from a sample. In addition, a microdialysis system coupled to an electrochemical reading cuvette could be used for continuous monitoring of 3-HB in a sample or subject. The working electrode of the reading cell could be prepared with the 3-HBDH immobilized in a film of oxidoreductive polymer on the surface of the electrode. Coupling an electrochemical sensor with 3-HB directly with microdialysis eliminates the need to transfer sample aliquots to a liquid chromatography system with a post-column oxidase enzyme reactor. The 3-HB in the dialysate of the microdialysis probe can be selectively detected on the enzyme electrode without any significant interference from other oxidizable species. Furthermore, enzyme-coupled biosensors have been described in the art. Accordingly, 3-HBDH can be coupled to a surface (for example, by printing a mixture of 3-HBDH and graphite on electrodeposited graphite pads or by adsorption or immobilization of the mutant 3-HBDH on carbon particles, particles carbon dioxide, carbon / manganese dioxide particles, glassy carbon or by mixing it with carbon paste electrodes, etc.)

A test strip or test item is an analytical or diagnostic device used to determine the presence and / or amount of a target substance within a sample. A standard test strip can comprise one or more different reaction zones or pads comprising reagents that react (eg, change color) when contacted with a sample. Test strips are known in many embodiments, for example, from US 6,541,216, EP 262445 and US 4816224. It is commonly known that one or more reagents (eg, enzymes) necessary to carry out testing procedures determination are present on or in layers of solid support. As backing layers, there are particularly preferred absorbent and / or swellable materials that are moistened with the sample liquid to be analyzed. Examples include layers of gelatin, cellulose, and synthetic wool (fleece).

The 3-HBDH of the present invention can also be part of a liquid test. A liquid test is a test in which the test components react in a liquid medium. Typically, in the field of laboratory analysis, liquid reagents are water-based, for example a buffered saline solution to provide the activity of the enzyme (s) involved. The liquid is normally adapted to the specific intended use. To carry out a liquid test, all the components of the test are dissolved in a liquid and combined (or vice versa). Typical containers for conducting such tests include vials, multiwell plates, cuvettes, containers, reagent cups, tubes, etc.

In one embodiment of the present invention, the 3-HBDH of the present invention can be immobilized. Typical immobilization procedures include covalent attachment, for example, to a membrane, encapsulation in a polymer, cross-linking to a support matrix, or immobilization in a sol-gel matrix (for example, glasses such as silicate glasses ) or adsorption on porous substrates. Suitable procedures for immobilizing enzymes are known in the art (see, for example, Lillis et al., 2000, Sensors and Actuators B 68: 109-114).

In a preferred embodiment of the present invention, the method further comprises determining the amount or concentration of glucose. In another embodiment of the present invention, the method comprises determining the amount or concentration of acetone and / or acetoacetate; and / or determining the amount or concentration of glucose. The determination of these compounds is of particular relevance in the diagnosis of the foregoing diseases and medical conditions. Procedures for determining these compounds are well known in the art. Additionally, systems and procedures for the analysis of multiple analytes are known from WO 2014/068024.

Consequently and preferably, the process of the present invention is further characterized in that

a) wherein the determination of the change in the redox state of NAD or the derivative thereof includes the determination of the concentration of (i) NAD or the derivative thereof and / or (ii) NADH or the derivative thereof; I

b) wherein the determination of the change in the redox state of NAD or the derivative thereof is electrochemical or optical; I

c) wherein the method further comprises determining the amount or concentration of acetoacetate and / or acetone; I

d) wherein the method further comprises determining the amount or concentration of glucose; I

e) wherein the derivative of NAD is carba-NAD; I

f) wherein the mutant 3-HBDH is part of a sensor, test strip, test item, test strip device, or liquid test; I

g) in which the sample is a body fluid, in particular a blood sample or a urine sample.

In yet another aspect, the present invention relates to the use of the mutant 3-HBDH of the present invention to determine the amount or concentration of 3-hydroxybutyrate in a sample.

With respect to the use of the present invention, it refers to specific terms, examples, and embodiments used in the context of the other aspects of the present disclosure, which are also applicable to this aspect. In particular, the mutant 3-HBDH according to the present invention can be used as detailed with respect to the methods of the present invention.

In still another aspect, the present invention relates to a device for determining the amount or concentration of 3-hydroxybutyrate in a sample comprising the mutant 3-HBDH of the present invention and optionally an additional component required for said determination.

