Biocatalysts as core components of enzymatic redox systems for biocatalytic reduction of cystine

Abstract

The present invention relates to an enzyme for the reduction of cystine to cysteine, characterized in that the enzyme is a fusion protein comprising the protein activity of a thio-oxido-reductase (protein i) having the KEGG database number EC 1.8.4.8 or EC 1.8.4.10 and a thio-oxido-reductase reductase (protein ii) having the KEGG database number EC 1.8.1.9, the activity of the fusion protein being at least 100% of the activity of a mixture of the same but unbound individual proteins i and ii. The present invention also relates to a method for the enzymatic reduction of cystine to cysteine by said fusion protein in the presence of a co-factor.

Description

Technical Field

[0001] This invention relates to an enzyme for reducing cysteine ​​to cysteine, characterized in that the enzyme is a fusion protein comprising the protein activity of a thioreductase protein (protein i) having KEGG database number EC1.8.4.8 or EC1.8.4.10 and a thioreductase protein reductase (protein ii) having KEGG database number EC1.8.1.9, wherein the activity of the fusion protein is at least 100% of the activity of a mixture of identical but unfused individual proteins i and ii. The invention further relates to a method for enzymatically reducing cysteine ​​to cysteine ​​using the fusion protein in the presence of a cofactor. Background Technology

[0002] Cysteine ​​(abbreviated as Cys or C) is an α-amino acid with a side chain -CH2-SH. Because the naturally occurring enantiomer is L-cysteine, and because this is the only proteogenic amino acid, in the context of this invention, L-cysteine ​​refers to the term cysteine ​​when no descriptive term is used. Oxidation of the thiol group can lead to the formation of a disulfide bond between two cysteine ​​residues, subsequently forming cystine; the same statement applies here, i.e., In the absence of a descriptive term, it refers to the L-enantiomer (or R,R-cysteine) as understood in this invention. L-cysteine ​​is a semi-essential amino acid in humans because 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 (especially N-acetylcysteine ​​and S-carboxymethylcysteine), and is therefore of economic importance. The use of cysteine ​​as a flavoring agent in the food industry (e.g., as a chicken or meat flavoring agent) is considered particularly important. L-cysteine ​​is also used to modify the rheology of raw dough and as an antioxidant, for example, in fruit juices.

[0004] L-cysteine ​​plays a crucial role in sulfur metabolism in all organisms. It is used to synthesize proteins, methionine, biotin, lipoic acid, glutathione, and other sulfur-containing metabolites. L-cysteine ​​is also involved in the biosynthesis of coenzyme A. In enzymes, thiol groups typically play an important role in the catalysis of reactions or in stabilizing proteins by forming intramolecular or intermolecular disulfide bonds.

[0005] L-cysteine ​​can be obtained by hydrolyzing keratin-containing materials such as hair, mane, nails, hooves, feathers, and horn. However, such methods always raise concerns about biosafety and environmental protection. They also have low productivity. As alternatives, methods for producing L-cysteine ​​via biotransformation from precursors and for fermentation have been developed (US 6218168 B1, US 5972663 A, US 2004-038352 A, CA 2386539 A2, EP 2138585, EP1769080, and EP 2726625 B1). A disadvantage of fermentation production is the low yield of L-cysteine ​​(even after optimizing fermentation control), which is also because cysteine ​​is oxidized in the presence of atmospheric oxygen and then exists primarily as disulfide L-cysteine, which can be obtained in this form.

[0006] Oxidation is reversible, so L-cysteine ​​can be selectively reduced back to L-cysteine. However, if L-cysteine ​​is electrolytically reduced back to L-cysteine ​​after separation from cells (e.g., using a decanter), this chemical conversion means that, according to flavor regulations, such L-cysteine ​​may not be declared natural.

[0007] According to the EU Flavor Regulation (Article 22 of 1334 / 2008), natural flavoring agents are defined as follows: "Natural" flavoring agents are chemically defined substances that have flavoring properties that occur naturally and have been detected in nature. They are obtained from starting materials of plant, animal, or microbial origin by suitable physical, enzymatic, or microbial methods, which are used as is or prepared by one or more conventional food preparation methods for human consumption.

[0008] The term "natural" is also used in this sense in this application. There is great interest in using natural ingredients in the production of flavorings.

[0009] Enzymatic cleavage allows for the production of native cysteine, resulting in high fermentation cysteine ​​yields. For such an enzymatic process to occur, a suitable oxidoreductase cascade system is required. Examples of disulfide lyases described include thioreductin, pentylene dioxide reductase, and disulfide isomerases. However, these enzymes most frequently act on disulfides already formed within polypeptides or between two protein subunits. Cleavage of free cysteine ​​has been described only for a few proteins (e.g., bacterial thioreductin), which are subsequently regenerated by thioreductin reductases, for which NADPH is required as a cofactor. Such oxidoreductases or enzyme cascades typically require NADH or NADPH as cofactors to provide the corresponding electrons for the reduction reaction.

[0010] Therefore, cystine reductase is characterized by using the substrate cystine and transferring two electrons with the help of cofactors such as NADPH or NADH to form two cysteine ​​residues as the final product.

[0011] This means that the cofactor must therefore be added to the reaction in equimolar amounts, which is cost-intensive and therefore economically disadvantageous. This can be avoided by coupling it with a second reaction to regenerate the cofactor. As a regeneration system, 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 in correspondingly high yields via fermentation.

[0012] Since the effort involved in producing the required enzymes increases with the number of enzymes, it is economically feasible to genetically engineer individual enzymes to fuse them. This can also have the advantage that substrates can migrate more easily from one enzyme to the next due to close spatial proximity. However, such enzyme fusions often result in poor fermentative producibility, or the fusion protein may otherwise have reduced activity or even no activity at all. To prevent mutual inhibition between the two enzymes in the fusion protein, longer linker sequences connecting / separating the two proteins are often used instead. When using very short linker sequences or no linker sequences at all, it is rare for the fusion protein to show comparable activity compared to the mixture of starting proteins.

[0013] In *Mycobacterium leprae* (Wieles B. et al. 1995, J. Biol. Chem. 270, pp. 25604-25606), a protein active as both a sulforeductase and a sulforeductase was discovered. The N-terminus of this protein is homologous to sulforeductase, and the C-terminus is homologous to sulforeductin. When comparing these two protein units with homologous proteins from *Escherichia coli*, these are linked in *Mycobacterium leprae* via a 22-amino acid spacer / linker. Summary of the Invention

[0014] The object of this invention is to provide a protein for the biocatalytic reduction of cysteine ​​to cysteine, which can be produced inexpensively and has activity at least as high as that of a mixture of sulfur redox proteins and sulfur redox protein reductases known in the art. Using this protein in the enzymatic reduction of cysteine ​​to cysteine ​​provides a particularly efficient and economical method.

[0015] This objective is achieved by providing an enzyme that reduces cysteine ​​to cysteine, characterized in that the enzyme is a fusion protein comprising the protein activity of a thioreduction protein (protein i) having KEGG database number EC1.8.4.8 or EC1.8.4.10 and a thioreduction protein reductase (protein ii) having KEGG database number EC1.8.1.9, wherein the activity of the fusion protein is at least 100% of the activity of a mixture of identical but non-fused individual proteins, the thioreduction protein and the thioreduction protein reductase.

[0016] According to the present invention, the enzyme for reducing cysteine ​​to cysteine ​​is a fusion protein comprising the protein activities of proteins i and ii. This means that the coding sequences (cds) for the activities of protein i (Trx, described in KEGG database number EC 1.8.4.8 or EC 1.8.4.10) and protein ii (TR, described in KEGG database number EC 1.8.1.9) are fused together. By removing the stop codon present after the first cds, the two cds are expressed together as a single cds.

[0017] Trx is an oxidoreductase with a disulfide bond at its active site and can exist in either a reduced or oxidized state.

[0018] In this reaction, two electrons are transferred from two thiol groups to disulfide bonds (intramolecular or intermolecular). Intramolecular disulfide bonds are thus formed in Trx. The reduced state of Trx is then restored by the catalytic activity of the oxidoreductase TR. This requires electrons that are transferred to Trx through the breaking of disulfide bonds.

[0019] Whether a protein is active against cystine as Trx and / or TR (hereinafter referred to as cystine reductase / CR activity when a protein has both activities) can be checked using the following test:

[0020] First, NADPH consumption can be measured spectrophotometrically at 340 nm. In a mixture containing the fusion protein, the substrate cysteine, and the cofactor NADPH, electrons are transferred from NADPH to cysteine ​​to form cysteine. NADPH consumption can then be monitored spectrophotometrically.

[0021] Alternatively, the cysteine ​​residues formed can be measured in a mixture containing the fusion protein, cystine, and NADPH. This utilizes 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 time-dependent nature of this reaction thus allows for the measurement of the enzymatic activity of the fusion protein against cystine.

[0022] Proteins i and ii are preferably microbial sequences.

[0023] The sulfur redox protein (protein i) is preferably the protein activity of sulfur redox protein 1 from Escherichia coli (abbreviated as TrxA in the context of this invention).

[0024] The thioreductase (protein ii) is preferably a thioreductase derived from Escherichia coli with protein activity (abbreviated as TrxB in the context of this invention).

[0025] Particularly preferred is that the fusion protein comprises two amino acid sequences, wherein one of these amino acid sequences has at least 50%, preferably at least 70%, and particularly preferably at least 90% identity with SEQ ID No. 7, and the other amino acid sequence has at least 50%, preferably at least 70%, and particularly preferably at least 90% identity with SEQ ID No. 8, and wherein the fusion protein has CR activity.

[0026] The fusion protein is therefore designated as TrxAB or TrxBA / TrxB5A, wherein in fusion protein TrxAB, protein TrxA is located at the N-terminus of TrxB. Conversely, in fusion proteins TrxBA or TrxB5A, protein TrxB is located at the N-terminus of TrxA. The preferred fusion protein is TrxAB, TrxBA, or TrxB5A. The fusion protein is particularly preferred to be a fusion of the amino acid sequences of sulfur redox protein A and sulfur redox protein B from *Escherichia coli*, especially preferably one of the amino acid sequences selected from the group consisting of SEQ ID No. 9, SEQ ID No. 10, and SEQ ID No. 28.

