Biocatalysts as core components of enzymatically catalyzed redox systems for the biocatalytic reduction of cystine

Abstract

The present invention relates to an enzyme for reducing cystine to cysteine, which is a fusion protein comprising the protein activity of thioredoxin (protein i) having KEGG database number EC 1.8.4.8 or EC 1.8.4.10 and thioredoxin reductase (protein ii) having KEGG database number EC 1.8.1.9, wherein the activity of the fusion protein is at least 100% of the activity of a mixture of the individual proteins identical to proteins i and ii but not fused to each other. The present invention also relates to a method for enzymatically reducing cystine to cysteine ​​using the fusion protein in the presence of a cofactor.

Description

[Technical field]

[0001] The present invention relates to an enzyme for reducing cystine to cysteine, which is a fusion protein comprising the protein activity of thioredoxin (protein i) with KEGG database number EC 1.8.4.8 or EC 1.8.4.10 and thioredoxin reductase (protein ii) with KEGG database number EC 1.8.1.9, characterized in that the activity of the fusion protein is at least 100% of the activity of a mixture of the individual proteins identical to but not fused to said proteins i and ii. The present invention further relates to a method for the enzymatic reduction of cystine to cysteine ​​in the presence of cofactors using said fusion protein. [Background technology]

[0002] Cysteine, abbreviated as Cys or C, is an α-amino acid with the side chain -CH2-SH. The naturally occurring enantiomeric form is L-cysteine, and since this is the only proteinogenic amino acid, in the context of the present invention, when cysteine ​​is used without a descriptor, it means L-cysteine. Oxidation of the sulfhydryl group can result in two cysteine ​​residues forming a disulfide bond together, resulting in the formation of cystine. The same applies to cystine, i.e., when there is no descriptor, it means the L-enantiomer (or R,R-cystine) in the present invention. L-cysteine ​​is a semi-essential amino acid for humans, since it can be formed from the amino acid methionine.

[0003] The amino acid L-cysteine ​​is used, for example, as a food additive (particularly in the baking industry), as a raw material in cosmetics, and as a starting material for the production of active pharmaceutical ingredients (particularly N-acetylcysteine ​​and S-carboxymethylcysteine) and is therefore economically important. The use of cysteine ​​as a flavoring agent in the food industry (e.g. chicken or meat flavoring) appears to be of particular importance. L-cysteine ​​is also used to modify the rheology of doughs and as an antioxidant, for example in fruit juices.

[0004] In all living organisms, L-cysteine ​​plays a key role in sulfur metabolism. It is used for the synthesis of proteins, methionine, biotin, lipoic acid, glutathione and other sulfur-containing metabolites. L-cysteine ​​is also used in the biosynthesis of coenzyme A. In enzymes, thiol groups often play an important role in catalysis of reactions or stabilize proteins through intra- or intermolecular disulfide bonds.

[0005] L-cysteine ​​can be obtained by hydrolysis from keratin-containing materials such as hair, wool, nails, hooves, feathers and horns. However, such methods always raise issues regarding biosafety and environmental protection. They also have low productivity. As alternatives, methods for biotransformation production from precursors and methods for fermentative production of L-cysteine ​​have been developed (US6218168B1, US5972663A, US2004-038352A, CA2386539A2, EP2138585, EP1769080, and EP2726625B1).

[0006] A disadvantage of fermentative production is the low yield of L-cysteine ​​(even after optimizing the fermentation control), also because cysteine ​​exists mainly as disulfide L-cystine and can be obtained in this form by oxidation in the presence of atmospheric oxygen.

[0007] The oxidation reaction is reversible, so L-cystine can be converted back to L-cysteine ​​by selective reduction, but if L-cystine is reduced back to L-cysteine ​​by electrolysis after separation from the cells (e.g. by decantation), this chemical conversion means that such L-cysteine ​​cannot be declared natural according to the Regulation on Flavorings.

[0008] According to the EU Regulation on Flavorings (Article 22 of Regulation 1334 / 2008 on the reorganization of food labelling rules), natural flavours are defined as follows: "Natural" flavourings are chemically defined substances having flavouring properties which occur in nature and have been detected in nature. They are obtained from starting materials of vegetable, animal or microbial origin by suitable physical, enzymatic or microbial processes and are used as such or prepared for human consumption by one or more conventional food manufacturing methods.

[0009] The term "natural" is used in this application in this sense. There is a great deal of interest in the use of natural raw materials in the manufacture of flavour additives.

[0010] Enzymatic cleavage allows the production of natural cysteine ​​with a high fermentative cystine yield. For the implementation of such an enzymatic process, a suitable oxidoreductase cascade system is required. Examples of disulfide cleavage enzymes known in the literature include thioredoxin, glutaredoxin, and disulfide isomerase. However, these enzymes often act on disulfides formed in polypeptides or between two subunits of a protein. The cleavage of free cystine has only been described in the literature for a few proteins (e.g., bacterial thioredoxin, which is then regenerated by thioredoxin reductase, for which NADPH is required as a cofactor). Such oxidoreductases or enzyme cascades often require NADH or NADPH as cofactors, which provide the corresponding electrons for the reduction reaction.

[0011] Thus, cystine reductase is characterized by using the substrate cystine with the assistance of a cofactor such as NADPH or NADH, transferring two electrons to form two cysteines as end products.

[0012] This therefore means that the cofactor must be added in equimolar amounts to this reaction, which is costly and therefore economically undesirable. This can be avoided by combining it with a second reaction that regenerates the cofactor. As regenerating systems, dehydrogenases such as alcohol dehydrogenase or glucose-6-phosphate dehydrogenase can be used. From an economic point of view, it must also be possible to produce these enzymes by fermentation with correspondingly high yields.