With respect to the device of the present invention, reference is made to specific terms, examples, and embodiments used in the context of the other aspects of the present disclosure, which are also applicable to this aspect. In particular, the mutant 3-HBDH according to the present invention can be used as detailed above.

The 3-HBDH of the present invention can be part of a device for determining 3-HB in a sample. The device can be any device suitable for this purpose. The device can be a machine or tool that can be used to determine 3-HB. Preferably the device is a sensor, preferably an electrochemical sensor or a test strip. Exemplary devices are described above and below:

The device can be a sensor, for example a biosensor, which is an analytical device for the detection of an analyte that combines a biological component (here, 3-HBDH according to the present invention) with a detector component, in particular a physicochemical sensing component.

Biosensors are particularly useful for determining the concentration of various analytes (including 3-HB) in biological samples, particularly blood. Exemplary biosensors based on an electrochemical test strip format are described in US Patent Nos. 5,413,690; 5,762,770 and 5,997,817.

In the (bio) sensor of the present invention, the 3-HB converted to acetoacetate in the presence of the 3-HBDH and NAD or derivative and the change of the redox state of NAD or derivative can be monitored by the transducer or detector element.

In particular, 3-HB sensors have been combined with other sensors, for example to determine glucose, acetone, acetoacetate, cholesterol, triglycerides, urea, blood gases or electrolytes, etc. Obviously, the mutant 3-HBDH of the present invention could also be used in these multi-analyte devices.

As detailed above, the sensor is preferably an electrochemical or optical sensor. An electrochemical sensor is based on the translation of a chemical signal (here, the presence of 3-HB) into an electrical signal (for example, current). A suitable electrode can measure the 3-HB mediated production of NADH or derivative thereof as an electrical signal.

A suitable optical sensor can measure the 3-HBDH-mediated change in the redox state of NAD or derivative thereof. The signal may be NAD / NADH mediated absorbance / light emission.

The device of the present invention may comprise, in addition to the 3-HBDH of the present invention, one or more additional components, such as other reagents, required for or useful in said determination. The components can be any of those described in the context of the methods and devices of the present invention. Additionally, this may include an instruction manual, a scalpel device, a capillary pipette, an additional enzyme, a substrate and / or a control solution, etc.

Preferably, the device of the present invention is characterized in that the device is or comprises a sensor, preferably an electrochemical sensor or an optical sensor, or a test strip, in particular a test strip and / or allows the amount or concentration of glucose to be determined. in the sample. With respect to the device of the present invention, reference is also made to the specific terms, examples and embodiments described above.

Unless defined otherwise, all technical and scientific terms and any acronyms used herein document have the same meaning commonly understood by one skilled in the art in the field of the invention. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). The invention is not limited to the particular methodology, protocols, and reagents described herein because they may vary. Although any methods and materials similar or equivalent to those described herein may be used in the practice of the present invention, preferred methods and materials are described herein. Furthermore, the terminology used herein is for the purpose of describing only particular embodiments and is not intended to limit the scope of the present invention.

As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly indicates otherwise. Similarly, the words "comprise", "contain" and "encompass" should be interpreted inclusively rather than exclusively. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "plurality" refers to two or more.

The following figures and examples are intended to illustrate various embodiments of the invention.

FIGURES

Figure 1 shows the amino acid sequence and domains of the natural 3-HBDH of Alcaligenes faecalis. EXAMPLES

Example 1 Establishment of a collection of mutant 3-HBDH enzymes

The gene for 3-hydroxybutyrate dehydrogenase (3-HBDH) from Alcaligenes faecalis (UniProtKB database - D0VWQ0) was synthesized, cloned into the vector pKKt5, and transformed into E. coli strain XL-1 Blue by known procedures. of molecular biology.

Saturation mutagenesis was applied at many amino acid positions of the enzyme. Mutagenesis was achieved by applying randomly synthesized primers with the QuikChange site directed mutagenesis kit (Agilent Technologies Cat. 200518).

The 5 'primer and the 3' primer used for mutagenesis were complementary to each other and contained NNN (randomly synthesized nucleotides) for amino acid exchange at a central position. This randomly created codon was flanked by 12 to 16 nucleotides at each end. The sequences of these nucleotides were identical to the cDNA strand or the complementary cDNA strand flanking the codon for amino substitution. The collection of mutant enzymes was created by transforming mutated genes in E. coli strain XI-Blue and culturing on agar plates overnight at 37 ° C.