[0027] In a particularly preferred embodiment, the fusion protein is TrxBA, and more preferably has the amino acid sequence of SEQ ID NO. 10.

[0028] 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 are used for the algorithm parameters used to align two or more nucleotide sequences. The default general parameters are: Max targetsequences = 100; Short queries = "Automatically adjust parameters for short input sequences"; ExpectThreshold = 10; Word size = 28; Automatically adjust parameters for short input sequences = 0. The corresponding default scoring parameters are: Match / Mismatch Scores = 1, -2; Gap Costs = Linear.

[0029] Use the "Protein Blast" program (at http: / / blast.ncbi.nlm.nih.gov / ) to compare protein sequences. This program uses the BLASTP algorithm. Default parameters are used for the algorithm parameters when aligning two or more protein sequences. The default general parameters are: Maximum Target Sequence = 100; Short Query = Auto-adjust parameters for short input sequences; Expected Threshold = 10; Word Size = 3; Auto-adjust parameters for short input sequences = 0. Default scoring parameters are: Matrix = BLOSUM62; Gap Cost = Existence: 11; Extension: 1; Compositional Adjustments = Conditional compositional score matrix adjustment.

[0030] In the context of this invention, proteins such as TrxA, TrxB, TrxAB, TrxBA, TrxB5A, or MI-TrxBA begin with an uppercase letter, while the sequences encoding these proteins (also abbreviated as cds) are indicated by lowercase letters (trxA, trxB, trxAB, trxBA, trxB5A, or MI-trxBA).

[0031] In the context of this invention, the term fusion protein refers to a protein comprising two separate proteins: a thioreductase protein (Trx) and a thioreductase protein (TR). It is encoded by a gene whose coding region contains both a Trx coding region and a TR coding region, resulting in them being transcribed and translated together as a single unit into a polypeptide.

[0032] The specific advantages of using a fusion protein containing Trx and TR compared to using two separate proteins, Trx and TR, stem from the spatial position of the two proteins: TR transfers electrons to Trx, and Trx and TR do not need to first meet each other during three-dimensional diffusion; rather, they are linked together within the fusion protein. In other words, spatial proximity means that electrons can be more easily transferred from one enzyme to the other. While individual proteins must be purified separately, the enzyme activity in the fusion protein is isolated together, which is also a significant economic advantage.

[0033] Preferably, the fusion protein further includes a sequence as a purification aid, for example, particularly preferably, a polyhistidine tag (His-tag). A His-tag is a protein tag that can be used for protein purification and for detecting labeled 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 of the N-terminus after the start methionine codon or the C-terminus before the stop codon. This produces a fusion protein with a polyhistidine tag. Cloning only one His-tag allows for the purification of the fusion protein composed of proteins Trx and TR. This not only means fewer reaction steps required for purification but also simplifies the cloning strategy.

[0034] In addition, it is possible to insert a cleavage site of a protease or inteptide between the polypeptide sequence and the His-tag so that the His-tag can be cleaved after protein purification.

[0035] Preferably, the amino acid sequence of the N-terminal protein is shortened by 1 to 5 amino acids at the C-terminus of the fusion protein. Particularly preferred is that the amino acid sequence of the N-terminal protein is shortened by 1 amino acid at the C-terminus of the fusion protein.

[0036] Preferably, the amino acid sequence of the C-terminus protein is shortened by one to five amino acids at the N-terminus of the fusion protein, particularly preferably by one amino acid. Particularly preferably, the start codon is not present in the protein located at the C-terminus of the fusion protein. This latter feature has the advantage of eliminating the risk of restarting translation at that point.

[0037] In addition, the amino acid sequences of the preferred fusion protein activity of sulfur redox protein (protein i) and sulfur redox protein reductase (protein ii) are linked in the fusion protein by a linker sequence of 1 to 5 amino acids, particularly preferably 1 amino acid.

[0038] The amino acid sequences of the fusion protein thioreductase (protein i) and thioreductase (protein ii) are particularly preferred to follow each other directly in the fusion protein, i.e. there is no linker sequence.

[0039] As mentioned above, it is advantageous to combine synergistic proteins in fusion proteins to improve their cloning, purification, and activity. However, using long linker sequences would seem preferable to avoid hindering the folding of the two fusion proteins or the necessary interactions between them in this case. Unexpectedly, however, it was found that the direct fusion of the two proteins (Trx, TR) in this case yields a functional enzyme pair. This is highlighted by the finding that the activity of the fusion protein depends on the fusion sequence. Therefore, the fusion protein TrxAB exhibits activity comparable to a mixture of the individual proteins.

[0040] In the fusion protein TrxAB (SEQ ID No. 9), the stop codon is absent from the TrxA sequence (SEQ ID No. 7), while in TrxB (SEQ ID No. 8), the first amino acid (methionine, M) has been deleted. Furthermore, the protein carries a His-tag (amino acids 1-12 in SEQ ID NO: 9). After removing the start codon, there is no linker sequence between the last amino acid (Ala) of TrxA and the first amino acid (Gly) of TrxB.

[0041] In the fusion protein TrxBA (SEQ ID NO.10), the last amino acid (lysine) and the stop codon are absent in the TrxB sequence (SEQ ID NO.8), while the first amino acid (methionine, M) is missing in TrxA (SEQ ID NO.7). Furthermore, the protein carries a His-tag (amino acids 1-12 in SEQ ID NO.10). The linker sequence for the amino acid (Gly) is located between the penultimate amino acid (Ala) of TrxB and the first amino acid (Ser) of TrxA.

[0042] For the fusion protein TrxB5A (SEQ ID No. 28), it is the same as that described for TrxBA, except that there is a five-amino acid (Gly-Pro-Ala-Pro-Gly) linker sequence between TrxB and TrxA.

[0043] The region between the start and stop codons of the amino acid sequence encoding a fusion protein is called the coding sequence (cds). The cds are surrounded by non-coding regions. A gene is a portion of DNA containing all the essential information for producing a biologically functional protein. A gene contains the portion of DNA from which a single-stranded RNA copy is produced through transcription, along with expression signals involved in regulating this copying process. Expression signals include, for example, at least one promoter, transcription initiation, translation initiation, and a ribosome binding site. Furthermore, expression regulation is possible through terminators and one or more operons.

[0044] For a functional promoter, CDs regulated by that promoter are transcribed into RNA.

[0045] The DNA portion encoding protein i (thioreductase), the fusion protein, can be amplified first by PCR using oligonucleotides as primers and a DNA matrix encoding thioreductase (e.g., genomic DNA isolated from *E. coli*), and then coupled (in a manner that induces in-frame fusion) to a DNA molecule in each case (which contains the sequence of protein ii (thioreductase) and is generated in a similar manner) using standard molecular biology techniques. The second start codon of the C-terminus-localized fusion partner in the fusion protein between proteins i and ii can be deleted to avoid alternative reading frames. At the transition points of the two fusion partners, adapter sequences of different lengths can be inserted.

[0046] As an alternative to fusion at a cloning site, the entire DNA molecule can be generated through gene synthesis. This DNA molecule can then be introduced into a vector (e.g., a plasmid) or directly integrated into the chromosome of a bacterial strain using known methods. Preferably, the DNA molecule is introduced into a plasmid, such as a derivative of a known expression vector, like pJF118EH, pKK223-3, pUC18, pBR322, pACYC184, pASK-IBA3, pGJ3477, or pET.

[0047] Preferably, the DNA molecule primarily carries the coding sequence of the fusion protein and a few other nucleotides, and is thus cloned into the plasmid such that cloning occurs at distances defined by the promoter and terminator, specifically at the 3' position of the region encoding the promoter and the 5' position of the region encoding the terminator, in the plasmid.

[0048] Suitable promoters are all promoters known to those skilled in the art, such as constitutive promoters like the GAPDH promoter, or inducible promoters like lac, tac, trc, lambda PL, araB, cumate, or tet promoters or sequences derived therefrom. The fusion protein is particularly preferred to be expressed by the arabinose-inducible araB promoter (P... BAD Expression under the control of ).

[0049] The plasmids used can carry selection markers. Suitable selection markers are genes encoding resistance to antibiotics such as ampicillin, tetracycline, chloramphenicol, kanamycin, or other antibiotics. The plasmid preferably contains the gene that confers ampicillin resistance. Auxotrophic markers are also suitable as selection markers because they encode essential metabolic genes that are missing in the corresponding bacterial strains containing the plasmid.

[0050] Plasmids can be introduced into the cells of a bacterial strain (transformation) using methods known to those skilled in the art. The bacterial strain is preferably characterized as being a Gram-negative bacterium, particularly preferably a strain of the genus *Enterobacteriaceae*, and especially preferably a strain of the species *Escherichia coli* (E. coli).

[0051] Bacterial strains containing plasmids can be used in the fermentation process, whereby bacterial cells can be propagated in a culture medium, preferably in selective LB medium (corresponding to the selection marker present in the plasmid), and after cultivation, separated from the medium by precipitation, discarding the supernatant, and lysed. The expressed fusion protein can then be separated, for example, by affinity chromatography (e.g., via His-tag, see above).

[0052] The enzyme of the present invention has the following advantages: it can be produced in high yield and therefore inexpensive through fermentation. Its production is particularly efficient and advantageous because it requires the production, purification, and isolation of only one fusion protein instead of two separate proteins.

[0053] Advantageously, the enzyme can be regenerated by cofactors such as NADPH or NADH as electron donors and thus as reducing agents, since systems for regenerating NADPH or NADH already exist.

[0054] The enzymatic activities of Trx and TR can be determined by cofactor conversion, for example, by photometric measurement at 340 nm. This is possible because the extinction coefficient at 340 nm changes significantly when changing from a reducing (NADPH / NADH) state to an oxidizing (NADP / NAD) state.

[0055] When measuring the enzyme activity of various constructs (fusion proteins) or the enzyme activity of various mixing ratios of individual proteins, it is surprising and unexpected to those skilled in the art that the activity of the fusion protein is found to be at least 100%, preferably at least 150%, and particularly preferably at least 200% of the activity of a mixture of identical but unfused individual proteins, thioreductase and thioreductase. This means that the activity of the fusion protein is as high as or even higher than that of a mixture of identical but unfused individual proteins i and ii.