[0013] Since the effort for producing the required enzymes increases with the number of enzymes, it makes economic sense to fuse individual enzymes by genetic engineering. This has the added advantage that the substrate can be transferred more easily from one enzyme to the next due to spatial proximity. However, such enzyme fusions often result in attenuation of fermentation productivity, or else the fusion protein has reduced or no activity. To avoid the two enzymes having mutual inhibitory effects in the fusion protein, a longer linker sequence is often used instead to connect / separate the two proteins. If a very short linker sequence or no linker sequence is used at all, it is rare for a fusion protein to show comparable activity compared to the mixture of starting proteins.

[0014] In Mycobacterium leprae (Wieles B. et al. 1995, J. Biol. Chem. 270, pp. 25604-25606), a protein with N-terminal homology to thioredoxin reductase and C-terminal homology to thioredoxin has been found to be active as thioredoxin and thioredoxin reductase. Comparing the two protein units with the homologous protein from E. coli, they are linked in M. leprae via a 22 amino acid spacer / linker. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] U.S. Patent No. 6,218,168 [Patent Document 2] U.S. Pat. No. 5,972,663 [Patent Document 3] US Patent Application Publication No. 2004-038352 [Patent Document 4] Canadian Patent No. 2386539 [Patent Document 5] European Patent No. 2138585 [Patent Document 6] European Patent No. 1769080 [Patent Document 7] European Patent No. 2726625 [Non-patent literature]

[0016] [Non-Patent Document 1] Wieles B.et al.1995,J.Biol.Chem.270,pp.25604-25606 Summary of the Invention [Problem to be solved by the invention]

[0017] The object of the present invention is to provide a protein for the biocatalytic reduction of cystine to cysteine ​​which can be produced cheaply and has an activity at least as high as the mixture of thioredoxin and thioredoxin reductase proteins known in the prior art. The use of this protein in a process for the enzymatic reduction of cystine to cysteine ​​provides a particularly efficient and economical process. [Means for solving the problem]

[0018] This object is achieved by providing an enzyme for reducing cystine to cysteine, which is a fusion protein comprising the protein activity of thioredoxin (protein i) having KEGG database number EC 1.8.4.8 or EC 1.8.4.10 and thioredoxin reductase (protein ii) having KEGG database number EC 1.8.1.9, characterized in that the activity of the fusion protein is at least 100% of the activity of a mixture of the individual proteins identical to thioredoxin and thioredoxin reductase but not fused thereto. [Brief description of the drawings]

[0019] [Figure 1] The plasmid map of pGJ3477 is shown. [Diagram 2] 1 shows a scheme for the reduction of cystine to cysteine, accompanied by the oxidation of the cofactor NADPH to NADP+, catalyzed by cystine reductase. [Diagram 3] FIG. 1 shows a scheme for the combined reduction of cysteine ​​(Cys-SH) with DTNB (5,5′-dithiobis-2-nitrobenzoic acid; Ellman's reagent), with release of Cys-TNB and TNB, catalyzed by cystine reductase. [Figure 4] The relative cystine reductase activity of the various enzymes is shown. [Diagram 5] FIG. 1 shows chromatographic detection of cysteine ​​formation by TrxBA and TrxAB using the DTNB assay. [Figure 6] Cysteine ​​formation by TrxBA with or without a regeneration system - measured using the DTNB assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] According to the present invention, the enzyme for reducing cystine to cysteine ​​is a fusion protein with the protein activity of proteins i and ii. This means that the coding sequences (cds) responsible for the activity of protein i (Trx as described in KEGG database number EC1.8.4.8 or EC1.8.4.10) and the activity of protein ii (TR as described in KEGG database number EC1.8.1.9) are fused. The two cds are expressed together as one cds by removing the stop codon present after the first cds.

[0021] The protein activity of Trx is an oxidoreductase with a disulfide bond in the active site and can exist in a reduced or oxidized state.

[0022] In this reaction, two electrons are transferred (intramolecularly or intermolecularly) from two sulfhydryl groups to a disulfide bond. Here, in Trx, an intramolecular disulfide bond is formed. The reduced state of Trx is then reversed by the catalytic activity of oxidoreductase TR, which requires electrons to be transferred to Trx to cleave the disulfide bond.

[0023] Whether a protein is active towards cystine as Trx and / or TR (hereinafter referred to as cystine reductase / CR activity if the protein has both activities) can be confirmed by the following test:

[0024] First, the consumption of NADPH can be measured photometrically at 340 nm. In a mixture containing the fusion protein, the substrate cystine and the cofactor NADPH, electrons are transferred from NADPH to cystine to form cysteine. The consumption of NADPH can then be monitored photometrically.

[0025] Alternatively, the cysteine ​​formed may also be measured in such a mixture (containing the fusion protein, cystine and NADPH) using the ability of the free SH group of cysteine ​​to react with DTNB (5,5'-dithiobis-2-nitrobenzoic acid; Ellman's reagent) to form a dye. This colored compound, Cys-TNB, can be measured at 412 nm. The course of this reaction over time therefore makes it possible to measure the enzymatic activity of the fusion protein towards cystine.

[0026] Protein i and protein ii are preferably microbial sequences.

[0027] The thioredoxin (protein i) is preferably the protein activity of thioredoxin 1 from E. coli (abbreviated as TrxA in the context of the present invention).

[0028] The thioredoxin reductase (protein ii) is preferably the protein activity of thioredoxin reductase from E. coli (abbreviated as TrxB in the context of the present invention).

[0029] Particularly preferred is a fusion protein comprising two amino acid sequences, where one of these amino acid sequences is at least 50%, preferably at least 70%, and particularly preferably at least 90%, identical to SEQ ID NO: 7, and the other amino acid sequence is at least 50%, preferably at least 70%, and particularly preferably at least 90% identical to SEQ ID NO: 8, and where this fusion protein has CR activity.

[0030] The fusion protein is therefore called TrxAB or TrxBA / TrxB5A (when in the fusion protein TrxAB the protein TrxA is arranged N-terminally to TrxB). Conversely, in the fusion protein TrxBA or TrxB5A the protein TrxB is arranged N-terminally to TrxA. The fusion protein is preferably TrxAB, TrxBA or TrxB5A. It is particularly preferred that the fusion protein is a fusion of the amino acid sequences of thioredoxin A and thioredoxin B from E. coli, especially preferred is one selected from the group consisting of amino acid sequence SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 28.