Example 2 Determination of the properties of the mutant 3-HBDH enzymes of the first round of mutagenesis (mutant enzymes with individual amino acid substitutions)

A collection of 3-HBDH mutant enzymes produced as described in Example 1 was examined for the following enzymatic properties:

- Thermal stability

- Affinity for 3-hydroxybutyrate

- Affinity for c-NAD (carba-NAD = artificial cofactor, see document US20120130062A1)

Mutant colonies on the agar plates described above were picked into microtiter plates (pmv) containing 200 µl of LB-ampicillin medium / well and incubated at 37 ° C overnight. These plates were called foundation plates. For each amino acid position, two critical plaques were selected to ensure that all possible exchanges were included.

From each core plate 40 µl sample / well was transferred to a pmv containing 200 µl 0.1% Triton X-100; 500 mM NaCl; 200 mM Hepes at pH 9.0; 2% B-Per / well (B-Per = Pierce Bacterial Protein Extraction Reagent # 78248) and incubated for cell disruption at 40 ° C for 30 minutes. This plate was called the work plate.

4 x 20 µl sample / well were transferred from the working plate to four empty pmv. One of these underwent 62.22 mM 3-hydroxybutyrate test; 4.15 mM cNAD; 0.1% Triton X-100; 200 mM Hepes, pH 9.0 at room temperature and is called a reference measurement. The other pmv were tested under different conditions and the values obtained were compared with the reference plate in percentage.

The following parameters were measured:

- Thermal stability: The pmv was incubated for 30 min at 64 ° C and subsequently tested with 62.22 mM 3-hydroxybutyrate; 4.15 mM cNAD; 0.1% Triton X-100; 200 mM Hepes at pH 9.0

- Affinity for 3-hydroxybutyrate: Activity test with a reduced amount of substrate (ie below substrate saturation). Measurement with 3-hydroxybutyrate 1.94 mM; 4.15 mM cNAD; 0.1% Triton X-100; 200 mM Hepes at pH 9.0

- Affinity for cNAD: Assay for activity with a reduced amount of cofactor (ie below saturation). Measurement with 3-hydroxybutyrate 62.22 mM; 0.032 mM cNAD; 0.1% Triton X-100; 200 mM Hepes at pH 9.0

The enzymatic reaction was monitored at room temperature at 340 nm for 5 minutes and the dE / min was calculated for each working plate. The baseline measurement value was established as 100% activity. The values obtained with the other three plates (thermal stability, affinity for 3-hydroxybutyrate or for cNAD) were compared with the reference and the percentage of activity was calculated ((dE / min Parameter / dE / min Reference) * 100). Each fundamental plate contained, in addition to the mutant enzymes, the natural enzyme as a control to better estimate the improvements or deterioration of the properties.

Thermal stability expressed as remaining activity was calculated as follows:

dE / min sample in cond. extreme (i.e. in the example 2:30 min. 64 ° C)

* 100 remaining activity in percentage dE / min sample in cond. not extreme

A value obtained with a mutant enzyme that is higher than the value obtained with the natural enzyme represents an increase in the thermal stability of the mutant enzyme.

The affinity for the substrate expressed as activity ratio was calculated as follows:

dE / min obtained with less substrate

* 100 = activity in percentage

.dE / min obtained with saturated substrate

A mutant enzyme with higher affinity for the substrate will show higher activity when reacting with less substrate (below substrate saturation) than a mutant enzyme with lower affinity for the substrate. A value obtained with a mutant enzyme that is greater than the value obtained with the natural enzyme represents an increase in the affinity of the substrate for the mutant enzyme.

The affinity for the cofactor expressed as a proportion of activity was calculated accordingly:

dE / min obtained with less cofactor

* 100 = activity in percentage

dE / min obtained with cofactor in saturation ^

Data below 0.001 dE / min were set to zero, resulting in "zero" values, as in Tables 1B and 1C.

The results for the natural enzyme are summarized in Tables 1A, 1B and 1C.

Table 1A: Thermal Stability and Affinity of Various Individual Mutant Enzymes Relative to Natural 3-HBDH Named AFDH1

(+ = improved; or = similar; - = decreased)

Table 1B: Thermal Stability and Affinity of Various Individual 3-HBDH Mutant Enzymes

* given as activity ratio (%) with 3-HB 1.94 / 62.22 mM and 0.032 / 4.15 mM cNAD

** given as remaining activity (%) after 30 min incubation at 64 ° C

Table 1C: Various individual mutant enzymes with highly improved thermal stability relative to natural 3-HBDH

* given as activity ratio (%) with 3-HB 1.94 / 62.22 mM and 0.032 / 4.15 mM

* given as remaining activity (%) after 30 min incubation at 64 ° C

An exemplary mutant enzyme with L253I exchange was chosen for further optimization by combining additional found positions.