[0056] More precisely, this means that, except in the absence of a negative control (without cofactor or substrate), the same reaction mixture consisting of substrate, cofactor, and buffer system is initially loaded in each case. After adding the same amount of fusion protein to one mixture, or the same amount of individual proteins i (Trx) and ii (TR) to another mixture, the cofactor conversion rate is determined at the same time point and under the same reaction conditions. Alternatively, a decrease in substrate amount or an increase in product amount can also be detected.

[0057] The enzyme is preferably characterized in that the gene of the fusion protein contains a DNA sequence that is at least 50%, preferably at least 70%, and particularly preferably at least 90% identical to SEQ ID No. 2, and another DNA sequence that is at least 50%, preferably at least 70%, and particularly preferably at least 90% identical to SEQ ID No. 3.

[0058] In a particularly preferred embodiment, the DNA sequence encoding the fusion protein is SEQ ID NO.5.

[0059] The present invention further provides a method for enzymatically reducing cysteine ​​to homocysteine, characterized in that homocysteine ​​is reduced by the enzyme of the present invention in the presence of a cofactor.

[0060] In the enzymatic reduction of cystine to cysteine, the disulfide (-SS-) is converted into two thiol (-SH) groups, meaning that two molecules of L-cysteine ​​are formed from one molecule of the compound cysteine. The fusion protein of this invention catalyzes the reduction of the disulfide and transfers electrons to cysteine.

[0061] Surprisingly, the activity of the fusion protein TrxBA was significantly higher than that of a mixture of the same but unfused individual proteins TrxA and TrxB (see Example 4). Figure 4 ).

[0062] A particular advantage of the method of the present invention is that the cysteine ​​produced by the method can be declared as natural cysteine ​​according to the Regulation on Flavorings.

[0063] Another challenge of economical approaches is the poor solubility of cystine in buffer systems with a neutral pH range (or physiological pH range, i.e., around 7.4).

[0064] The method is preferably carried out in a pH range of 4-11, particularly preferably 5-10, and especially preferably 6-9.

[0065] This is particularly advantageous and therefore especially preferred when the enzyme of the present invention is used in a method for the enzymatic reduction of cystine to cysteine ​​at physiological pH.

[0066] The method is preferably carried out at a temperature of 20°C to 40°C, and particularly preferably from 25°C to 30°C.

[0067] The method is preferably characterized in that the cofactor is a substance selected from the group consisting of NADPH and NADH. NADPH is particularly preferred as the cofactor.

[0068] The method is preferably characterized in that, after reduction, the resulting cysteine ​​residues are separated. To further purify the target product, the following steps / procedures can be employed:

[0069] - Separation of L-cysteine ​​via ion exchange adsorption

[0070] -Precipitation and crystallization.

[0071] Such methods are known from the prior art (see, for example, WO 2013 / 000864).

[0072] During the reduction of cysteine ​​to cysteine, the enzyme of the present invention is oxidized, i.e., it transfers electrons to cysteine ​​through the formation of disulfide bonds. Since electrons from the present NADPH or NADH cofactors are transferred to the fusion protein (Trx moiety) through the activity of the fusion protein (TR moiety), NADP is formed respectively. + or NAD + Therefore, the process preferably includes a cofactor regenerating enzyme. Particularly preferably, the cofactor regenerating enzyme is a dehydrogenase, wherein the reduction occurs additionally in the presence of a separate electron donor.

[0073] 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, wherein isopropanol is used as an electron donor. Attached Figure Description

[0074] Figure 1 Image of pGJ3477 plasmid.

[0075] Figure 2 By oxidizing the cofactor NADPH to NADP + The scheme of reducing cysteine ​​to cysteine ​​catalyzed by cysteine ​​reductase.

[0076] Figure 3 A scheme involving the release of Cys-TNB and TNB, catalyzing the reduction of cysteine ​​(Cys-SH) and DTNB (5,5'-dithiobis-2-nitrobenzoic acid; Ellman's reagent) by cysteine ​​reductase.

[0077] Figure 4 : Relative cystine reductase activities of various enzymes.

[0078] Figure 5 Cysteine ​​formation was detected by DTNB assay using TrxBA and TrxAB chromatography.

[0079] Figure 6 The formation of cysteine ​​via TrxBA was measured using the DTNB assay in the presence and absence of a regeneration system.

[0080] Abbreviations used in the attached diagram

[0081] AraC: AraC gene (inhibitor gene)

[0082] pAraC: Promoter of the AraC gene (repressor gene; rev orientation against PBAD)

[0083] pBAD (also referred to as PBAD in the context of this invention): an arabinose-inducible promoter for expressing the (downstream) inserted target protein sequence.

[0084] 6HIS: The encoding area of ​​the His-tag

[0085] term: transcription terminator

[0086] Amp: Ampicillin resistance marker

[0087] CR: Cystine reductase

[0088] rel.: relative

[0089] bps: base pairs

[0090] t: time. Detailed Implementation

[0091] The invention will now be described in more detail with reference to exemplary embodiments, but the invention is not limited thereto.

[0092] Example

[0093] Example 1: Production of the cystine reductase system TrxA, TrxB, TrxBA, and TrxAB

[0094] Preparation of expression vectors:

[0095] The expression plasmid pGJ3477 was selected as the vector for expressing the DNA sequences encoding the corresponding candidate proteins TrxA, TrxB, TrxAB, TrxBA, TrxB5A, or MI-TrxBA. This is a medium- to high-copy plasmid (50-60 copies / cell) based on the ColE1 origin of replication. The plasmid diagram is in... Figure 1The image shows the location of a common, single-cleavage restriction enzyme (with a 6-base recognition sequence) on the plasmid map. This sequence is specified in SEQ ID No. 11.

[0096] In this plasmid, the coding sequence of the corresponding candidate is placed in the arabinose-inducible promoter P. BAD Under their control.

[0097] First, the entire expression plasmid was amplified by reverse PCR:

[0098] -50 ng of pGJ3477 DNA, 0.5 pmol of the corresponding primers 3477-fwrd (SEQ ID No. 12) and 3477-rev (SEQ ID No. 13), Reaction buffer (New England Biolabs, NEB), 1 unit DNA polymerase (NEB), final volume 50 μl.

[0099] -PCR program: 30 cycles of 1 min at 98°C, followed by 30 s at 98°C, 30 s at 65°C (annealing), and 2 min at 72°C (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 at 37°C for 1 hour. This was followed by chromatographic purification of the DNA (Macherey & Nagel). Gel and PCRClean-up-Kit).

[0101] Clones of trxA and trxB:

[0102] To clone the protein-coding sequences trxA (SEQ ID NO.2) and trxB (SEQ ID NO.3), oligonucleotide primers were defined with target gene-specific sequences extended by at least 15 nucleotides, overlapping the sequences at the ends of the vector DNA. These genes were amplified from the genome of *E. coli* BL21 by PCR (cloning PCR).

[0103] Select the following mixtures for PCR amplification:

[0104] -50 ng of E. coli BL21 genomic DNA (NEB), 0.5 pmol of the corresponding 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), Reaction buffer (NEB), 1 unit DNA polymerase (NEB), final volume 50 μl.

[0105] -PCR program: 30 cycles of 1 min at 98°C, followed by 30 s at 98°C, 30 s at 60°C (annealing), and 15 s at 72°C (trxA) or 30 s at trxB (synthesis).

[0106] At the end of the reaction, the restriction enzyme DpnI (10 units, NEB) was added to the reaction mixture and the mixture was incubated at 37°C for 1 hour. This was followed by chromatographic purification of the DNA (Macherey & Nagel). Gel and PCRClean-up-Kit).

[0107] LIC-PCR:

[0108] Typically, the DNA sequence bases encoding the protein are precisely introduced into the basic expression vector pGJ3477 via LIC-PCR (ligation-independent cloning of PCR products) as described in Aslanidis C. and de Jong PJ, Nucleic Acids Res. 18, pp. 6069-6074.

[0109] For this purpose, 50 ng of purified encoding DNA of the corresponding candidate protein and 50 ng of prepared vector DNA were used in the LIC-PCR reaction. The LIC-PCR mixture was then transformed into *E. coli* XL1 Blue cells using standard methods and plated on selective LB medium (LB + 100 mg / L ampicillin) and incubated at 37°C for 18 h. To identify the correct clone, plasmid DNA was isolated from the obtained colonies and the expression cassette was fully sequenced.

[0110] The plasmid obtained by combining the expression vector pGJ3477 and the encoding DNA sequence trxA or trxB is referred to as the trxA or trxB expression vector below.

[0111] Clones of trxAB and trxBA:

[0112] The DNA clones encoding the corresponding fusion proteins TrxAB or TrxBA were cloned in a manner similar to that used for cloning trxA and trxB, respectively. The TrxA sequence was inserted into the trxB expression vector via LIC-PCR between the N-terminal his tag and the trxB sequence encoding the protein TrxB, or the TrxB sequence was inserted into the trxA expression vector via LIC-PCR between the his tag and the trxA sequence encoding the protein TrxA.

[0113] Select the following mixtures for PCR amplification:

[0114] - Amplify the vector DNA (trxA or trxB expression vector) by reverse 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), selecting the following PCR program: 30 cycles of 98°C for 2 minutes, followed by 45 seconds at 98°C, 30 seconds at 60°C, and 2.5 minutes at 72°C.

[0115] - The trxA or trxB gene fragment of the fusion protein was 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), with the following PCR program selected: 30 cycles of 98°C for 2 minutes, followed by 45 seconds at 98°C, 30 seconds at 60°C, and 15 seconds at 72°C. BL21 genomic DNA was used as a template.

[0116] At the end of the PCR reaction, restriction enzyme DpnI (10 units, NEB) was added to the corresponding reaction mixture, and the mixture was incubated at 37°C for 1 hour. This was followed by chromatographic purification of the inserted DNA (Macherey & Nagel). Gel and PCR Clean-up Kit).