[0031] In a particularly preferred embodiment, the fusion protein is TrxBA, especially preferred is the amino acid sequence having SEQ ID NO:10.

[0032] The degree of DNA identity is determined by the "nucleotide blast" program, which can be found at http: / / blast.ncbi.nlm.nih.gov / and is based on the blastn algorithm. Default parameters were used as algorithm parameters for the alignment of two or more nucleotide sequences. Default general parameters are: maximum target sequence=100; short query="auto-correction parameters for short input sequences"; prediction threshold=10; word size=28; auto-correction parameters for short input sequences=0. Corresponding default scoring parameters are match / mismatch score=1,-2; gap cost=linear.

[0033] Protein sequences are compared using the "protein blast" program at http: / / blast.ncbi.nlm.nih.gov / . This program uses the blastp algorithm. Default parameters were used as algorithm parameters for alignment of two or more protein sequences. Default general parameters are: max target sequence=100; short query="auto-correction parameters for short input sequences"; prediction threshold=10; word size=28; auto-correction parameters for short input sequences=0. Corresponding default scoring parameters are: matrix=BLOSUM62; gap cost=presence:11 extension:1; composition correction=tentative correction score matrix correction.

[0034] In the context of the present invention, proteins such as TrxA, TrxB, TrxAB, TrxBA, TrxB5A or Ml-TrxBA are written in capital letters, whereas the sequences encoding these proteins (also abbreviated as cds) are identified by lower case letters (trxA, trxB, trxAB, trxBA, trxB5A or Ml-trxBA).

[0035] In the context of the present invention, the term fusion protein refers to a protein that comprises two separate proteins, thioredoxin (Trx) and thioredoxin reductase (TR), that are encoded by a gene, the coding region of which comprises the Trx coding region and the TR coding region, such that they are transcribed together as a unit and translated into a polypeptide.

[0036] The particular advantage of using a fusion protein containing Trx and TR compared to using two individual proteins Trx and TR comes from the position of the two proteins in space: TR transfers electrons to Trx, where Trx and TR do not necessarily have to face each other in the first place in the process of three-dimensional diffusion, but are linked to each other in the fusion protein. In other words, the spatial proximity means that electrons can be more easily transferred from one enzyme to the other.

[0037] Although the individual proteins must be purified separately, the enzymatic activities in the fusion protein are co-isolated, which is also a great economic advantage.

[0038] Preferably, the fusion protein further comprises a sequence, particularly preferably a polyhistidine tag (His tag), to aid in purification. The His tag is a protein tag that can be used for protein purification and for detecting targeted proteins. The amino acid sequence of the polyhistidine tag is a sequence of at least six histidines (6HIS), whose gene sequence is cloned into the cds at the N-terminus after the initiation methionine codon or at the C-terminus before the stop codon. This results in a fusion protein with a polyhistidine tag. The cloning of a single His tag makes it possible to purify a fusion protein composed of Trx and TR proteins. This means that not only fewer reaction steps are required for purification, but also simplifies the cloning strategy.

[0039] In addition, a cleavage site for a protease or intein can be inserted between the polypeptide sequence and the His tag, allowing the His tag to be cleaved off after protein purification.

[0040] In the fusion protein, the amino acid sequence of the protein located on the N-terminus is preferably truncated on the C-terminus by 1 to a maximum of 5 amino acids. In the fusion protein, it is particularly preferred that the amino acid sequence of the protein located on the N-terminus is truncated on the C-terminus by 1 amino acid.

[0041] In the fusion protein, the amino acid sequence of the protein located at the C-terminus is preferably shortened at the N-terminus by 1 to a maximum of 5 amino acids, particularly preferably by 1 amino acid. Particularly preferably, in the fusion protein, there is no start codon in the protein located at the C-terminus. This latter feature has the advantage that there is no retranslation in this respect.

[0042] Furthermore, it is preferred that the amino acid sequences of the fusion protein activity of thioredoxin (protein i) and thioredoxin reductase (protein ii) are linked in the fusion protein by a linker sequence of 1 to a maximum of 5 amino acids, particularly preferably 1 amino acid.

[0043] It is particularly preferred that the amino acid sequences of the thioredoxin (protein i) and thioredoxin reductase (protein ii) fusion protein directly follow each other in the fusion protein, ie there are no linker sequences present.

[0044] As mentioned above, it is beneficial to bring together proteins in a fusion protein that work in concert to improve their cloning, purification and activity. However, a long linker sequence would be desirable in order not to disturb the folding of the two fusion proteins or the interaction between them, which is essential in this case. However, in this case, it was surprisingly found that the direct fusion of the two proteins (Trx, TR) results in a functional enzyme pair. This fact is further emphasized by the finding that the activity of the fusion protein depends on the order of fusion. Thus, the fusion protein TrxAB showed an activity comparable to the mixture of the individual proteins.

[0045] In the fusion protein TrxAB (SEQ ID NO: 9), the stop codon in the TrxA sequence (SEQ ID NO: 7) is absent, and the first amino acid (methionine, M) in TrxB (SEQ ID NO: 8) is deleted. In addition, this protein has a His tag (amino acids 1-12 in SEQ ID NO: 9). There is no linker sequence between the last amino acid of TrxA (Ala) and the first amino acid after removal of the start codon of TrxB (Gly).

[0046] In the fusion protein TrxBA (SEQ ID NO: 10), the last amino acid (lysine) and the stop codon in the TrxB sequence (SEQ ID NO: 8) are absent, whereas the first amino acid (methionine, M) in TrxA (SEQ ID NO: 7) is deleted. Furthermore, this protein has a His tag (amino acids 1-12 in SEQ ID NO: 10). A linker sequence of one amino acid (Gly) is present between the penultimate amino acid (Ala) of TrxB and the first amino acid (Ser) of TrxA.