Example 3 Screening for mutant 3-HBDH enzymes from the second round of mutagenesis (mutant enzymes with L253I amino acid substitution and optionally one or more additional amino acid substitutions) Unless otherwise indicated, experiments have been carried out as is detailed in Example 2. In a second round of mutagenesis, selected mutations were introduced into the AFDH1 L253I variant. The results are summarized in Table 2A:

Table 2A: Thermal stability (68 ° C, 30 min) and affinity of various mutant enzymes with amino acid substitution L253I

* given as activity ratio (%) with 3-HB 1.94 / 62.22 mM and 0.032 / 4.15 mM cNAD

* given as remaining activity (%) after 30 min incubation at 68 ° C

The exemplary variant AFDH1 L253I G233K A234T was further combined with other found positions. The results are summarized in Table 2B:

Table 2B: Thermal stability (75 ° C, 30 min) and affinity of various mutant enzymes with L253I, G233K and A234T amino acid substitutions

* given as activity ratio (%) with 3-HB 1.94 / 62.22 mM

* given as remaining activity (%) after 30 min incubation at 75 ° C

The selected mutant enzymes were chosen and subjected to extreme conditions at 80 ° C for 30 minutes. The results are summarized in Table 2C:

Table 2C: Thermal stability (80 ° C, 30 min) and affinity of selected mutant enzymes

* given as activity ratio (%) with 3-HB 1.94 / 62.22 mM

** given as remaining activity (%) after 30 min incubation at 80 ° C

Example 4 Screening for mutant 3-HBDH enzymes with amino acid substitutions A62V + A143V + A148G + G233K + A234T + L253I and optionally one or more additional amino acid substitutions)

The mutant enzymes selected from Example 3 were measured at pH 7.5 and the best variant was selected for further mutagenesis.

Unless otherwise indicated, the experiments have been carried out as detailed in Example 2. The mutant enzymes found were selected analogously to Example 3 but with different solutions at pH 7.5. The reference pmv was tested with 150 mM 3-hydroxybutyrate; 5 mM cNAD; 0.1% Triton X-100; Mops 70 mM at pH 7.5.

The following parameters were measured:

- The pmv was incubated for thermal stability for 30 min at 60-90 ° C (depending on the stability of the enzyme variant) and subsequently tested with 150 mM 3-hydroxybutyrate; 5 mM cNAD; 0.1% Triton X-100; Mops 70 mM at pH 7.5

- The pmv for 13-hydroxybutyrate affinity was measured with 5 mM 3-hydroxybutyrate; 5 mM cNAD; 0.1% Triton X-100; Mops 70 mM at pH 7.5

- The pmv for cNAD affinity was measured with 150 mM 3-hydroxybutyrate; 0.5 mM cNAD; 0.1% Triton X-100; Mops 70 mM at pH 7.5

Table 2D: Thermal stability (various temperatures, 30 min) and affinity of selected mutant enzymes

* given as activity ratio (%) with 5/150 mM 3-HB and 0.5 / 5 mM cNAD

** given as remaining activity (%) after a 30 minute incubation at the indicated temperature

AFDH1: natural

AFDH2: AFDH1 L253I

AFDH3: AFDH1 G233K + A234T + L253I

AFDH4: AFDH1 A62V + G233K + A234T + L253I

AFDH4 147R: AFDH1 A62V + V147R + G233K + A234T + L253I

AFDH5: AFDH1 P39G + A62R + A143V + A148G + G233K + A234T + L253I

AFDH6: AFDH1 A62V + A143V + A148G + G233K + A234T + L253I

AFDHx: AFDH1 A62V + E111L + I140M A148G

Based on mutant AFDH6 (i.e., AFDH1 plus A62V A143V A148G G233K A234T L253I mutations) additional rounds of mutagenesis were performed with the following mutant enzymes listed in Table 3

Table 3: Thermal stability (87 ° C and 90 ° C, 30 min) and affinity of selected mutant enzymes

* given as activity ratio (%) with 5/150 mM 3-HB and 0.5 / 5 mM cNAD

** given as remaining activity (%) after a 30 minute incubation at the indicated temperature

Note: The values in () are AFDH6 data measured in the same series of tests as the control.