[0117] LIC-PCR was performed using 50 ng of amplified vector DNA of the trxA or trxB expression vector and 75 ng of insert DNA (trxB or trxA).

[0118] The LIC-PCR mixture was then transformed into *E. coli* XL1 Blue cells using standard methods and plated on selective LB medium (LB + 100 mg / L ampicillin) and incubated at 37°C for 18 h. To identify the correct clone, plasmid DNA was isolated from the obtained colonies and the expression cassette was fully sequenced.

[0119] SEQ ID NO.9 and SEQ ID NO.10 specify the amino acid sequences of the fusion proteins TrxBA and TrxAB obtained from the expression of sequences SEQ ID NO.4 and SEQ ID NO.5, respectively.

[0120] Example 2: Cloning of cystine reductase MI-TrxBA

[0121] As mentioned in the introduction, *Mycobacterium leprae* possesses a gene fragment encoding a protein homologous to thioredoxin (Trx) and thioredoxin reductase (TR) (Wieles B. et al. 1995, J. Biol. Chem. 270, pp. 25604-25606). This sequence can be obtained from a publicly available database (NCBI Reference Sequence: WP_010909042.1).

[0122] A custom sequence derived from a public database was used to target the codons of the host *E. coli*. This was accomplished using the IDT web server (www.idtdna.com). To clone into the target vector pGJ3477, the coding region was extended at the 5' and 3' ends by sequences overlapping with the vector sequence (see also cloning of trxA and trxB). The resulting total DNA sequence was synthetically produced by Geneart (www.thermofisher.com) (as specified in SEQ ID No. 24) and named MI-trxBA (also known as mI-trxBA).

[0123] The synthesized MI-trxBA sequence was cloned into the vector pGJ3477 in a manner similar to that used for cloning trxA and trxB expression vectors via LIC-PCR.

[0124] To clone the protein-coding sequence MI-trxBA (SEQ ID NO.24), oligonucleotide primers were defined with a target gene-specific sequence extended by at least 15 nucleotides, overlapping the sequence at the end of the vector DNA. The synthetic gene served as a template.

[0125] Select the following mixtures for PCR amplification:

[0126] -20 ng template (Geneart), 0.5 pmol corresponding primers MI-trxBA-fwrd (SEQ ID No. 25) and MI-trxBA-rev (SEQ ID No. 26), Reaction buffer, 1 unit DNA polymerase (NEB), final volume 50 μl.

[0127] -PCR program: 30 cycles of 1 min at 98°C, followed by 30 s at 98°C, 30 s at 60°C (annealing), and 60 s at 72°C (synthesis).

[0128] At the end of the reaction, the restriction enzyme DpnI (10 units, NEB) was added to the reaction mixture and the mixture was incubated at 37°C for 1 hour. This was followed by chromatographic purification of the DNA (Macherey & Nagel). Gel and PCRClean-up-Kit).

[0129] LIC-PCR:

[0130] As described in Aslanidis C. and de Jong PJ, Nucleic Acids Res. 18, pp. 6069-6074, the DNA sequence bases encoding the protein were precisely introduced into the basic expression vector pGJ3477 by LIC-PCR (ligation-independent cloning of PCR products).

[0131] For this purpose, 50 ng of purified coding DNA and 60 ng of prepared vector DNA were used together in a LIC-PCR reaction.

[0132] The LIC-PCR mixture was then transformed into *E. coli* XL1 Blue cells using standard methods and plated on selective LB medium (LB + 100 mg / L ampicillin) and incubated at 37°C for 18 h. To identify the correct clone, plasmid DNA was isolated from the obtained colonies and the expression cassette was fully sequenced.

[0133] The plasmid obtained by combining the expression vector pGJ3477 and the encoding DNA sequence MI-trxBA is referred to below as the MI-trxBA expression vector.

[0134] Example 3: Cloning of cystine reductase TrxB5A (with adapter sequence)

[0135] Cloning of trxB5A:

[0136] The DNA encoding the corresponding fusion protein TrxB5A was cloned in a manner similar to that used for cloning trxBA, by inserting a TrxB sequence (hereinafter referred to as TrxB5, and the cds are also referred to as trxB5) extending 5 amino acids from the C-terminus between the his-tag and the sequence encoding TrxA into the trxA expression vector via LIC-PCR.

[0137] Select the following mixtures for PCR amplification:

[0138] - The vector DNA (trxA expression vector) was amplified by reverse PCR using primers vtrxA5-fwrd (SEQ ID No. 29) and 3477-rev (SEQ ID No. 13), and the following PCR program was selected for it: 30 cycles of 2 min at 98°C, followed by 45 s at 98°C, 30 s at 60°C, and 2.5 min at 72°C.

[0139] - trxB5 was amplified by PCR using primers ftrxB-fwrd (SEQ ID No. 19) and ftrxB5-rev (SEQ ID No. 30), and the following PCR program was selected for it: 30 cycles of 2 min at 98°C, followed by 45 s at 98°C, 30 s at 60°C, and 15 s at 72°C. BL21 genomic DNA was used as a template.

[0140] At the end of the PCR reaction, restriction enzyme DpnI (10 units, NEB) was added to the corresponding reaction mixture, and the mixture was incubated at 37°C for 1 hour. This was followed by chromatographic purification of the inserted DNA (Macherey & Nagel). Gel and PCR Clean-up Kit).

[0141] LIC-PCR was performed using 50 ng of amplified vector DNA from the trxA expression vector and 75 ng of insert DNA (trxB5).

[0142] The LIC-PCR mixture was then transformed into *E. coli* XL1 Blue cells using standard methods and plated on selective LB medium (LB + 100 mg / L ampicillin) and incubated at 37°C for 18 h. To identify the correct clone, plasmid DNA was isolated from the obtained colonies and the expression cassette was fully sequenced.

[0143] SEQ ID NO.28 specifies the amino acid sequence of the fusion protein TrxB5A generated by the expression of the sequence SEQ ID NO.27.

[0144] Example 4: Enzymatic activity of cystine reductase

[0145] For recombinant expression of proteins TrxA, TrxB, TrxBA, TrxB5A, TrxAB, and MI-TrxBA, expression plasmids encoding the corresponding proteins were introduced into *Escherichia coli* strain TOP10 (Thermo Fisher Scientific, Massachusetts, USA). Bacterial cells were plated on LB medium containing 100 mg / L ampicillin. Single colonies were then inoculated into 25 ml LB medium containing 100 mg / L ampicillin and 0.2% arabinose (w / v) and incubated in a shaker at 28°C for 18 h.

[0146] To isolate the expressed protein, cells were precipitated by centrifugation (4000g for 10 min), the culture supernatant was removed, and the biomass was resuspended in lysis buffer (PBS + 10% BugBuster (Sigma)). Cells were completely lysed at room temperature for 15 min according to the manufacturer's instructions in the BugBuster kit (Sigma) manual, and then insoluble material was removed by centrifugation (9500 rpm for 20 min according to the manufacturer's instructions). To purify the supernatant (cell lysate) by affinity chromatography, the obtained cell lysate was loaded into a PBS-equilibrated Protino IDA 2000 column (Macherey & Nagel). After a washing step (7 ml PBS), the sample was eluted with 5 ml elution buffer (PBS + 200 mM imidazole). The protein concentration was determined using a Bradford assay (Thermo Fisher).

[0147] The cystine reductase activities of different enzymes TrxA, TrxB, TrxBA, TrxB5A, TrxAB, or MI-TrxBA were determined using two analytical methods described in detail below.

[0148] 1. The first method consists of a photometric detection method, which is based on the depletion of NADPH and the decrease in absorbance at 340 nm during the reduction reaction. For example... Figure 2 As illustrated in the diagram, the enzymatic conversion of cysteine ​​to cysteine ​​is accompanied by the consumption of the cofactor NADPH.

[0149] The following detection mixture was used for photometric determination, with a final sample volume of 1 ml:

[0150] • 100mM phosphate buffer, pH 7.4

[0151] 2mM EDTA

[0152] ·0.2mM NADPH (Sigma)

[0153] ·1mM Cystine (Wacker Chemie AG)

[0154] • After mixing all detection components except the enzyme, the reaction is initiated by adding the following enzyme:

[0155] 10 μg of enzymes TrxA, TrxB, a mixture of TrxA and TrxB, TrxBA, TrxB5A, TrxAB, or MI-TrxBA. Incubate the assay mixture at room temperature (approximately 25°C) until the measurements described below.

[0156] The NADPH concentration in different 1 ml assay mixtures was measured at different time points using an Evolution 201 UV-vis spectrophotometer (Thermo Fisher Scientific). The absorbance at 340 nm was used to calculate the enzyme activity of different samples using Thermo INSIGHT software from Thermo Fisher Scientific. Samples containing heat-inactivated enzyme, without enzyme, without cysteine ​​as a substrate, or without NADPH as a cofactor were analyzed in parallel as negative controls in each case.

[0157] Enzyme activity is determined by the linear slope of the recorded curve, where the change (decrease) in absorbance over time indicates the rate of NADPH consumption. Since the same amount of enzyme is always used, these rates can be compared.

[0158] 2. In the second method, cysteine, formed from cystine by cystine reductase activity, is directly detected via the free SH group through a reaction with the compound DTNB (5,5'-dithiobis-2-nitrobenzoic acid; Ellman's reagent). Figure 3 As illustrated, and also referred to in the context of this invention as DTNB determination.

[0159] The following detection mixture was used for photometric determination, with a final sample volume of 1 ml:

[0160] • 100mM phosphate buffer, pH 7.4

[0161] 2mM EDTA

[0162] ·0.2mM NADPH (Sigma)

[0163] ·1mM Cystine (Wacker Chemie AG)

[0164] • After mixing all detection components except the enzyme, the reaction is initiated by adding the following enzyme:

[0165] 10 μg of enzymes TrxA, TrxB, a mixture of TrxA and TrxB, TrxBA, TrxB5A, TrxAB, or MI-TrxBA. Incubate the assay mixture at room temperature (approximately 25°C) until the measurements described below.