[0047] For the fusion protein TrxB5A (SEQ ID NO: 28), the same applies as described for TrxBA, except that there is a 5 amino acid linker sequence (Gly-Pro-Ala-Pro-Gly) between TrxB and TrxA.

[0048] The region between the start and stop codons that codes for the amino acid sequence of the fusion protein is called the coding sequence (cds). The cds are surrounded by non-coding regions. What is called a gene is a piece of DNA that contains all the basic information to produce a biologically functional protein. A gene comprises a piece of DNA from which a single-stranded RNA copy is generated by transcription, and furthermore expression signals involved in the regulation of this replication process. The expression signals comprise, for example, at least one promoter, a transcription initiation site, a translation initiation site and a ribosome binding site. Furthermore, expression regulation by a terminator and one or more operators is possible.

[0049] For a functional promoter, the cds under the control of this promoter are transcribed into RNA.

[0050] The portion of DNA coding for protein i (thioredoxin) of the fusion protein can be first amplified by PCR using oligonucleotides as primers and a DNA matrix coding for thioredoxin, for example a genome isolated from E. coli, and then linked in frame fusion by standard molecular biology techniques to a DNA molecule containing the sequence of protein ii (thioredoxin reductase) of the fusion protein, which is produced in a similar manner in each case. The second initiation codon of the fusion partner located at the C-terminus, which is present between proteins i and ii in the fusion protein, can be deleted to avoid alternative reading frames. At the transition point of the two fusion partners, a linker sequence of various lengths can be inserted.

[0051] Instead of fusion via a cloning site, the entire DNA molecule may be produced by gene synthesis. The DNA molecule may then be introduced into a vector, such as a plasmid, or directly integrated into the chromosome of a bacterial strain by known methods. Preferably, the DNA molecule is introduced into a plasmid, such as a derivative of a known expression vector, such as pJF118EH, pKK223-3, pUC18, pBR322, pACYC184, pASK-IBA3, pGJ3477 or pET.

[0052] This DNA molecule is first cloned into a plasmid carrying the coding sequence of the fusion protein and only a few other nucleotides, such that the cloning occurs at a predetermined distance from the promoter and terminator, i.e., the DNA molecule is cloned into the plasmid 3' of the region encoding the promoter and 5' of the region encoding the terminator.

[0053] Suitable promoters are all promoters known to those skilled in the art, such as constitutive promoters, for example the GAPDH promoter, or inducible promoters, for example the lac, tac, trc, lambda PL, araB, cumate or tet promoters or sequences derived therefrom. The fusion protein is particularly preferably driven by the arabinose-inducible araB promoter (P BAD ) is expressed under the control of

[0054] The plasmid used may carry a selection marker. Suitable selection markers are genes encoding resistance to antibiotics such as ampicillin, tetracycline, chloramphenicol, kanamycin, etc. The plasmid preferably contains a gene whose expression confers resistance to ampicillin. Also suitable as selection markers are auxotrophic markers, since they code for metabolically essential genes that are deleted in the respective bacterial strains that contain the plasmid.

[0055] The plasmid may be introduced (transformed) into cells of a bacterial strain, preferably a Gram-negative bacterium, particularly preferably a strain of the Enterobacteriaceae family, especially preferably a strain of the Escherichia coli species, using methods known to those skilled in the art.

[0056] The bacterial strain containing the plasmid may be used in a fermentation process, i.e. the bacterial cells may be grown in a medium, preferably a selective LB medium (corresponding to the selection marker present in the plasmid), separated from the medium by a precipitation method involving discarding the supernatant after cultivation, and lysed. The expressed fusion protein may then be isolated, for example, by affinity chromatography (e.g. via the His tag, see above).

[0057] The enzyme of the invention has the advantage that it can be produced by fermentation in high yield and therefore at low cost, which production is particularly economically efficient and also beneficial since instead of isolating two separate proteins, only one fusion protein needs to be produced, purified and isolated.

[0058] It is advantageous that the enzyme can be regenerated with a cofactor such as NADPH or NADH as an electron donor, i.e., as a reducing agent, since the system for regenerating NADPH or NADH already exists.

[0059] The enzymatic activity of Trx and TR can be determined photochemically, for example at 340 nm, via the conversion of cofactors, since the extinction coefficient at 340 nm changes significantly when going from the reduced (NADPH / NADH) to the oxidized (NADP / NAD) state.

[0060] When measuring the enzymatic activity of different constructs (fusion proteins) or different mixture ratios of the separate proteins, it was surprisingly and unexpectedly found by the person skilled in the art that the activity of this fusion protein is at least 100%, preferably at least 150%, and particularly preferably at least 200% of the activity of a mixture of the same but unfused separate proteins thioredoxin and thioredoxin reductase, which means that the activity of this fusion protein is as high or even higher than the activity of a mixture of the same but unfused separate proteins i and ii.

[0061] More precisely, this means that in each case, except for the negative control without cofactor or substrate, the same reaction mixture consisting of substrate, cofactor and buffer system was initially introduced. After adding the same amount of fusion protein to the mixture, or after adding the individual proteins i (Trx) and ii (TR) to another mixture, the cofactor conversion was measured under the same reaction conditions and at the same time. Alternatively, it is equally possible to detect a decrease in the amount of substrate or an increase in the amount of product.

[0062] This enzyme is preferably characterized in that the gene of the fusion protein comprises one DNA sequence which is at least 50%, preferably at least 70%, and particularly preferably at least 90% identical to SEQ ID NO: 2, and another DNA sequence which is at least 50%, preferably at least 70%, and particularly preferably at least 90% identical to SEQ ID NO: 3.

[0063] In a particularly preferred embodiment, the DNA sequence encoding the fusion protein is SEQ ID NO:5.

[0064] The present invention further provides a method for the enzymatic reduction of cystine to cysteine, which is characterized in that cysteine ​​is reduced by an enzyme of the invention in the presence of a cofactor.

[0065] In the enzymatic reduction of cystine to cysteine, the disulfide (-SS-) is converted to two sulfhydryl (-SH) groups, i.e., two molecules of L-cysteine ​​are formed from one molecule of the chemical compound cystine. The fusion protein of the invention catalyzes the reduction and transfers electrons to the disulfide of cystine.