Table 4: Amino Acid Substitutions Found to Improve the Yield of 3-HBDH, Particularly When Combined with AFDH6 (i.e. AFDH1 Plus A62V A143V A148G G233K A234T L253I Mutations)

SEQUENCES

AFDH1: natural 3-HBDH from Alcaligenes faecalis (SEQ ID NO: 1)

MLKGKKAWT GSTSGIGLAM ATELAKAGAD WINGFGQPE DIERERSTLE SKFGVKAYYL 60 NADLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH 120 GTAAALPIMQ KQGWGRIINI ASAHGLVASV NKSAYVAAKH GWGLTKVTA LENAGKGITC 180 NAICPGWVRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGGAAVFLSS 240 260 AAADQMTGTT LSLDGGWTAR

Extended central sequence of A FDH1 (SEQ ID NO: 2)

ADLSDAQATR DFIAKAAEAL GGLDILVNNA GIQHTAPIEE FPVDKWNAII ALNLSAVFHG 60 TAAALPIMQK QGWGRIINIA SAHGLVASVN KSAYVAAKHG WGLTKVTAL ENAGKGITCN 120AICPGWVRTP SLQEAPKGWVRTP SLQEAPGWQIVRTP SLQEAPKGWVRTP SLQLAVE QLAVGWVRTP SLQLAVE QGWQITQLEV120

AFDH2: AFDH1 L253I (SEQ ID NO: 3)

DIERERSTLE SKFGVKAYYL60 NADLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH120 GTAAALPIMQ KQGWGRIINI ASAHGLVASV NKSAYVAAKH

LENAGKGITC180 NAICPGWVRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGGAAVFLSS240 AAADQMTGTT LSIDGGWTAR 260 MLKGKKAWT GSTSGIGLAM ATELAKAGAD

DIERERSTLE SKFGVKAYYL60 NADLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH120 GTAAALPIMQ KQGWGRIINI ASAHGLVASV NKSAYVAAKH

LENAGKGITC180 NAICPGWVRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGGAAVFLSS240 AAADQMTGTT LSIDGGWTAR 260

AFDH3: AFDH1 G233K + A234T + L253I (SEQ ID NO: 4)

MLKGKKAWT GSTSGIGLAMATELAKAGAD WINGFGQPE DIERERSTLE SKFGVKAYYL 60 NADLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH 120 GTAAALPIMQ KQGWGRIINI ASAHGLVASV NKSAYVAAKH GWGLTKVTA LENAGKGITC 180 NAICPGWVRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGKTAVFLSS 240 260 AAADQMTGTT LSIDGGWTAR

AFDH4: AFDH1 A62V + G233K + A234T + L253I (SEQ ID NO: 5)

MLKGKKAWT GSTSGIGLAM ATELAKAGAD WINGFGQPE DIERERSTLE SKFGVKAYYL 60 NVDLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH 120 GTAAALPIMQ KQGWGRIINI ASAHGLVASV NKSAYVAAKH GWGLTKVTA LENAGKGITC 180 NAICPGWVRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGKTAVFLSS 240 260 AAADQMTGTT LSIDGGWTAR

AFDH4 147R: AFDH1 A62V + V147R G233K + A234T + L253I (SEQ ID NO: 6)

MLKGKKAWT GSTSGIGLAM ATELAKAGAD WINGFGQPE DIERERSTLE SKFGVKAYYL 60 NVDLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH 120 GTAAALPIMQ KQGWGRIINI ASAHGLRASV NKSAYVAAKH GWGLTKVTA LENAGKGITC 180 NAICPGWVRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGKTAVFLSS 240 260 AAADQMTGTT LSIDGGWTAR

AFDH5: AFDH1 P39G + A62R + A143V + A148G + G233K + A234T + L253I (SEQ ID NO: 7)

MLKGKKAWT GSTSGIGLAM ATELAKAGAD WINGFGQGE DIERERSTLE SKFGVKAYYL 60 NRDLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH 120 GTAAALPIMQ KQGWGRIINI ASVHGLVGSV NKSAYVAAKH GWGLTKVTA LENAGKGITC 180 NAICPGWVRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGKTAVFLSS 240 260 AAADQMTGTT LSIDGGWTAR

AFDH6: AFDH1 A62V + A143V + A148G + G233K + A234T + L253I (SEQ ID NO: 8)

