Process for preparing galactaric acid

Abstract

The object is achieved according to the invention by oxidising galacturonic acid in an aqueous solution in vitro with a dehydrogenase having NAD(P)+ as cofactor to form reduced cofactor NAD(P)H, wherein the reduced cofactor NAD(P)H is oxidised with an NAD(P)H oxidase, characterised in that the NAD(P)H oxidase for the oxidation of NAD(P)H to NAD(P)+ comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having at least 80% identity to SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16 or SEQ ID No. 18, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15 or SEQ ID No. 17, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, or SEQ ID No. 17.

Description

[0001] Methods for the production of galactaric acid

[0002] The present invention relates to a process for the production of galactaric acid by reacting galacturonic acid in an aqueous solution in vitro with a dehydrogenase containing NAD(P) + as a cofactor to form reduced cofactor NAD(P)H, whereby the reduced cofactor NAD(P)H is oxidized with an NAD(P)H oxidase.

[0003] Background of the invention

[0004] Galactaric acid, also known as mucinic acid or mucoic acid, is a sugar dicarboxylic acid (aladaric acid). It serves as a building block for polycondensates (e.g., polyamides), for SBAPs (sugar-based amphiphilic polymers), which can be used as drug carriers for hydrophobic cancer drugs or gene therapies or for the treatment of atherosclerosis, for metal-organic frameworks (MOFs) (Sakuta & Nakamura, 2019), and for the production of platform chemicals adipic acid (Li et al., 2014) or 2,5-furandicarboxylic acid (US 9,701,652 B2; US 9,994,539 B2).

[0005] Galactaric acid and its anion galactarate occur in nature, for example in fruits (Anet & Reynolds, 1954) or sugar beets (Stark et al., 1950), only in small quantities, making synthetic production unavoidable. D-galactose can be oxidized to galactaric acid using nitric acid (Pigman et al., 1949), but this process leads to low yields and the formation of byproducts and nitrogen oxides (Sakuta & Nakamura, 2019).

[0006] Other methods utilize the precursor D-galacturonic acid (or its anion D-galacturonate), which, instead of two terminal carboxylic acids, possesses only one carboxylic acid and one aldehyde functional group. D-Galacturonic acid occurs naturally as the main component of the heteropolysaccharide pectin. Pectin is found in the cell walls of plant cells—especially in fruits such as apples and citrus fruits, in sugar beet pulp, and in potato peels. In total, more than 1.5 million tons of D-galacturonic acid (bound in pectin) are available worldwide (van der Klis et al., 2017; Roman-Benn et al., 2023).

[0007] Depending on their structure, pectin can be classified as homogalacturonan, rhamnogalacturonan I, rhamnogalacturonan II, and xylogalacturonan. The monomers can be released through the action of pectinases, a group of pectin-degrading enzymes such as polygalacturonase (Kuivanen et al., 2019; Roman-Benn et al., 2023).

[0008] The aldehyde group in D-galacturonic acid can be oxidized either chemically-catalytically or biocatalytically (in vivo or in vitro). Rautiainen et al. (2015) used an Au / ALOs catalyst to oxidize D-galacturonic acid to galactaric acid at pH 8–10 and 40–60 °C with atmospheric oxygen. EP 2 836 498 Bl and EP 3 204 155 Bl describe similar procedures and list sugar beet pulp hydrolysates and citrus fruit hydrolysates as sources of D-galacturonic acid.

[0009] Van der Klis et al. (2018) also used a gold catalyst for oxidation and achieved higher oxidation productivity by switching from a batch reactor (see van der Klis et al. (2013)) to a plug-flow fixed-bed reactor. Since the process is carried out at elevated pH values ​​(pH > 9), traces of 5-keto-L-galactonate, which is formed by the base-catalyzed isomerization of D-galacturonate, were found. The authors postulated D-talaric acid / D-talarate as a further byproduct, an oxidation product of D-taluronic acid / D-taluronate (the C2 epimer of D-galacturonate) (van der Klis et al., 2018).

[0010] A general disadvantage of the methods mentioned here is the need for gold as a catalyst, which is both expensive and only occurs in limited quantities in nature.

[0011] As an alternative, biocatalytic processes are available, which are highly selective and take place under mild reaction conditions, using biodegradable catalysts such as cells or enzymes.

[0012] Mojzita et al. (2010) used engineered fungal strains (Hypocrea jecorina and Aspergillus niger) to oxidize D-galacturonate to galactarate. For this purpose, the gene encoding D-galacturonate reductase (D-galacturonate) was modified. The gene encoding L-galactonate was deleted, and the bacterial udh gene, which encodes a NAD-dependent D-galacturonate dehydrogenase (a uronate dehydrogenase (UDH), EC 1.1.1.203), was introduced. The resulting strains were able to oxidize D-galacturonate, with the H. jecorina strain producing galactarate at higher conversion rates (15.8 g / L after 24 h). Galactaric acid was isolated from the culture supernatants in high purity (>94%) by acidification.