[0166] L-cysteine ​​was quantified using the assay described in Lee S.-H. et al. 1995 (Biochemical and Biophysical Research Communications 213, pp. 837-844) with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB). Cys-TNB quantification was performed by measuring DTNB-mediated absorbance at 412 nm. HPLC analysis was also possible.

[0167] In each case, parallel analyses were performed as negative controls on samples containing heat-inactivated enzymes, without enzymes, without cystine as a substrate, or without NADPH as a cofactor.

[0168] Figure 4 The relative cystine reductase activities of enzymes TrxBA, TrxB5A, TrxAB, and MI-Trx, as well as a (9:1) mixture of TrxA and TrxB, are shown by photometric determination as described in point 1 below. The activity of the (9:1) mixture of TrxA and TrxB is normalized to 1, along with all other associated activity sets. The (9:1) mixture of TrxA and TrxB is an estimated mixing ratio of the individual enzymes that has been shown to be the most active by comparative testing.

[0169] Figure 5 The results show that cysteine ​​formation was detected by DTNB and by HPLC using TrxBA and TrxAB as described in point 2 above.

[0170] The use of clones containing a longer linker sequence than TrxB5A in a region of ≥60 nucleotides between the cds of TrxA and TrxB unexpectedly resulted in significantly lower activity in the fusion protein.

[0171] Example 5: Activity of the combination of cystine reductase and ADH enzyme

[0172] For cofactors NADPH and NADP +Comparison of enzyme specificity revealed that cystine reductases TrxBA and TrxAB are highly specific for the cofactor NADPH. In these tests, NADP... + No enzyme activity targeting cystine was detected.

[0173] A joint experiment investigated the ability of alcohol dehydrogenase (ADH) to convert NADP... + The extent to which NADPH is converted back for use in the reaction.

[0174] For this purpose, the following conditions shall be used:

[0175] • 100mM phosphate buffer, pH 7.4

[0176] 5 μl of isopropanol

[0177] 2mM EDTA

[0178] 15-50 μM NADPH or NADP +

[0179] 1mM Cystine

[0180] 5 μg of enzyme TrxBA

[0181] • 50 μl of crude cell lysate extract or, as a negative control, 50 μl of phosphate-buffered saline (0.5 ml of cells in 1.5 ml of 4x phosphate-buffered saline (pH 7.4) for rapid preparative digestion. (Cell production is described in EP 1 832 658 B1)

[0182] The reaction was incubated at 30°C for 60 minutes. Samples were taken at 5-minute intervals, and the released cysteine ​​was derivatized with DTNB as described in WO 2013 / 000864 A1. Quantification was performed by measuring DTNB-mediated absorbance at 412 nm.