MLKGKKAWT GSTSGIGLAM ATELAKAGAD WINGFGQPE DIERERSTLE SKFGVKAYYL 60 NVDLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH 120 GTAAALPIMQ KQGWGRIINI ASVHGLVGSV NKSAYVAAKH GWGLTKVTA LENAGKGITC 180 NAICPGWVRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGKTAVFLSS 240 260 AAADQMTGTT LSIDGGWTAR

AFDH A: AFDH6 33L + 125G + 175V + 187F (SEQ ID NO: 9) MLKGKKAWT GSTSGIGLAM ATELAKAGAD WLNGFGQPE DIERERSTLE SKFGVKAYYL 60

NVDLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH 120

GTAAGLPIMQ KQGWGRIINI ASVHGLVGSV NKSAYVAAKH GWGLTKVTA LENAVKGITC 180

NAICPGFVRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGKTAVFLSS 240

AAADQMTGTT LSIDGGWTAR 260

AFDH B: AFDH6 125G + 175T + 187F (SEQ ID NO: 10)

MLKGKKAWT GSTSGIGLAM ATELAKAGAD WINGFGQPE DIERERSTLE SKFGVKAYYL 60

NVDLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH 120

GTAAGLPIMQ KQGWGRIINI ASVHGLVGSV NKSAYVAAKH GWGLTKVTA LENATKGITC 180

NAICPGFVRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGKTAVFLSS 240

AAADQMTGTT LSIDGGWTAR 260

AFDH C: AFDH6 170G + 175T (SEQ ID NO: 11)

MLKGKKAWT GSTSGIGLAM ATELAKAGAD WINGFGQPE DIERERSTLE SKFGVKAYYL 60

NVDLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH 120

GTAAALPIMQ KQGWGRIINI ASVHGLVGSV NKSAYVAAKH GWGLTKVTG LENATKGITC 180

NAICPGWVRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGKTAVFLSS 240

AAADQMTGTT LSIDGGWTAR 260

AFDH D: AFDH6 18E + 125G + 187F (SEQ ID NO: 12)

MLKGKKAWT GSTSGIGEAM ATELAKAGAD WINGFGQPE DIERERSTLE SKFGVKAYYL 60

NVDLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH 120

GTAAGLPIMQ KQGWGRIINI ASVHGLVGSV NKSAYVAAKHGWGLTKVTA LENAGKGITC 180

NAICPGFVRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGKTAVFLSS 240

AAADQMTGTT LSIDGGWTAR 260

AFDH E: AFDH6 33L + 125G + 175I + 187F (SEQ ID NO: 13)

MLKGKKAWT GSTSGIGLAM ATELAKAGAD WLNGFGQPE DIERERSTLE SKFGVKAYYL 60

NVDLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIEEFPVDKWNAI IALNLSAVFH 120

GTAAGLPIMQ KQGWGRIINI ASVHGLVGSV NKSAYVAAKH GWGLTKVTA LENAIKGITC 180

NAICPGFVRT PLVEKQIEAISQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGKTAVFLSS 240

AAADQMTGTT LSIDGGWTAR 260

AFDH F: AFDH6 33L + 125G + 170G + 187F (SEQ ID NO: 14)

MLKGKKAWT GSTSGIGLAM ATELAKAGAD WLNGFGQPE DIERERSTLE SKFGVKAYYL 60

NVDLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH 120

GTAAGLPIMQ KQGWGRIINI ASVHGLVGSV NKSAYVAAKH GWGLTKVTG LENAGKGITC 180

NAICPGFVRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGKTAVFLSS 240

AAADQMTGTT LSIDGGWTAR 260

AFDH G: AFDH6 33L + 125G + 175V + 187F + 216V (SEQ ID NO: 15)

MLKGKKAWT GSTSGIGLAM ATELAKAGAD WLNGFGQPE DIERERSTLE SKFGVKAYYL 60

NVDLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH 120

GTAAGLPIMQ KQGWGRIINI ASVHGLVGSV NKSAYVAAKH GWGLTKVTA LENAVKGITC 180

NAICPGFVRT PLVEKQIEAI SQQKGIDIEA AARELVAEKQ PSLQFVTPEQ LGKTAVFLSS 240

AAADQMTGTT LSIDGGWTAR 260

AFDH H: AFDH6 33I + 38K + 39G + 125G + 175V + 187F (SEQ ID NO: 16)