[0066] It was now surprising to find that the activity of the fusion protein TrxBA was particularly significantly higher than the activity of a mixture of the identical but unfused individual proteins TrxA and TrxB (see Example 4, FIG. 4).

[0067] A particular advantage of the method of the present invention is that the cysteine ​​produced using this method can be claimed as naturally occurring cysteine ​​in accordance with the Regulation on Flavorings.

[0068] A further challenge for an economical process has been the low solubility of cystine in buffer systems within the neutral pH range (or physiological pH range, i.e., approximately pH 7.4).

[0069] The process preferably takes place within a pH range of 4 to 11, particularly preferably 5 to 10 and especially preferably 6 to 9.

[0070] It is of particular interest, and therefore even particularly preferred, when the enzyme of the invention is used in a method for the enzymatic reduction of cystine to cysteine ​​at physiological pH.

[0071] This process preferably takes place at a temperature of 20 to 40°C, particularly preferably 25 to 30°C.

[0072] This process is preferably characterized in that the cofactor is a substance selected from the group consisting of NADPH and NADH, this cofactor being particularly preferably NADPH.

[0073] The process is preferably characterized by the isolation of the cysteine ​​formed after reduction. For further purification of the target product the following steps / procedures can be used: Isolation of L-cysteine ​​by ion exchange adsorption Precipitation crystallization.

[0074] Such processes are known from the prior art (see, for example, WO2013 / 000864).

[0075] In the reduction of cystine to cysteine, the enzyme of the present invention is oxidized, i.e., transfers electrons to cystine to form a disulfide bond. NADP + or NAD + are formed, respectively. Therefore, it is preferred that the method comprises a cofactor regenerating enzyme. Particularly preferably, this cofactor regenerating enzyme is a dehydrogenase, and further, the reduction takes place in the presence of a further electron donor.

[0076] The cofactor regenerating enzyme may be glucose-6-phosphate dehydrogenase and / or alcohol dehydrogenase. In a particularly preferred embodiment, the dehydrogenase is an alcohol dehydrogenase and isopropanol is used as the electron donor.

[0077] The invention will be explained in more detail hereinafter with reference to exemplary embodiments, without however being limited thereto. EXAMPLES

[0078] Example 1: Generation of cystine reductase systems TrxA, TrxB, TrxBA, and TrxAB Preparation of expression vector: The expression plasmid pGJ3477 was selected as a vector for the expression of the DNA sequences coding for the corresponding candidate proteins TrxA, TrxB, TrxAB, TrxBA, TrxB5A or Ml-TrxBA. It is a medium-to-high copy plasmid (50-60 copies per cell) based on the ColE1 origin of replication. The plasmid map is shown in FIG. 1, on which the positions of the common single-cutting restriction enzymes (with six-base recognition sequences) are indicated. This sequence is identified in SEQ ID NO: 11.

[0079] In this plasmid, each candidate coding sequence was expressed by the arabinose-inducible promoter P BAD was placed under control.

[0080] First, the entire expression plasmid was amplified by inverse PCR: 50 ng pGJ3477 DNA, 0.5 pmol each of primers 3477-fwrd (SEQ ID NO: 12) and 3477-rev (SEQ ID NO: 13), Q5® reaction buffer (New England Biolabs, NEB), 1 unit of Q5® DNA polymerase (NEB) in a final volume of 50 μl. PCR program: 98° C. for 1 min, then 30 cycles of 98° C. for 30 s, 65° C. for 30 s (annealing), and 72° C. for 2 min (synthesis).

[0081] At the end of the reaction, the restriction enzyme DpnI (10 units, NEB) was added to the reaction mixture, and the mixture was incubated for 1 hour at 37° C. Then, the DNA was chromatographically purified (Macherey & Nagel: NucleoSpin® Gel and PCR Clean-up-Kit).

[0082] Cloning of trxA and trxB: For cloning the protein-coding sequences trxA (SEQ ID NO:2) and trxB (SEQ ID NO:3), oligonucleotide primers were designed that extend the target gene specific sequence by at least 15 nucleotides, which overlaps with sequences at the ends of the vector DNA. The genes were amplified from the E. coli genome of BL21 by PCR (colony PCR).

[0083] The following mixtures were selected for amplification by PCR: 50 ng genomic E. coli BL21 DNA (NEB), 0.5 pmol each of primers trxA-fwrd (SEQ ID NO: 14) and trxA-rev (SEQ ID NO: 15) or trxB-fwrd (SEQ ID NO: 16) and trxB-rev (SEQ ID NO: 17), Q5(R) reaction buffer (NEB), 1 unit of Q5(R) DNA polymerase (NEB) in a final volume of 50 μl. PCR program: 98° C. for 1 min, then 30 cycles of 98° C. for 30 s, 60° C. for 30 s (annealing), and 72° C. for 15 s (trxA) or 30 s (trxB) (synthesis).

[0084] At the end of the reaction, the restriction enzyme DpnI (10 units, NEB) was added to the reaction mixture, and the mixture was incubated for 1 hour at 37° C. Then, the DNA was chromatographically purified (Macherey & Nagel: NucleoSpin® Gel and PCR Clean-up-Kit).

[0085] LIC-PCR: In general, DNA sequences encoding proteins were introduced at the correct base into the basic expression vector pGJ3477 via LIC-PCR (ligation-dependent cloning of PCR products) as described in Aslanidis C. and de Jong PJ, Nucleic Acids Res. 18, pp. 6069-6074.

[0086] For this purpose, 50 ng of purified coding DNA of each candidate protein was used in a LIC-PCR reaction together with 50 ng of prepared vector DNA.

[0087] The LIC-PCR mixture was then transformed into E. coli XL1 Blue cells using standard methods and plated onto selective LB medium (LB+100 mg / L ampicillin) and incubated for 18 hours at 37° C. For identification of correct clones, plasmid DNA was isolated from the resulting colonies and the expression cassettes were fully sequenced.