[0013] The expression of UDH from Agrobacterium tumefaciens CS58 (Rhizobium radiobacter) in modified (deletion of D-galacturonate reductase) Aspergillus sp. or Hypocrea sp. is described in US 8895273 B2. Using an H. jecorina (= Trichoderma reese) mutant, 10 g / l D-galacturonate could be oxidized to 1-1.3 g / l galactarate, with 10 g / l D-xylose added to promote strain growth.

[0014] The direct conversion of pectin to galactarate using the modified fungus Trichoderma reesei was described by Paasikallio et al. (2017). 29.2 g / L of pectin were converted to 21 g / L of galactaric acid on a lOL scale in 185 h, with 85% of the product being isolated. Tamminen et al. (2022) also used a Trichoderma sp. transformant that produced up to 53 g / L of galactaric acid in a fed-batch reactor in 300 h.

[0015] Protzko et al. (2018) used an engineered Saccharomyces cerevisiae expressing GatA (A niger D-galacturonate transporter) and UDH to oxidize D-galacturonate from citrus peel waste to galactarate (titer 8 g / L after 80 h), with the addition of D-glucose. The procedure is also described in US 11332723 B2.

[0016] Analogous to Mojzita et al. (2010), Vidgren et al. (2020) engineered two marine fungi (Trichoderma sp. and Coniochaeta sp.) for the production of galactarate. The Trichoderma sp. transformant LF328 T2 produced up to 25 g / l galactarate in 200 h (with D-glucose as a cosubstrate). A Coniochaeta sp. transformant, in turn, was able to produce galactarate directly from pectin, but D-galacturonate metabolism was not completely inhibited.

[0017] Modified microorganisms for the production of galactarate from D-galacturonate are also mentioned in US 10,982,239 B2, EP 3486 323 Al and WO 2010 / 072902 Al.

[0018] Enzymatic methods for the production of galactarate are also known.

[0019] Wagner and Hollmann (1976) describe an enzymatic assay for the spectrophotometric determination of (free and conjugated) D-glucuronic acid and D-galacturonic acid, using UDH from Pseudomonas syringae as the enzyme.

[0020] The article by Chang and Feingold (1969) describes the production of galactaric acid using a hexuronic acid dehydrogenase from Agrobacterium tumefaciens. For this purpose, 7 mM (1.4 g / L) of D-galacturonic acid was reacted with the enzyme. A lactate dehydrogenase from rabbit muscle (EC 1.1.1.27) with potassium pyruvate (30 mM; 4.3 equivalents based on D-galacturonic acid) served as a co-substrate for the regeneration of the cofactor. Galactaric acid was isolated as the product after activated carbon filtration, ion-exchange chromatography, and subsequent crystallization; the product yield was not reported in the article.

[0021] Enzymatic methods are also known for the production of D-glucaric acid (C3 epimer of galactaric acid).

[0022] Su et al. (2019) describe the conversion of 50 mM sucrose to 34.8 mM D-glucaric acid in vitro over 70 h using a seven-enzyme cascade system. The D-glucuronic acid produced in the penultimate step is converted from Agrobacterium tumefaciens and NAD by means of UDH. + The resulting NADH is oxidized to D-glucaric acid, with the NADH produced being oxidized by an NADH oxidase from Lactobacillus rhamnosus. Petroll et al. (2020) used a similar enzyme cascade consisting of six enzymes to convert glucose-L-phosphate (G1P), which can be obtained from starch, sucrose, or cellulose using phosphorylases, to D-glucaric acid in vitro. The final step of the cascade also involved oxidation with UDH (from Fulvimarina pelagi) and NADH oxidase from Lactobacillus rhamnosus for cofactor regeneration. In this way, 40 mM G1P could be converted to 8.1 mM D-glucaric acid in 10 h.

[0023] Besides UDH, there are other enzymes that can be used to oxidize D-galacturonic acid.

[0024] Sakuta et al. (2016) developed an electrode with immobilized pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) that bioelectrochemically oxidizes D-galacturonate to galactarate. This electrode was also used by Nakagawa et al. (2022) for the conversion of D-galacturonate to galactarate in an enzymatic biofuel cell. A disadvantage is the use of the dye methylene green as an electron acceptor and the formation of galactarolactone as a byproduct (Vastano et al., 2019).

[0025] Glucose oxidase from A. niger also exhibits uronic acid oxidase activity, as Kobayashi et al. (1999) were able to show; however, in addition to uronic acids, aldoses (D-glucose, D-galactose, D-mannose and D-xylose) are also oxidized.

[0026] Vastano et al. (2019) tested three enzymes for the oxidation of D-galacturonate to galactarate: a commercial laccase, a laccase from Myceliophthora thermophila (both with the stabilized radical 2,2,6,6-tetramethylpiperidinyloxyl (TEMPO) as a mediator), and PQQ-GDH (with methylene green as a mediator). Similar conversion rates (approximately 40% after 12 days at substrate concentrations up to 500 mM (97 g / L)) were observed for all enzymes.

[0027] Uronic acid oxidase, which can convert D-galacturonic acid and D-glucuronic acid into the corresponding aldaric acids, is known from the Shamouti orange (Citrus sinensis L. Osbeck) (Riov, 1975).

