Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1998-12-02
2001-06-05
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S090000, C435S106000, C435S128000, C435S189000, C435S440000
Reexamination Certificate
active
06242234
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to mutants of formate dehydrogenase from
Candida boidinii
(DSM 32195). The invention also relates to new gene sequences encoding these mutants and use of the formate dehydrogenases according to the invention in a process for preparing chiral compounds.
DISCUSSION OF THE PRIOR ART
To prepare L-amino acids, biocatalysts, inter alia, have been successfully used. One approach to the problem is to convert prochiral alpha-ketoacids by reductive amination. The amino acid dehydrogenases used for this purpose require stoichiometric amounts of NADH or NADPH as a coenzyme in order to convert the alpha-ketoacids. These coenzymes are very expensive and make the process mentioned above economically non-viable for use on an industrial scale.
One possibility of avoiding high costs due to the coenzyme comprises regenerating the coenzyme in situ. NAD-dependent formate dehydrogenase from the yeast
Candida boidinii
is currently used, inter alia, in the enzyme reactor for coenzyme regeneration on an industrial scale.
In situ regeneration of NADH with NAD-dependent formate dehydrogenase during the reductive amination of trimethyl pyruvate to give L-tert-leucine (Bommarius et al. Tetrahedron Asymmetry 1995, 6, 2851-2888).
A disadvantage of using FDH from
Candida boidinii
in a production process is the necessity of having to continue to add FDH during the process, since it becomes inactive as a result of lack of stability. This inactivation can be affected by a variety of factors:
pH
temperature
mechanical stress
ionic strength of and type of ion in the substrate solution
traces of heavy metals
oxidation of sulfhydryl groups by oxygen in the air
cross-linking due to thiol/disulfide exchange.
Tishkov et al. showed that targeted mutation of recombinant FDH from Pseudomonas sp. 101 could increase its stability towards mercury salts, whereas, however, the thermal stability was lowered by mutagenesis (Biochem, Biophys. Res. Commun. 1993, 192, 976-981).
Sakai et al. elucidated the gene sequence of FDH from the methylotrophic yeast
Candida boidinii
(J. Bacteriol. 1997, 179, 4480-4485). The protein sequence derived agreed 100% with the amino acid sequence of the basic recombinant FDH from
Candida boidinii
in this work.
SUMMARY OF THE INVENTION
In view of the prior art outlined and discussed above, it was also the object of the present invention to modify the FDH from
Candida boidinii
used in the industrial process in such a way that this has greater resistance to oxidation than recombinant FDH and the wild type and thus make costly and complicated post-addition of FDH during the process unnecessary.
Specifically, the invention is directed to stable mutants of rec-FDH from
Candida boidinii
having a higher level of stability to aggregation and oxidation than rec-FDH and the wild type enzyme. In these mutants one or more of the sulfur-containing amino acids in the rec-FDH are replaced by non sulfur containing amino acids. In particular those mutants wherein at least one of the cysteines at positions 23 and 262 is replaced by an amino acid selected from the group consisting of by serine, alanine or valine. The invention also includes the novel genes that encode these new mutants, in particular those genes, the DNA of which are set forth in sequences 1,3,5,7,9,11,13,15,17,19,21,23,25,27 and 29 hereof. The invention also includes the process of converting alpha keto acids into the corresponding chiral alpha amino acids in the presence of these mutants. Suitably this process is one wherein the conversion is carried out in the further presence of NAD
+
.H
2
O, preferably one wherein the conversion is carried out in the further presence of leucine dehydrogenase. Also included in the scope of the present invention is the process of preparing these mutant genes by means of targeted mutagenesis.
As a result of modifying the recombinant formate dehydrogenase from
Candida boidinii
by means of targeted mutagenesis, it has been possible in a very advantageous, and nevertheless surprising manner, to generate mutants which are not sensitive to aggregation and oxidation. These are unlike rec-FDH and the wild type enzyme, and thus to enable a longer working lifetime for this enzyme in a production process. Surprisingly, other advantageous properties of FDH, such as e.g. catalytic activity, conformational stability, thermal stability, etc. are only marginally affected so the new advantage is not negated by introducing different additional disadvantages. This could not have been predicted since, in such a complex molecule, even the smallest modification frequently leads to the complete loss of activity of the enzyme.
The recombinant formate dehydrogenase being considered is preferably modified in such a way that the sulfur-containing amino acids in the enzyme are replaced, independently, and separately or together, by amino acids which do not contain sulfur.
The cysteine units at positions 23 and 262 in FDH appear to be the particularly preferred targets of targeted mutation. Targeted mutagenesis may take place either at only one of these positions or at both. The sulfur-containing amino acids at positions 23 and/or 262 are advantageously replaced, independently, and separately or together, by amino acids without a sulfhydryl group. Replacement with serine, alanine or valine is particularly preferred.
The success of this modification, at the time when the invention was discovered, was neither predictable nor obvious, for the reasons given above.
The enzymes with improved stability encoded by the new gene sequences are preferably used in an enzymatic process for preparing chiral compounds, including the type mentioned at the beginning.
Enzymes with formate dehydrogenase activity according to the invention can advantageously be produced by means of targeted mutagenesis on the basis of the recombinant FDH gene and expressed in
E. coli.
Working with recombinant FDH offers the advantage that a standardised gene sequence and thus a standardised gene product is present, in which mutations can be produced. In order to be able effectively to compare the effects of the mutation in the mutants with the wild type enzyme, however, it is a critical advantage to be able to start from a standardised enzyme. There are probably several isoforms of the enzyme present in
Candida boidinii
itself and these are difficult to separate preparatively. In any case the wild enzyme exhibits microheterogeneities at the protein level.
In addition, all the advantages of
Escherichia coli
which are known to a person skilled in the art and relate to the parent organism, such as multiplication and expression, can be used.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The gene and amino acid sequences according to the invention can be prepared by biochemical and microbiological methods, which are known per se.
Thus, genomic DNA from
Candida boidinii
can be obtained by cultivation, lysis and precipitation using Ferbeyre et al.'s method (Bio Techniques 1993, 14. 386) The FDH gene can then be amplified by means of a polymerase chain reaction (PCR). The primers that were required were derived from protein sequence data. The FDH gene obtained was ligated in a cloning vector and transformed in
E. coli.
After isolating the recombinant plasmid DNA from the
E. coli
cells using a commercially available preparation kit (e.g. Qiagen Plasmid Tip 20), both DNA strands were sequenced. The sequence is shown in Sequences 31 and 32.
The recombinant plasmid DNA also acts as a template for PCR-promoted mutagenesis using Ho et al.'s method (Gene, 1989, 77, 52-59). The primers used contain the modified codon (in brackets) for replacement at the corresponding amino acid position: C23 (TGT bp 67-69) for S23 (TCT); C262 (TGT. bp 784-786) for V262 (GTT) or A262 (GCT). The amplified, mutated FDH genes were cloned in expression vector pBTac2 (Boehringer) (FIG. 2) and expressed in
E. coli.
The mutants were obtained from cells in the form of a crude cell-free extract by lysing the cultivated
E. coli.
Kula Maria-Regina
Pohl Martina
Slusarczyk Heike
Degussa - Aktiengesellschaft
Selitto Behr & Kim
Slobodyansky Elizabeth
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