[0088] The resulting plasmid consisting of the expression vector pGJ3477 and the coding DNA sequence trxA or trxB is hereinafter referred to as the trxA or trxB expression vector.

[0089] Cloning of trxAB and trxBA: Cloning of the DNAs trxAB or trxBA encoding the corresponding fusion proteins TrxAB or TrxBA, respectively, was performed in a similar manner to the cloning of trxA and trxB described above, by inserting the trxA sequence into the trxB expression vector between the N-terminal His tag and the sequence trxB encoding the protein TrxB by LIC-PCR, or by inserting the trxB sequence into the trxA expression vector between the His tag and the sequence trxA encoding the protein TrxA by LIC-PCR:

[0090] The following mixtures were selected for amplification by PCR: Vector DNA (trxA or trxB expression vector) was amplified by inverse PCR using primers vtrxA-fwrd (SEQ ID NO: 18) and 3477-rev (SEQ ID NO: 13) or vtrxB-fwrd (SEQ ID NO: 21) and 3477-rev (SEQ ID NO: 13). For this purpose, the following PCR program was selected: 98°C for 2 min, followed by 30 cycles of 98°C for 45 sec / 60°C for 30 sec / 72°C for 2.5 min. The trxA or trxB gene segments for the fusion proteins were amplified by PCR using primers ftrxA-fwrd (SEQ ID NO: 22) and ftrxA-rev (SEQ ID NO: 23) or ftrxB-fwrd (SEQ ID NO: 19) and ftrxB-rev (SEQ ID NO: 20). For this purpose, the following PCR program was selected: 98°C for 2 min, followed by 30 cycles of 98°C for 45 sec / 60°C for 30 sec / 72°C for 15 sec. BL21 genomic DNA was used as the template.

[0091] At the end of the PCR reaction, the restriction enzyme DpnI (10 units, NEB) was added to each reaction mixture, and the mixture was incubated for 1 hour at 37° C. Then, the DNA insert was purified by chromatography (Macherey & Nagel: NucleoSpin® Gel and PCR Clean-up-Kit).

[0092] LIC-PCR reactions were performed with 50 ng of amplified vector DNA of trxA or trxB expression vector and 75 ng of DNA insert (trxB or trxA).

[0093] The LIC-PCR mixture was then transformed into E. coli XL1 Blue cells using standard methods and plated onto selective LB medium (LB+100 mg / L ampicillin) and incubated for 18 hours at 37° C. For identification of correct clones, plasmid DNA was isolated from the resulting colonies and the expression cassettes were fully sequenced.

[0094] SEQ ID NO:9 and SEQ ID NO:10 were identified as the amino acid sequences of the fusion proteins TrxBA and TrxAB resulting from expression of the sequences SEQ ID NO:4 and SEQ ID NO:5, respectively.

[0095] Example 2: Cloning of cystine reductase Ml-TrxBA As explained in the introduction, M. leprae possesses gene segments encoding proteins with homology to thioredoxin (Trx) and thioredoxin reductase (TR) (Wieles B. et al. 1995, J. Biol. Chem. 270, pp. 25604-25606). The sequences are available from public databases (NCBI reference sequence: WP_010909042.1).

[0096] The sequences obtained from public databases were computer customized for the codon usage of the host E. coli. This was done using the IDT web server (www.idtdna.com). For cloning into the target vector pGJ3477, the coding region was extended at the 5' and 3' ends by sequences overlapping with vector sequences (see also cloning of trxA and trxB). The entire resulting DNA sequence (shown in SEQ ID NO: 24) was synthetically generated by Geneart (www.thermofisher.com) and called Ml-trxBA (also called ml-trxBA).

[0097] The synthetic Ml-trxBA sequence was cloned into the vector pGJ3477, similar to the cloning by LIC-PCR of the trxA and trxB expression vectors.

[0098] For the cloning of the protein coding sequence Ml-trxBA (SEQ ID NO: 24), oligonucleotide primers were determined that extended this target gene specific sequence by at least 15 nucleotides overlapping the sequence at the ends of the vector DNA. The synthetic gene served as a template.

[0099] The following mixtures were selected for amplification by PCR: 20 ng template (Geneart), 0.5 pmol each of primers Ml-trxBA-fwrd (SEQ ID NO: 25) and Ml-trxBA-rev (SEQ ID NO: 26), Q5(R) reaction buffer, 1 unit of Q5(R) DNA polymerase (NEB) in a final volume of 50 μl. PCR program: 98° C. for 1 min, then 30 cycles of 98° C. for 30 s, 60° C. for 30 s (annealing), and 72° C. for 60 s (synthesis).

[0100] At the end of the reaction, the restriction enzyme DpnI (10 units, NEB) was added to the reaction mixture, and the mixture was incubated for 1 hour at 37° C. Then, the DNA was chromatographically purified (Macherey & Nagel: NucleoSpin® Gel and PCR Clean-up-Kit).

[0101] LIC-PCR: The DNA sequences encoding the proteins were introduced at the correct bases into the basic expression vector pGJ3477 via LIC-PCR (ligation-dependent cloning of PCR products) as described in Aslanidis C. and de Jong PJ, Nucleic Acids Res. 18, pp. 6069-6074.

[0102] For this purpose, 50 ng of purified coding DNA was used in a LIC-PCR reaction together with 60 ng of prepared vector DNA.

[0103] The LIC-PCR mixture was then transformed into E. coli XL1 Blue cells using standard methods and plated onto selective LB medium (LB+100 mg / L ampicillin) and incubated for 18 hours at 37° C. For identification of correct clones, plasmid DNA was isolated from the resulting colonies and the expression cassettes were fully sequenced.

[0104] The resulting plasmid consisting of the expression vector pGJ3477 and the coding DNA sequence Ml-trxBA was hereafter referred to as the Ml-trxBA expression vector.