[0183] Figure 6 The results of cysteine ​​formation measured by TrxBA using DTNB detection are shown in the presence (with) or absence (without) of ADH as a regeneration system. sequence list <110> Wacker Chemie AG <120> Biocatalysts are a core component of an enzyme-catalyzed redox system for the biocatalytic reduction of cystine. <130> CO11913 <160> 30 <170> PatentIn version 3.5 <210> 1 <211> 36 <212> DNA <213> artificial <220> <223> his-tag <400> 1 atgacacaga gggcccacca tcaccatcac cattcc 36 <210> 2 <211> 330 <212> DNA <213> E. coli <220> <221> trxA <222> (1)..(330) <400> 2 atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60 gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120 ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180 atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240 ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300 aaagagttcc tcgacgctaa cctggcgtaa 330 <210> 3 <211> 966 <212> DNA <213> Escherichia coli <220> <221> trxB <222> (1)..(966) <400> 3 atgggcacga ccaaacacag taaactgctt atcctgggtt caggcccggc gggatacacc 60 gctgctgtct acgcggcgcg cgccaacctg caacctgtgc tgattaccgg catggaaaaa 120 ggcggccaac tgaccaccac cacggaagtg gaaaactggc ctggcgatcc aaacgatctg 180 accggtccgt tattaatgga gcgcatgcac gaacatgcca ccaagtttga aactgagatc 240 atttttgatc atatcaacaa ggtggatctg caaaaccgtc cgttccgtct gaatggcgat 300 aacggcgaat acacttgcga cgcgctgatt attgccaccg gagcttctgc acgctatctc 360 ggcctgccct ctgaagaagc ctttaaaggc cgtggggttt ctgcttgtgc aacctgcgac 420 ggtttcttct atcgcaacca gaaagttgcg gtcatcggcg gcggcaatac cgcggttgaa 480 gaggcgctgt atctgtctaa catcgcttcg gaagtgcatc tgattcaccg ccgtgacggt 540 ttccgcgcgg aaaaaatcct cattaagcgc ctgatggata aagtggagaa cggcaacatc 600 attctgcaca ccaaccgtac gctggaagaa gtgaccggcg atcaaatggg tgtcactggc 660 gttcgtctgc gcgatacgca aaacagcgat aacatcgagt cactcgacgt tgccggtctg 720 tttgttgcta tcggtcacag cccgaatact gcgattttcg aagggcagct ggaactggaa 780 aacggctaca tcaaagtaca gtcgggtatt catggtaatg ccacccagac cagcattcct 840 ggcgtctttg ccgcaggcga cgtgatggat cacatttatc gccaggccat tacttcggcc 900 ggtacaggct gcatggcagc acttgatgcg gaacgctacc tcgatggttt agctgacgca 960 aaataa 966 <210> 4 <211> 1326 <212> DNA <213> Artificial <220> <223> histrxAB (Fusion gene of his-tag and Escherichia coli genes trxA and trxB) <220> <221> histrxAtrxB <222> (1)..(1326) <400> 4 atgacacaga gggcccacca tcaccatcac cattccatga gcgataaaat tattcacctg 60 actgacgaca gttttgacac ggatgtactc aaagcggacg gggcgatcct cgtcgatttc 120 tgggcagagt ggtgcggtcc gtgcaaaatg atcgccccga ttctggatga aatcgctgac 180 gaatatcagg gcaaactgac cgttgcaaaa ctgaacatcg atcaaaaccc tggcactgcg 240 ccgaaatatg gcatccgtgg tatcccgact ctgctgctgt tcaaaaacgg tgaagtggcg 300 gcaaccaaag tgggtgcact gtctaaaggt cagttgaaag agttcctcga cgctaacctg 360 gcgggcacga ccaaacacag taaactgctt atcctgggtt caggcccggc gggatacacc 420 gctgctgtct acgcggcgcg cgccaacctg caacctgtgc tgattaccgg catggaaaaa 480 ggcggccaac tgaccaccac cacggaagtg gaaaactggc ctggcgatcc aaacgatctg 540 accggtccgt tattaatgga gcgcatgcac gaacatgcca ccaagtttga aactgagatc 600 atttttgatc atatcaacaa ggtggatctg caaaaccgtc cgttccgtct gaatggcgat 660 aacggcgaat acacttgcga cgcgctgatt attgccaccg gagcttctgc acgctatctc 720 ggcctgccct ctgaagaagc ctttaaaggc cgtggggttt ctgcttgtgc aacctgcgac 780 ggtttcttct atcgcaacca gaaagttgcg gtcatcggcg gcggcaatac cgcggttgaa 840 gaggcgctgt atctgtctaa catcgcttcg gaagtgcatc tgattcaccg ccgtgacggt 900 ttccgcgcgg aaaaaatcct cattaagcgc ctgatggata aagtggagaa cggcaacatc 960 attctgcaca ccaaccgtac gctggaagaa gtgaccggcg atcaaatggg tgtcactggc 1020 gttcgtctgc gcgatacgca aaacagcgat aacatcgagt cactcgacgt tgccggtctg 1080 tttgttgcta tcggtcacag cccgaatact gcgattttcg aagggcagct ggaactggaa 1140 aacggctaca tcaaagtaca gtcgggtatt catggtaatg ccacccagac cagcattcct 1200 ggcgtctttg ccgcaggcga cgtgatggat cacatttatc gccaggccat tacttcggcc 1260 ggtacaggct gcatggcagc acttgatgcg gaacgctacc tcgatggttt agctgacgca 1320 aaataa 1326 <210> 5 <211> 1326 <212> DNA <213> Artificial <220> <223> histrxBA (Fusion gene of his-tag and Escherichia coli genes trxB and trxA) <220> <221> histrxBtrxA <222> (1)..(1326) <400> 5 atgacacaga gggcccacca tcaccatcac cattccatgg gcacgaccaa acacagtaaa 60 ctgcttatcc tgggttcagg cccggggggga tacaccgctg ctgtctacgc ggcgcgcgcc 120 aacctgcaac ctgtgctgat taccggcatg gaaaaaggcg gccaactgac caccaccacg 180 gaagtggaaa actggcctgg cgatccaaac gatctgaccg gtccgttatt aatggagcgc 240 atgcacgaac atgccaccaa gtttgaaact gagatcattt ttgatcatat caacaaggtg 300 gatctgcaaa accgtccgtt ccgtctgaat ggcgataacg gcgaatacac ttgcgacgcg 360 ctgattattg ccaccggagc ttctgcacgc tatctcggcc tgccctctga agaagccttt 420 aaaggccgtg gggtttctgc ttgtgcaacc tgcgacggtt tcttctatcg caaccagaaa 480 gttgcggtca tcggcggcgg caataccgcg gttgaagagg cgctgtatct gtctaacatc 540 gcttcggaag tgcatctgat tcaccgccgt gacggtttcc gcgcggaaaa aatcctcatt 600 aagcgcctga tggataaagt ggagaacggc aacatcattc tgcacaccaa ccgtacgctg 660 gaaagtga ccgggggatca aatgggtgtc actggcgttc gtctgcgcga tacgcaaaac 720 agcgataaca tcgagtcact cgacgttgcc ggtctgtttg ttgctatcgg tcacagcccg 780 aatactgcga ttttcgaagg gcagctgggaa ctggaaaacg gctacatcaa agtacagtcg 840 ggtattcatg gtaatgccac ccagaccagc attcctggcg tctttgccgc aggcgacgtg 900 atggatcaca tttatcgcca ggccattact tcggccggta caggctgcat ggcagcactt 960 gatgcggaac gctacctcga tggtttagct gacgcaggta gcgataaaat tattcacctg 1020 actgacgaca gttttgacac ggatgtactc aaagcggacg gggcgatcct cgtcgatttc 1080 tgggcagagt ggtgcggtcc gtgcaaaatg atcgccccga ttctggatga aatcgctgac 1140 gaatatcagg gcaaactgac cgttgcaaaa ctgaacatcg atcaaaaccc tggcactgcg 1200 ccgaaatatg gcatccgtgg tatcccgact ctgctgctgt tcaaaaacgg tgaagtggcg 1260 gcaaccaaag tgggtgcact gtctaaaggt cagttgaaag agttcctcga cgctaacctg 1320 gcgtaa 1326 <210> 6 <211> 12 <212> PRT <213> Artificial <220> <223> His-tag <220> <221> His-tag <222> (1)..(12) <400> 6 Met Thr Gln Arg Ala His His His His His His Ser 1 5 10 <210> 7 <211> 109 <212> PRT <213> Escherichia coli <220> <221> TrxA <222> (1)..(109) <400> 7 Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp 1 5 10 15 Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45 Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50 55 60 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 65 70 75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala 100 105 <210> 8 <211> 321 <212> PRT <213> Escherichia coli <220> <221> TrxB <222> (1)..(321) <400> 8 Met Gly Thr Thr Lys His Ser Lys Leu Leu Ile Leu Gly Ser Gly Pro 1 5 10 15 Ala Gly Tyr Thr Ala Ala Val Tyr Ala Ala Arg Ala Asn Leu Gln Pro 20 25 30 Val Leu Ile Thr Gly Met Glu Lys Gly Gly Gln Leu Thr Thr Thr Thr 35 40 45 Glu Val Glu Asn Trp Pro Gly Asp Pro Asn Asp Leu Thr Gly Pro Leu 50 55 60 Leu Met Glu Arg Met His Glu His Ala Thr Lys Phe Glu Thr Glu Ile 65 70 75 80 Ile Phe Asp His Ile Asn Lys Val Asp Leu Gln Asn Arg Pro Phe Arg 85 90 95 Leu Asn Gly Asp Asn Gly Glu Tyr Thr Cys Asp Ala Leu Ile Ile Ala 100 105 110 Thr Gly Ala Ser Ala Arg Tyr Leu Gly Leu Pro Ser Glu Glu Ala Phe 115 120 125 Lys Gly Arg Gly Val Ser Ala Cys Ala Thr Cys Asp Gly Phe Phe Tyr 130 135 140 Arg Asn Gln Lys Val Ala Val Ile Gly Gly Gly Asn Thr Ala Val Glu 145 150 155 160 Glu Ala Leu Tyr Leu Ser Asn Ile Ala Ser Glu Val His Leu Ile His 165 170 175 Arg Arg Asp Gly Phe Arg Ala Glu Lys Ile Leu Ile Lys Arg Leu Met 180 185 190 Asp Lys Val Glu Asn Gly Asn Ile Ile Leu His Thr Asn Arg Thr Leu 195 200 205 Glu Glu Val Thr Gly Asp Gln Met Gly Val Thr Gly Val Arg Leu Arg 210 215 220 Asp Thr Gln Asn Ser Asp Asn Ile Glu Ser Leu Asp Val Ala Gly Leu 225 230 235 240 Phe Val Ala Ile Gly His Ser Pro Asn Thr Ala Ile Phe Glu Gly Gln 245 250 255 Leu Glu Leu Glu Asn Gly Tyr Ile Lys Val Gln Ser Gly Ile His Gly 260 265 270 Asn Ala Thr Gln Thr Ser Ile Pro Gly Val Phe Ala Ala Gly Asp Val 275 280 285 Met Asp His Ile Tyr Arg Gln Ala Ile Thr Ser Ala Gly Thr Gly Cys 290 295 300 Met Ala Ala Leu Asp Ala Glu Arg Tyr Leu Asp Gly Leu Ala Asp Ala 305 310 315 320 Lys <210> 9 <211> 441 <212> PRT <213> artificial <220> <223> HisTrxAB (a fusion protein of His-tag and E. coli proteins TrxA and TrxB) <220> <221> HisTrxAB <222> (1)..(441) <400> 9 Met Thr Gln Arg Ala His His His His His Ser Met Ser Asp Lys 1 5 10 15 Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala 20 25 30 Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys 35 40 45 Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly 50 55 60 Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala 65 70 75 80 Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn 85 90 95 Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu 100 105 110 Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Thr Thr Lys His Ser Lys 115 120 125 Leu Leu Ile Leu Gly Ser Gly Pro Ala Gly Tyr Thr Ala Ala Val Tyr 130 135 140 Ala Ala Arg Ala Asn Leu Gln Pro Val Leu Ile Thr Gly Met Glu Lys 145 150 155 160 Gly Gly Gln Leu Thr Thr Thr Thr Glu Val Glu Asn Trp Pro Gly Asp 165 170 175 Pro Asn Asp Leu Thr Gly Pro Leu Leu Met Glu Arg Met His Glu His 180 185 190 Ala Thr Lys Phe Glu Thr Glu Ile Ile Phe Asp His Ile Asn Lys Val 195 200 205 Asp Leu Gln Asn Arg Pro Phe Arg Leu Asn Gly Asp Asn Gly Glu Tyr 210 215 220 Thr Cys Asp Ala Leu Ile Ile Ala Thr Gly Ala Ser Ala Arg Tyr Leu 225 230 235 240 Gly Leu Pro Ser Glu Glu Ala Phe Lys Gly Arg Gly Val Ser Ala Cys 245 250 255 Ala Thr Cys Asp Gly Phe Phe Tyr Arg Asn Gln Lys Val Ala Val Ile 260 265 270 Gly Gly Gly Asn Thr Ala Val Glu Glu Ala Leu Tyr Leu Ser Asn Ile 275 280 285 Ala Ser Glu Val His Leu Ile His Arg Arg Asp Gly Phe Arg Ala Glu 290 295 300 Lys Ile Leu Ile Lys Arg Leu Met Asp Lys Val Glu Asn Gly Asn Ile 305 310 315 320 Ile Leu His Thr Asn Arg Thr Leu Glu Glu Val Thr Gly Asp Gln Met 325 330 335 Gly Val Thr Gly Val Arg Leu Arg Asp Thr Gln Asn Ser Asp Asn Ile 340 345 350 Glu Ser Leu Asp Val Ala Gly Leu Phe Val Ala Ile Gly His Ser Pro 355 360 365 Asn Thr Ala Ile Phe Glu Gly Gln Leu Glu Leu Glu Asn Gly Tyr Ile 370 375 380 Lys Val Gln Ser Gly Ile His Gly Asn Ala Thr Gln Thr Ser Ile Pro 385 390 395 400 Gly Val Phe Ala Ala Gly Asp Val Met Asp His Ile Tyr Arg Gln Ala 405 410 415 Ile Thr Ser Ala Gly Thr Gly Cys Met Ala Ala Leu Asp Ala Glu Arg 420 425 430 Tyr Leu Asp Gly Leu Ala Asp Ala Lys 435 440 <210> 10 <211> 441 <212> PRT <213> Artificial <220> <223> HisTrxBA (His-tag fused with E. coli proteins TrxB and TrxA) <220> <221> His-TrxBA <222> (1)..(441) <400> 10 Met Thr Gln Arg Ala His His His His His His Ser Met Gly Thr Thr 1 5 10 15 Lys His Ser Lys Leu Leu Ile Leu Gly Ser Gly Pro Ala Gly Tyr Thr 20 25 30 Ala Ala Val Tyr Ala Ala Arg Ala Asn Leu Gln Pro Val Leu Ile Thr 35 40 45 Gly Met Glu Lys Gly Gly Gln Leu Thr Thr Thr Thr Glu Val Glu Asn 50 55 60 Trp Pro Gly Asp Pro Asn Asp Leu Thr Gly Pro Leu Leu Met Glu Arg 65 70 75 80 Met His Glu His Ala Thr Lys Phe Glu Thr Glu Ile Ile Phe Asp His 85 90 95 Ile Asn Lys Val Asp Leu Gln Asn Arg Pro Phe Arg Leu Asn Gly Asp 100 105 110 Asn Gly Glu Tyr Thr Cys Asp Ala Leu Ile Ile Ala Thr Gly Ala Ser 115 120 125 Ala Arg Tyr Leu Gly Leu Pro Ser Glu Glu Ala Phe Lys Gly Arg Gly 130 135 140 Val Ser Ala Cys Ala Thr Cys Asp Gly Phe Phe Tyr Arg Asn Gln Lys 145 150 155 160 Val Ala Val Ile Gly Gly Gly Asn Thr Ala Val Glu Glu Ala Leu Tyr 165 170 175 Leu Ser Asn Ile Ala Ser Glu Val His Leu Ile His Arg Arg Asp Gly 180 185 190 Phe Arg Ala Glu Lys Ile Leu Ile Lys Arg Leu Met Asp Lys Val Glu 195 200 205 Asn Gly Asn Ile Ile Leu His Thr Asn Arg Thr Leu Glu Glu Val Thr 210 215 220 Gly Asp Gln Met Gly Val Thr Gly Val Arg Leu Arg Asp Thr Gln Asn 225 230 235 240 Ser Asp Asn Ile Glu Ser Leu Asp Val Ala Gly Leu Phe Val Ala Ile 245 250 255 Gly His Ser Pro Asn Thr Ala Ile Phe Glu Gly Gln Leu Glu Leu Glu 260 265 270 Asn Gly Tyr Ile Lys Val Gln Ser Gly Ile His Gly Asn Ala Thr Gln 275 280 285 Thr Ser Ile Pro Gly Val Phe Ala Ala Gly Asp Val Met Asp His Ile 290 295 300 Tyr Arg Gln Ala Ile Thr Ser Ala Gly Thr Gly Cys Met Ala Ala Leu 305 310 315 320 Asp Ala Glu Arg Tyr Leu Asp Gly Leu Ala Asp Ala Gly Ser Asp Lys 325 330 335 Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala 340 345 350 Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys 355 360 365 Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly 370 375 380 Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala 385 390 395 400 Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn 405 410 415 Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu 420 425 430 Lys Glu Phe Leu Asp Ala Asn Leu Ala 435 440 <210> 11 <211> 4100 <212> DNA <213> Artificial <220> <223> pGJ3477 (vector sequence) <220> <221> pGJ3477 <222> (1)..