MLKGKKAWT GSTSGIGLAM ATELAKAGAD WLNGFGKGE DIERERSTLE SKFGVKAYYL 60

NVDLSDAQAT RDFIAKAAEA LGGLDILVNN AGIQHTAPIE EFPVDKWNAI IALNLSAVFH 120

GTAAGLPIMQ KQGWGRIINI ASVHGLVGSV NKSAYVAAKH GWGLTKVTA LENAVKGITC 180

NAICPGFVRT PLVEKQIEAI SQQKGIDIEA AARELLAEKQ PSLQFVTPEQ LGKTAVFLSS 240

AAADQMTGTT LSIDGGWTAR 260

Claims (15)

1. A mutant 3-hydroxybutyrate dehydrogenase (3-HBDH) from Alcaligenes faecalis with improved performance relative to wild-type 3-HBDH, wherein the mutant enzyme comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 ( Alcaligenes faecalis 3-HBDH; wild-type 3-HBDh) or the amino acid sequence of SEQ ID NO: 2 (wild-type 3-HBDH extended core sequence) and wherein the mutant enzyme has at least three amino acid substitutions relative to natural 3-HBDH, wherein the amino acid at the position corresponding to - position 253 of SEQ ID NO: 1 is replaced with Ile (253Ile), Ala (253Ala) or Cys (253Cys), - position 233 of SEQ ID NO: 1 is replaced with Ser (233Ser), Ile (233Ile), Ala (233Ala), Pro (233Pro), Arg (233Arg), Thr (233Thr), Lys (233Lys) or Cys (233Cys), and - position 234 of SEQ ID NO: 1 is replaced with Thr (234Thr). 2. The mutant 3-HBDH of claim 1, wherein the amino acid at the position corresponding to position 253 of SEQ ID NO: 1 is substituted with Ile (253Ile) and / or in which the amino acid in the position corresponding to position 233 of SEQ ID NO: 1 is substituted with Lys (233Lys). 3. The mutant 3-HBDH of claim 1 or 2, wherein the mutant enzyme has at least one additional amino acid substitution at one or more of the positions corresponding to positions 18, 19, 33, 38, 39, 62 , 125, 143, 148, 170, 175, 187 and / or 216 of SEQ ID NO: 1, in particular at least positions 62, 143 and / or 148 of SEQ ID NO: 1. 4. The mutant 3-HBDH of any of claims 1 to 3, wherein the amino acid at the position corresponding to - position 18 of SEQ ID NO: 1 is replaced with Arg (18Arg), Lys (18Lys), Glu (18Glu) or Pro (18Pro); - position 19 of SEQ ID NO: 1 is replaced with Gly (19Gly); - position 33 of SEQ ID

substitute Ala (33Ala), Leu (33Leu) or Thr (33Thr); - position 38 of SEQ ID

replace with Lys (38 Lys); - position 39 of SEQ ID

replace with Gly (39Gly); - position 62 of SEQ ID NO: 1 is replaced with Phe (62Phe), Met (62Met),