[0105] Example 3: Cloning of cystine reductase TrxB5A (including linker sequence) Cloning of trxB5A: Cloning of the DNA encoding the corresponding fusion protein TrxB5A, trxB5A, was performed in a similar manner to the cloning of trxBA described above, by inserting the TrxB sequence with a C-terminal extension of 5 amino acids (hereafter referred to as TrxB5, cds referred to as trxB5) into the trxA expression vector between the His tag and the sequence trxA encoding the protein TrxA by LIC-PCR:

[0106] The following mixtures were selected for amplification by PCR: Vector DNA (trxA expression vector) was amplified by inverse PCR using primers vtrxA5-fwrd (SEQ ID NO: 29) and 3477-rev (SEQ ID NO: 13), for which the following PCR program was selected: 98°C for 2 min, followed by 30 cycles of 98°C for 45 sec / 60°C for 30 sec / 72°C for 2.5 min. trxB5 was amplified by PCR using primers ftrxB-fwrd (SEQ ID NO: 19) and ftrxB5-rev (SEQ ID NO: 30), for which the following PCR program was selected: 98° C. for 2 min, followed by 30 cycles of 98° C. for 45 sec / 60° C. for 30 sec / 72° C. for 15 sec. BL21 genomic DNA was used as the template.

[0107] At the end of the PCR reaction, the restriction enzyme DpnI (10 units, NEB) was added to each reaction mixture, and the mixture was incubated for 1 hour at 37° C. Then, the DNA insert was purified by chromatography (Macherey & Nagel: NucleoSpin® Gel and PCR Clean-up-Kit).

[0108] LIC-PCR reactions were performed with 50 ng of amplified vector DNA of the trxA expression vector and 75 ng of the DNA insert (trxB5).

[0109] The LIC-PCR mixture was then transformed into E. coli XL1 Blue cells using standard methods and plated onto selective LB medium (LB+100 mg / L ampicillin) and incubated for 18 hours at 37° C. For identification of correct clones, plasmid DNA was isolated from the resulting colonies and the expression cassettes were fully sequenced.

[0110] SEQ ID NO:28 specifies the amino acid sequence of the fusion protein TrxB5A obtained by expression of the sequence of SEQ ID NO:27.

[0111] Example 4: Enzyme activity of cystine reductase For recombinant expression of proteins TrxA, TrxB, TrxBA, TrxB5A, TrxAB, and Ml-TrxBA, the expression plasmids under study encoding the corresponding proteins were introduced into E. coli strain TOP10 (Thermo Fisher Scientific, MA / USA). The bacterial cells were plated on LB medium containing 100 mg / L ampicillin. Then, 25 ml of LB medium containing 100 mg / L ampicillin and 0.2% arabinose (w / v) was inoculated with one colony and incubated at 28° C. in a culture shaker cabinet for 19 hours.

[0112] For isolation of the expressed proteins, cells were pelleted by centrifugation (4000 g for 10 min), the medium supernatant was removed, and the cells were resuspended in lysis buffer (PBS + 10% BugBuster (Sigma)). According to the manufacturer's instructions in the BugBuster kit (Sigma) manual, cells were completely lysed for 15 min at RT, and then insoluble material was removed by centrifugation (9500 rpm for 20 min, as per manufacturer's instructions). For purification of the supernatant (i.e. cell lysate) by affinity chromatography, the obtained cell lysate was loaded onto a PBS-equilibrated Protino IDA2000 column (Macherey & Nagel). After a washing step (7 ml PBS), the sample was eluted with 5 ml elution buffer (PBS + 200 mM imidazole). The obtained protein concentration was determined by Bradford assay (Thermo Fisher).

[0113] The cystine reductase activity of the various enzymes TrxA, TrxB, TrxBA, TrxB5A, TrxAB or Ml-TrxBA was determined using two analytical methods described in detail below.

[0114] 1. The first method consists of a photometric detection method, which is based on the consumption of NADPH and the decrease in absorbance at 340 nm that occurs during the reduction reaction. As shown diagrammatically in Figure 2, the enzymatic conversion of cystine to cysteine ​​in the reduction is achieved by the consumption of the cofactor NADPH.

[0115] The following assay mixture was used for the photometric measurements. The final volume of the sample is 1 ml: 100mM phosphate buffer pH 7.4 2mM EDTA 0.2 mM NADPH (Sigma) 1mM cystine (Wacker Chemie AG) After mixing all assay components except the enzyme, the reaction is initiated by the addition of the enzyme: 10 μg of enzymes TrxA, TrxB, a mixture of TrxA and TrxB, TrxBA, TrxB5A, TrxAB or Ml-TrxBA. The assay mixtures were incubated at room temperature (approximately 25° C.) until the following measurements.

[0116] The NADPH concentration in the various 1 ml assay mixtures was measured at various times in the form of absorbance values ​​at 340 nm using a spectrophotometer (Evolution 201 UV-vis spectrophotometer, Thermo Fisher Scientific). The absorbance values ​​at 340 nm were used to calculate the enzyme activity of the various samples using Thermo INSIGHT software from the manufacturer Thermo Fisher Scientific. In each case, samples either with heat-inactivated enzyme, without enzyme, without cystine as substrate, or without NADPH as cofactor were measured in parallel as negative controls.

[0117] The change (decrease) in absorbance over time was used to determine the enzyme activity from the linear slope at the beginning of the recorded curve, which indicates the rate of NADPH consumption. The rates can be compared since the same amount of enzyme is always used.

[0118] 2. In the second method, cysteine ​​formed from cystine by cystine reductase activity was determined directly via the free SH group by reaction with the compound DTNB (5,5'-dithiobis-2-nitrobenzoic acid; Ellman's reagent), as shown diagrammatically in Figure 3. In the context of the present invention, it is also called DTNB assay.

[0119] The following assay mixture was used for the photometric measurements. The final volume of the sample is 1 ml: 100mM phosphate buffer pH 7.4 2mM EDTA 0.2 mM NADPH (Sigma) 1 mM cystine (Wacker Chemie AG) After mixing all components except the enzyme, the reaction is started by the addition of the enzyme: 10 μg of enzymes TrxA, TrxB, a mixture of TrxA and TrxB, TrxBA, TrxB5A, TrxAB or Ml-TrxBA. The assay mixtures were incubated at room temperature (approximately 25° C.) until the following measurements.