(4100) <400> 11 aaaccaattg tccatattgc atcagacatt gccgtcactg cgtcttttac tggctcttct 60 cgctaaccaa accggtaacc ccgcttatta aaagcattct gtaacaaagc gggaccaaag 120 ccatgacaaa aacgcgtaac aaaagtgtct ataatcacgg cagaaaagtc cacattgatt 180 atttgcacgg cgtcacactt tgctatgcca tagcattttt atccataaga ttagctgatc 240 ctacctgacg ctttttatcg caactctcta ctgtttctcc atacccgttt aaataatttt 300 gtttaacttt aagaaggaga tatacccatg acacagaggg cccaccatca ccatcaccat 360 tccggatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga 420 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa 480 aggaggaact atatccggcc ggatatccac aggacgggtg tggtcgccat gatcgcgtag 540 tcgatagtgg ctccaagtag cgaagcgagc aggactgggc ggcggccaaa gcggtcggac 600 agtgctccga gaacgggtgc gcatagaaat tgcatcaacg catatagcgc tagcagcacg 660 ccatagtgac tggcgatgct gtcggaatgg acgatatccc gcaagaggcc cggcagtacc 720 ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat gacgatgagc 780 gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg tgataaacta 840 ccgcattaaa gcttatcgat gataagctgt caaacatgag aattcttgaa gacgaaaggg 900 cctcgtgata cgcttttt tataggttaa tgtcatgcat gagaataa ccctgataaa 960 tgcttcaata atattgaaaa aggaaagta tgagtattca acatttccgt gtcgcccctta 1020 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 1080 taaaagatgc tgaagatcag ttgggtgcac gagtggggtta catcgaactg gatctcaaca 1140 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 1200 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc 1260 gccgcataca ctattctcag aatgacttgg ttgacgcgtc accagtcaca gaaaagcatc 1320 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 1380 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 1440 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 1500 1560 1620 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 1680 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 1740 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 1800 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 1860 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 1920 aggtgaagat cctttttgat aatctcatgc atgaccaaaa tcccttaacg tgagttttcg 1980 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 2040 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 2100 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 2160 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 2220 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 2280 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 2340 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2400 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2460 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2520 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2580 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2640 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 2700 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 2760 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt 2820 acgcatctgt gcggtatttc acaccgcata tatggtgcac tctcagtaca atctgctctg 2880 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc 2940 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 3000 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 3060 atcaccgaaa cgcgcgaggc agctggcacg acaggtttcc cgactggaat gtgcctgtca 3120 aatggacgaa gcagggattc tgcaaaccct atgctactcc gtcaagccgt caattgtctg 3180 attcgttacc aattatgaca acttgacggc tacatcattc actttttctt cacaaccggc 3240 acggaactcg ctcgggctgg ccccggtgca ttttttaaat acccgcgaga aatagagttg 3300 atcgtcaaaa ccaacattgc gaccgacggt ggcgataggc atccgggtgg tgctcaaaag 3360 cagcttcgcc tggctgatac gttggtcctc gcgccagctt aagacgctaa tccctaactg 3420 ctggcggaaa agatgtgaca gacgcgacgg cgacaagcaa acatgctgtg cgacgctggc 3480 gatatcaaaa ttgctgtctg ccaggtgatc gctgatgtac tgacaagcct cgcgtacccg 3540 attatccatc ggtggatgga gcgactcgtt aatcgcttcc atgcgccgca gtaacaattg 3600 ctcaagcaga tttatcgcca gcagctccga atagcgccct tccccttgcc cggcgttaat 3660 gatttgccca aacaggtcgc tgaaatgcgg ctggtgcgct tcatccgggc gaaagaaccc 3720 cgtattggca aatattgacg gccagttaag ccattcatgc cagtaggcgc gcggacgaaa 3780 gtaaacccac tggtgatacc attcgcgagc ctccggatga cgaccgtagt gatgaatctc 3840 tcctggcggg aacagcaaaa tatcacccgg tcggcaaaca aattctcgtc cctgattttt 3900 caccaccccc tgaccgcgaa tggtgagatt gagaataa cctttcattc ccagcggtcg 3960 gtcgataaaa aaatcgagat aaccgttggc ctcaatcggc gttaaacccg ccaccagatg 4020 ggcattaaac gagtatcccg gcagcagggg atcattttgc gcttcagcca tacttttcat 4080 actcccgcca ttcagagaag 4100 <210> 12 <211> twenty one <212> DNA <213> artificial <220> <223> 3477-fwrd (primer sequence vector pGJ3477-fwrd) <220> <221> 3477-fwrd <222> (1)..(21) <400> 12 ggatccggct gctaacaaag c 21 <210> 13 <211> twenty one <212> DNA <213> artificial <220> <223> 3477-rev (primer sequence vector pGJ3477-rev) <220> <221> 3477-rev <222> (1)..(21) <400> 13 ggaatggtga tggtgatggt g 21 <210> 14 <211> 48 <212> DNA <213> artificial <220> <223> trxA-fwrd (primer sequence trxA-fwrd) <220> <221> trxA-fwrd <222> (1)..(48) <400> 14 catcaccatc accattccat gagcgataaa attattcacc tgactgac 48 <210> 15 <211> 45 <212> DNA <213> artificial <220> <223> trxA-rev (primer sequence trxA-rev) <220> <221> trxA-rev <222> (1)..(45) <400> 15 ctttgttagc agccggatcc ttacgccagg ttagcgtcga ggaac 45 <210> 16 <211> 40 <212> DNA <213> artificial <220> <223> trxB-fwrd (primer sequence trxB-fwrd) <220> <221> trxB-fwrd <222> (1)..(40) <400> 16 caccatcacc attccatggg cacgaccaaa cacagtaaac 40 <210> 17 <211> 47 <212> DNA <213> artificial <220> <223> trxB-rev (primer sequence trxB-rev) <220> <221> trxB-rev <222> (1)..(47) <400> 17 ctttgttagc agccggatcc ttattttgcg tcagctaaac catcgag 47 <210> 18 <211> 38 <212> DNA <213> artificial <220> <223> vtrxA-fwrd (a primer vector containing trxA for the trxBA fusion gene fwrd) <220> <221> vtrxA-fwrd <222> (1)..(38) <400> 18 ctgacgcagg tagcgataaa attattcacc tgactgac 38 <210> 19 <211> 46 <212> DNA <213> artificial <220> <223> ftrxB-fwrd (a primer insert trxB used for the trxBA fusion gene fwrd) <220> <221> ftrxB-fwrd <222> (1)..(46) <400> 19 catcaccatc accattccat gggcacgacc aaacacagta aactgc 46 <210> 20 <211> 52 <212> DNA <213> artificial <220> <223> ftrxB-rev (a primer insert trxB for the trxBA fusion gene rev) <220> <221> ftrxB-rev <222> (1)..(52) <400> 20 gtcaggtgaa taattttatc gctacctgcg tcagctaaac catcgaggta gc 52 <210> twenty one <211> 37 <212> DNA <213> artificial <220> <223> vtrxB-fwrd (a primer vector containing trxB for the trxAB fusion gene fwrd) <220> <221> vtrxB-fwrd <222> (1)..(37) <400> twenty one cgctaacctg gcgggcacga ccaaacacag taaactg 37 <210> twenty two <211> 48 <212> DNA <213> artificial <220> <223> ftrxA-fwrd (a primer insert trxA used for the fwrd fusion gene of trxAB) <220> <221> ftrxA-fwrd <222> (1)..(48) <400> twenty two catcaccatc accattccat gagcgataaa attattcacc tgactgac 48 <210> twenty three <211> 37 <212> DNA <213> artificial <220> <223> ftrxA-rev (a primer insert trxA for the trxAB fusion gene rev) <220> <221> ftrxA-rev <222> (1)..(37) <400> twenty three gtgtttggtc gtgcccgcca ggttagcgtc gaggaac 37 <210> twenty four <211> 1377 <212> DNA <213> artificial <220> <223> Ml-trxBA (E. coli codon-optimized ml-trxBA gene) <220> <221> Ml-trxBA <222> (1)..(1377) <400> twenty four atgaacacaa cgcctagtgc gcatgagaca atccatgaag tgatcgtcat aggttccggt 60 cctgcgggtt atacagcagc gttgtacgca gctagagcac aactgacccc tctcgtcttt 120 gagggcacat catttggcgg ggctttaatg accacaactg aagtcgagaa ctacccgggt 180 tttcgcaacg gtattacagg tccggaactc atggacgaca tgcgtgaaca ggcattacgg 240 tttggagcgg agctgcgtac cgaagatgtt gagtcagtca gtctaagagg accgataaaa 300 tccgttgtca ctgccgaggg ccagacctat caagcgagag cggtgattct ggctatggga 360 acttctgtac gttacttaca aataccgggt gagcaggaac tgctcgggcg cggagtgtcc 420 gcttgcgcga cctgtgatgg tagtttcttc aggggccaag atatcgccgt gatcggtggt 480 ggcgacagcg ctatggagga agcgctgttt cttacccgat tcgccagatc tgtgactctc 540 gtacaccgac gcgacgagtt tcgtgcgagt aaaatcatgt tgggtcgcgc ccgtaataac 600 gataaaataa agttcataac taaccacacg gtggtggcag tcaatggcta taccacggtg 660 accggactgc gtttgcggaa tacgacgacc ggtgaagaaa ccacattagt cgtaaccggc 720 gtcttcgtgg ctattggcca cgaaccgaga agttctttag tgtcagatgt agtggatata 780 gatcccgacg gttatgtact ggtaaagggt cgaactactt ccaccagcat ggatggagtg 840 ttcgcggctg gggacttggt tgaccgcaca tatcgtcaag caattaccgc tgcgggttca 900 ggctgcgctg cggctataga cgcggaacgt tggttggcag agcatgcggg ttccaaggca 960 aatgaaacca ctgaagagac aggcgacgtg gattccaccg atacgacaga ttggtccaca 1020 gcgatgacag acgcaaagaa tgctggtgtg actatcgagg ttacggacgc ctctttcttc 1080 gccgacgttc taagctcaaa taagccggtc ctcgtcgact tctgggccac ttggtgtggc 1140 ccgtgtaaaa tggttgcacc cgtgttagag gagattgcat cggaacagcg caatcaatta 1200 acagttgcta agcttgatgt cgatacgaac ccggagatgg ctagagagtt tcaggtcgtg 1260 tccataccta caatgatatt atttcaaggt ggtcaaccgg tcaagcgcat cgtaggggcg 1320 aaagggaagg ccgcattact tcgcgacctc agcgatgttg ttcctaacct gaactga 1377 <210> 25 <211> 45 <212> DNA <213> Artificial <220> <223> Ml-trxBA-fwrd (Primer sequence ml-trxBA-fwrd) <220> <221> Ml-trxBA-fwrd <222> (1)..(45) <400> 25 catcaccatc accattccat gaacacaacg cctagtgcgc atgag 45 <210> 26 <211> 48 <212> DNA <213> Artificial <220> <223> Ml-trxBA-rev (Primer sequence ml-trxBA-rev) <220> <221> Ml-trxBArev <222> (1)..(48) <400> 26 ctttgttagc agccggatcc tcagttcagg ttaggaacaa catcgctg 48 <210> 27 <211> 1338 <212> DNA <213> Artificial <220> <223> histrxB5A (Fusion gene of His-tag and Escherichia coli proteins TrxB and TrxA) <220> <221> histrxB5A <222> (1)..(1338) <400> 27 atgacacaga gggcccacca tcaccatcac cattccatgg gcacgaccaa acacagtaaa 60 ctgcttatcc tgggttcagg cccggcggga tacaccgctg ctgtctacgc ggcgcgcgcc 120 aacctgcaac ctgtgctgat taccggcatg gaaaaaggcg gccaactgac caccaccacg 180 gaagtggaaa actggcctgg cgatccaaac gatctgaccg gtccgttatt aatggagcgc 240 atgcacgaac atgccaccaa gtttgaaact gagatcattt ttgatcatat caacaaggtg 300 gatctgcaaa accgtccgtt ccgtctgaat ggcgataacg gcgaatacac ttgcgacgcg 360 ctgattattg ccaccggagc ttctgcacgc tatctcggcc tgccctctga agaagccttt 420 aaaggccgtg gggtttctgc ttgtgcaacc tgcgacggtt tcttctatcg caaccagaaa 480 gttgcggtca tcggcggcgg caataccgcg gttgaagagg cgctgtatct gtctaacatc 540 gcttggaag tgcatctgat tcaccgccgt gacggtttcc gcgcggaaaa aatcctcatt 600 aagcgcctga tggataagt ggagaacggc aacatcattc tgcacaccaa ccgtacgctg 660 gaaagtga ccgggggatca aatgggtgtc actggcgttc gtctgcgcga tacgcaaaac 720 agcgataaca tcgagtcact cgacgttgcc ggtctgtttg ttgctatcgg tcacagcccg 780 aatactgcga ttttcgaagg gcagctgggaa ctggaaaacg gctacatcaa agtacagtcg 840 ggtattcatg gtaatgccac ccagaccagc attcctggcg tctttgccgc aggcgacgtg 900 atggatcaca tttatcgcca ggccattact tcggccggta caggctgcat ggcagcactt 960 gatgcggaac gctacctcga tggtttagct gacgcaggtc cggcccctgg cagcgataaa 1020 attattcacc tgactgacga cagttttgac acggatgtac tcaaagcgga cggggcgatc 1080 ctcgtcgatt tctgggcaga gtggtgcggt ccgtgcaaaa tgatcgcccc gattctggat 1140 gaaatcgctg acgaatatca gggcaaactg accgttgcaa aactgaacat cgatcaaaac 1200 cctggcactg cgccgaaata tggcatccgt ggtatcccga ctctgctgct gttcaaaaac 1260 ggtgaagtgg cggcaaccaa agtgggtgca ctgtctaaag gtcagttgaa agagttcctc 1320 gacgctaacc tggcgtaa 1338 <210> 28 <211> 445 <212> PRT <213> artificial <220> <223> HisTrxB5A (a fusion protein of His-tag and E. coli proteins TrxB and TrxA) <220> <221> His-TrxB5A <222> (1)..(445) <400> 28 Met Thr Gln Arg Ala His His His His His Ser Met Gly Thr Thr 1 5 10 15 Lys His Ser Lys Leu Leu Ile Leu Gly Ser Gly Pro Ala Gly Tyr Thr 20 25 30 Ala Ala Val Tyr Ala Ala Arg Ala Asn Leu Gln Pro Val Leu Ile Thr 35 40 45 Gly Met Glu Lys Gly Gly Gln Leu Thr Thr Thr Thr Thr Glu Val Glu Asn 50 55 60 Trp Pro Gly Asp Pro Asn Asp Leu Thr Gly Pro Leu Leu Met Glu Arg 65 70 75 80 Met His Glu His Ala Thr Lys Phe Glu Thr Glu Ile Ile Phe Asp His 85 90 95 Ile Asn Lys Val Asp Leu Gln Asn Arg Pro Phe Arg Leu Asn Gly Asp 100 105 110 Asn Gly Glu Tyr Thr Cys Asp Ala Leu Ile Ile Ala Thr Gly Ala Ser 115 120 125 Ala Arg Tyr Leu Gly Leu Pro Ser Glu Glu Ala Phe Lys Gly Arg Gly 130 135 140 Val Ser Ala Cys Ala Thr Cys Asp Gly Phe Phe Tyr Arg Asn Gln Lys 145 150 155 160 Val Ala Val Ile Gly Gly Gly Asn Thr Ala Val Glu Glu Ala Leu Tyr 165 170 175 Leu Ser Asn Ile Ala Ser Glu Val His Leu Ile His Arg Arg Asp Gly 180 185 190 Phe Arg Ala Glu Lys Ile Leu Ile Lys Arg Leu Met Asp Lys Val Glu 195 200 205 Asn Gly Asn Ile Ile Leu His Thr Asn Arg Thr Leu Glu Glu Val Thr 210 215 220 Gly Asp Gln Met Gly Val Thr Gly Val Arg Leu Arg Asp Thr Gln Asn 225 230 235 240 Ser Asp Asn Ile Glu Ser Leu Asp Val Ala Gly Leu Phe Val Ala Ile 245 250 255 Gly His Ser Pro Asn Thr Ala Ile Phe Glu Gly Gln Leu Glu Leu Glu 260 265 270 Asn Gly Tyr Ile Lys Val Gln Ser Gly Ile His Gly Asn Ala Thr Gln 275 280 285 Thr Ser Ile Pro Gly Val Phe Ala Ala Gly Asp Val Met Asp His Ile 290 295 300 Tyr Arg Gln Ala Ile Thr Ser Ala Gly Thr Gly Cys Met Ala Ala Leu 305 310 315 320 Asp Ala Glu Arg Tyr Leu Asp Gly Leu Ala Asp Ala Gly Pro Ala Pro 325 330 335 Gly Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp 340 345 350 Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 355 360 365 Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 370 375 380 Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 385 390 395 400 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 405 410 415 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser 420 425 430 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala 435 440 445 <210> 29 <211> 50 <212> DNA <213> artificial <220> <223> vtrxA5-fwrd (a primer vector containing trxA for the trxB5A fusion gene fwrd) <220> <221> vtrx5A-fwrd <222> (1)..(50) <400> 29 ctgacgcagg tccggcccct ggcagcgata aaattattca cctgactgac 50 <210> 30 <211> 64 <212> DNA <213> artificial <220> <223> ftrxB5-rev (a primer insert for the trxB5A fusion gene rev) <220> <221> ftrxB5-rev <222> (1)..(64) <400> 30 gtcaggtgaa taattttatc gctgccaggg gccggacctg cgtcagctaa accatcgagg 60 tagc 64