(62Lys), Arg (62Arg), Leu (62Leu) or (62Val), especially Val (62Val); - position 125 of SEQ ID NO: 1 is replaced with Gly (125Gly); - position 143 of SEQ ID NO: 1 is replaced with Val (143Val); - position 148 of SEQ ID NO: 1 is replaced with Gly (148Gly); - position 170 of SEQ ID NO: 1 is replaced with Gly (170Gly); - position 175 of SEQ ID NO: 1 is replaced with Thr (175Thr), Val (175Val) or Ile (175Ile); - position 187 of SEQ ID NO: 1 is replaced with Phe (187Phe); I - position 216 of SEQ ID NO: 1 is replaced with Val (216Val). 5. The mutant 3-HBDH of any of claims 1 to 4, wherein the mutant 3-HBDH has at least the mutations, corresponding to the positions of SEQ ID NO: 1 - 253Ile, 233Lys, 234Thr; - 253Ile, 62Val, 233Lys, 234Thr; - 253Ile, 62Val, 147Arg, 233Lys, 234Thr; - 253Ile, 39Gly, 62Val, 143Val, 148Gly, 233Lys, 234Thr; or - 253Ile, 62Val, 143Val, 148Gly, 233Lys, 234Thr, in particular where the mutant 3-HBDH has at least the mutations 253Ile, 62Val, 143Val, 148Gly, 233Lys, 234Thr, optionally in combination with - 19Gly; - 170Gly; - 125Gly, 187Phe; - 18Glu, 33Leu; - 33Leu, 125Gly, 187Phe; - 33Leu, 125Gly, 187Phe, 216Val; - 18Glu, 187Phe; - 33Leu, 125Gly, 175Val, 187Phe; - 175Val, 216Val; - 175Val, 187Phe, 216Val; - 19Gly, 125Gly, 187Phe; - 125Gly, 175Thr, 187Phe; - 19Gly, 33Leu; - 19Gly, 175Ile; - 19Gly, 175Thr; - 170Gly, 175Thr; - 18Glu, 125Gly, 187Phe; - 33Leu, 125Gly, 175Thr, 187Phe; - 33Leu, 125Gly, 175Ile, 187Phe; - 33Leu, 125Gly, 170Gly, 187Phe; - 18Glu, 175Ile, 187Phe; - 33Leu, 125Gly, 175Val, 187Phe; - 33Leu, 125Gly, 175Val, 187Phe, 216Val; - 33Leu, 125Gly, 175Thr, 187Phe; or - 33Leu, 38Lys, 39Gly, 125Gly, 175Val, 187Phe. 6. The mutant 3-HBDH of any of claims 1 to 5, wherein the mutant 3-HBDH comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97 %, 98% or 99% identical to the amino acid sequence of any of SEQ ID NO: 1 to 16, in particular wherein the mutant 3-HBDH comprises or consists of the sequence selected from the group consisting of SEQ ID NO: 4 to 16. 7. The mutant 3-HBDH of any of claims 1 to 6, wherein the improved performance relative to wild-type 3-HBDH is - increased stability, especially thermal stability, relative to natural 3-HBDH; I - an increased affinity for substrate, especially for 3-hydroxybutyrate, relative to natural 3-HBDH; and / or - an increased affinity for cofactor, especially for NAD or a derivative thereof, in particular where the derivative is carba-NAD, relative to natural 3-HBDH. 8. The mutant 3-HBDH of claim 7, characterized in that the mutant 3-HBDH has a stability increased by at least a factor 2 relative to the natural 3-HBDH, preferably a stability increased by at least a factor 3, preferably a stability increased by at least a factor 4, plus preferably a stability increased by at least a factor 5; and / or characterized in that the mutant 3-HBDH has an increased affinity for the substrate and / or cofactor relative to natural 3-HBDH, in particular an increased affinity for (i) 3-hydroxybutyrate and / or (ii) dinucleotide nicotinamide adenine (NAD) or a functionally active derivative thereof and / or in particular in which the affinity for the substrate and / or cofactor is increased by at least 5%, more in particular by at least 10%, even more particularly by at least 15% or 20%. 9. A nucleic acid encoding the mutant 3-HBDH of any of claims 1 to 8. 10. A cell comprising the mutant 3-HBDH of any of claims 1 to 8 or the nucleic acid of claim 9. 11. A method of determining the amount or concentration of 3-hydroxybutyrate in a sample, the method comprising a) contacting the sample with the mutant 3-HBDH of any of claims 1 to 8 under conditions conducive to the activity of 3-HBDH; b) reacting the 3-hydroxybutyrate with nicotinamide adenine dinucleotide (NAD) or a functionally active derivative thereof; Y c) determining the change in the redox state of NAD or the derivative thereof; thereby determining the amount or concentration of 3-hydroxybutyrate in the sample. 12. The method of claim 11, a) wherein the determination of the change in the redox state of NAD or the derivative thereof includes the determination of the concentration of (i) NAD or the derivative thereof and / or (ii) NADH or the derivative thereof; and / or b) wherein the determination of the change in the redox state of NAD or the derivative thereof is electrochemical or optical; I c) wherein the method further comprises determining the amount or concentration of acetoacetate and / or acetone; I d) wherein the method further comprises determining the amount or concentration of glucose; I e) wherein the functionally active derivative of NAD is carba-NAD; I f) wherein the mutant 3-HBDH is part of a sensor, test strip, test item, test strip device, or liquid test; I g) in which the sample is a body fluid, in particular a blood sample or a urine sample. 13. Use of the mutant 3-HBDH of any one of claims 1 to 8 to determine the amount or concentration of 3-hydroxybutyrate in a sample. A device for determining the amount or concentration of 3-hydroxybutyrate in a sample comprising the mutant 3-HBDH according to any of claims 1 to 8 and optionally an additional component required for said determination. The device according to claim 14, wherein the device is characterized in that the device is or comprises a sensor, preferably an electrochemical sensor or an optical sensor, or a test strip, in particular a test strip and / or wherein the device further allows the amount or concentration of glucose in the sample to be determined.