[0120] L-cysteine ​​was quantified using 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) according to the test described by Lee S.-H.et al.1995 (Biochemical and Biophysical Research Communications 213, pp.837-844). Quantification of Cys-TNB was by measuring the absorbance at 412 nm via DTNB. HPLC analysis is also possible.

[0121] In each case, samples either containing heat-inactivated enzyme, no enzyme, no cystine as substrate, or no NADPH as cofactor were measured in parallel as negative controls.

[0122] Figure 4 shows the relative cystine reductase activity of the enzymes TrxBA, TrxB5A, TrxAB, and Ml-Trx, as well as the 9:1 mixture of TrxA and TrxB, as determined photometrically, as described above in 1. The activity of the 9:1 mixture of TrxA and TrxB is normalized to 1, and all other activity sets are relative to it. The 9:1 mixture of TrxA and TrxB is assumed to be the mixture ratio of the individual enzymes, which comparative studies have shown to be the most active mixture ratio.

[0123] FIG. 5 shows the detection of cysteine ​​formation by TrxBA and TrxAB according to the DTNB assay and HPLC, as described above in 2.

[0124] Using clones containing longer linker sequences than TrxB5A, in the region of more than 60 nucleotides between the cds for TrxA and TrxB, surprisingly reduced activity in the fusion protein.

[0125] Example 5: Activity of the enzyme combination of cystine reductase and ADH Cofactors NADPH and NADP + A comparison of the enzyme specificities for cystine reductases TrxBA and TrxAB was found to be highly specific for the cofactor NADPH. + No enzyme activity against cystine was detected.

[0126] For this reaction, the enzyme alcohol dehydrogenase (ADH) converts NADP + The extent to which ATP can be converted back to NADPH was examined in combination experiments.

[0127] For this purpose, the following conditions were used: 100mM phosphate buffer pH 7.4 5 μl isopropanol 2mM EDTA 15-50μM NADPH or NADP + 1mM cystine 5 μg enzyme TrxBA 50 μl cell lysate crude extract or 50 μl phosphate buffer as negative control (Fast Prep digest of 0.5 ml cells in 1.5 ml 4× phosphate buffer pH 7.4. Cell purification is described in EP1832658B1)

[0128] The reaction was incubated for 60 min at 30° C. Samples were taken at 5 min intervals and the released cysteine ​​was derivatized with DTNB as described in WO2013 / 000864A1. Quantitation was by measurement of absorbance at 412 nm via DTNB.

[0129] FIG. 6 shows the results of measuring cysteine ​​formation by TrxBA in the presence (with) or absence (without) of ADH as a regeneration system using the DTNB assay.

[0130] Abbreviations used in the drawings AraC: AraC gene (repressor gene) pAraC: AraC gene (repressor gene; P BAD (in the rev direction) promoter pBAD (in the context of the present invention, BAD Also referred to as: an arabinose-inducible promoter for (downstream) expression of an inserted target protein sequence 6HIS: coding region for His tag term:transcription terminator Amp: ampicillin resistance marker CR: Cystine reductase rel.:Relative bps: base pair t: time

Claims

1. An enzyme for reducing cystine to cysteine, said enzyme comprising: Thioredoxin (protein i) having the protein activity of thioredoxin 1 from Escherichia coli, and Thioredoxin reductase (protein ii) having the protein activity of thioredoxin reductase from Escherichia coli wherein the fusion protein comprises the amino acid sequence of said thioredoxin (protein i) is at least 90% identical to SEQ ID NO:7; the amino acid sequence of said thioredoxin reductase (protein ii) is at least 90% identical to SEQ ID NO:8; the fusion protein has cystine reductase activity, The cystine reductase activity of the fusion protein is at least 100% of the cystine reductase activity of a mixture of unfused individual thioredoxins consisting of the amino acid sequence of SEQ ID NO:7 and thioredoxin reductase consisting of the amino acid sequence of SEQ ID NO:8; and the amino acid sequences of said proteins i and ii are fused; and The enzyme, characterized in that the amino acid sequences of the thioredoxin (protein i) and the thioredoxin reductase (protein ii) are linked by a linker sequence of 1 to a maximum of 5 amino acids in the fusion protein or directly follow each other in the fusion protein.

2. The enzyme described in claim 1, characterized in that in the fusion protein, the C-terminus of the amino acid sequence of the protein located on the N-terminal side is shortened by 1 to a maximum of 5 amino acids.

3. The enzyme according to any one of claims 1 to 2, characterized in that the amino acid sequence of the protein located on the C-terminal side is shortened by 1 to a maximum of 5 amino acids at the N-terminus in the fusion protein.

4. An enzyme described in any one of claims 1 to 3, characterized in that the amino acid sequences of the thioredoxin (protein i) and the thioredoxin reductase (protein ii) are linked in the fusion protein by a linker sequence of 1 to a maximum of 5 amino acids.

5. The enzyme according to any one of claims 1 to 4, characterized in that the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:

28.

6. A method for the enzymatic reduction of cystine to cysteine, comprising the steps of: reducing cystine by an enzyme according to any one of claims 1 to 5 in the presence of a cofactor; wherein two molecules of L-cysteine ​​are formed from one molecule of cystine.

7. The method of claim 6, wherein the reduction occurs at a pH of from 6 to 9.

8. A method according to any one of claims 6 to 7, wherein the cofactor is a substance selected from the group consisting of NADPH and NADH.

9. The method according to any one of claims 6 to 8, further comprising a cofactor regenerating enzyme.

10. 10. The method of claim 9, wherein the cofactor regenerating enzyme is a dehydrogenase and the reduction further occurs in the presence of an electron donor.

11. 11. The method according to claim 10, characterized in that the dehydrogenase is an alcohol dehydrogenase and isopropanol is used as the electron donor.

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