Claims

1. An enzyme that reduces cysteine ​​to cysteine, characterized in that... The enzyme is the following fusion protein: i) Protein i: Composed of the amino acid sequence shown in SEQ ID No. 7, and ii) Protein ii: Composed of the amino acid sequence shown in SEQ ID No. 8, In this fusion protein, the amino acid sequence of the N-terminal protein is shortened by at most one amino acid at the C-terminus, and the amino acid sequence of the C-terminal protein is shortened by at most one amino acid at the N-terminus. Wherein, the amino acid sequences of the fusion protein of protein i and protein ii are linked in the fusion protein by a linker sequence of one to five amino acids, or the amino acid sequences of the fusion protein of protein i and protein ii directly follow each other in the fusion protein, and The coding sequences responsible for the activities of protein i and protein ii are fused. The activity of the fusion protein is at least 100% of the activity of a mixture of identical but unfused individual proteins i and ii, the mixture being a mixture of thioredoxin composed of the amino acid sequence shown in SEQ ID NO: 7 and thioredoxin reductase composed of the amino acid sequence shown in SEQ ID NO:

8.

2. The enzyme according to claim 1, characterized in that, The fusion protein consists of an amino acid sequence selected from SEQ ID No. 9, SEQ ID No. 10 or SEQ ID No.

28.

3. A method for enzymatically reducing cystine to cysteine, characterized in that, Cystine is reduced by the enzyme according to any one of claims 1 to 2 in the presence of a cofactor, wherein one molecule of the compound cystine forms two molecules of L-cysteine.

4. The method as described in claim 3, characterized in that, The reduction occurs at pH values ​​from 6 to 9.

5. The method as described in claim 3 or 4, characterized in that, The cofactors are selected from the group consisting of NADPH and NADH.

6. The method as described in claim 3 or 4, characterized in that, The method includes cofactor regenerating enzyme.

7. The method as described in claim 6, characterized in that, The cofactor regenerating enzyme is a dehydrogenase in which the reduction occurs additionally in the presence of an electron donor.

8. The method as described in claim 7, characterized in that, The dehydrogenase is an alcohol dehydrogenase, wherein isopropanol is used as an electron donor.

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