[0028] Boverio et al. (2023) also characterized a uronic acid oxidase from Citrus sinensis that completely oxidized 0.35 wt% D-galacturonic acid to galactaric acid in 6 h. Similar conversions were observed for a mixture of D-galacturonic acid (0.32 wt%) and L-arabinose, D-galactose, and D-glucose (totaling 0.11 wt%) obtained by enzymatic hydrolysis of pectin from sugar beet pulp. L-arabinose, D-galactose, and D-glucose were not oxidized by the uronic acid oxidase. A significant disadvantage is the formation of enzyme-damaging hydrogen peroxide as a byproduct of uronic acid oxidation, which must be removed (e.g., by catalase).

[0029] EP 3 460 067 Al describes an alcohol dehydrogenase from Sphingomonas species Al, which can oxidize D-galacturonic acid to galactaric acid. NADH oxidase from Lactobacillus pentosus was added three times. Conversions and yields of the oxidation are not specified.

[0030] This is where the object of the present invention comes in, and it aims to provide an improved process for the production of galactaric acid from galacturonic acid.

[0031] Detailed description of the invention

[0032] The problem is solved according to the invention by reacting galacturonic acid in an aqueous solution in vitro with a dehydrogenase containing NAD(P) + as a cofactor to form reduced cofactor NAD(P)H, wherein the reduced cofactor NAD(P)H is oxidized with an NAD(P)H oxidase, characterized in that the NAD(P)H oxidase is used to oxidize NAD(P)H to NAD(P) +an amino acid sequence selected from the group consisting of: i) an amino acid sequence exhibiting at least 80% identity with SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, or SEQ ID No. 18; ii) an amino acid sequence encoded by a nucleic acid exhibiting at least 80% identity with SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, or SEQ ID No. 17; and iii) an amino acid sequence encoded by a nucleic acid that, under stringent conditions, is attached to a complementary strand of a binds to nucleic acid molecules with the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15 or SEQ ID No. 17.

[0033] It has been shown that the use of isolated dehydrogenases is indeed a good alternative to the use of oxidases (Boverio et al. 2023) when combined with a suitable cofactor regeneration system such as an NAD(P)H oxidase.

[0034] It has also been shown that by selecting NAD(P)H oxidases used according to the invention, the oxidation of galacturonic acid can be achieved at higher concentrations and with higher conversions compared to the conditions described in EP 3460067 A1. Furthermore, repeated addition of NAD(P)H oxidase is not necessary. Suitable NAD(P)H oxidases are known, for example, from WO 2011 / 157717A1.

[0035] A uronic acid / uronate dehydrogenase (EC 1.1.1.203) is preferably used as the dehydrogenase. Suitable uronic acid dehydrogenases are available, for example, from the organisms Pseudomonas putida or Streptomyces viridochromogenes (see Table 1).

[0036] In a particularly preferred embodiment of the process according to the invention, the galacturonic acid is D-galacturonic acid.

[0037] A preferred embodiment of the process according to the invention is schematically illustrated in the accompanying Figure 1, wherein the galacturonic acid is shown in the D-form. The designation A in Figure 1 stands for D-galacturonic acid, B for galactaric acid, 1 for uronic acid / uronate dehydrogenase, and 2 for NADH oxidase.

[0038] A NAD(P)H oxidase for the oxidation of NAD(P)H to NAD(P) +comprises or preferably consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence exhibiting at least 80% identity with SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16 or SEQ ID No. 18, ii) an amino acid sequence encoded by a nucleic acid exhibiting at least 80% identity with SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15 or SEQ ID No. 17, and iii) an amino acid sequence encoded by a nucleic acid adapted under stringent conditions to a complementary binds to a strand of a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15 or SEQ ID No. 17.

[0039] SEQ ID No. 1:

[0040] ATGAAAGTAGTAGTAGTAGGCTGTACACATGCAGGAACAGCGGCAGTTAAGACGATTTTAAATGAACATCCA GATGCATCAGTATCAGTATATGAGCGTAATGACAATGTCTCATTTCTATCTTGTGGGATTGCGTTGTATGTTGG TGGAGTTGTGAAAGATCCTGCAGGTTTGTTTTATTCAAGTCCAGAAGAACTTGCATCAATGGGCGCGAAAATT AACATGGAACACAATGTGAAAAATATAGATAATGAGAATAAGGTCGTAGTAATTGAGAATTTAAAAACAGGC GAAACATTTGAAGAAAGCTATGATAAGTTGGTAATGACAACTGGATCATGGCCAATTATTCCTCCAATTGATGG AATCAATAGTGAAAATATTCTTTTGTGTAAAAACTATAACCAAGCAAATGAAATTATTAAAGAATCAAAAAATG CTAAAAAGATTGTCATTGTTGGTGGTGGCTATATTGGAATTGAATTAGTTGAGGCATTTGCAGAATCTGGCAA GCAAGTGACGCTAGTTGATGGATTAGATCGTATTTTAAACAAATATTTAGATGCTGAATTCACTTCTGTTTTAGA GCATGATTTACAAGAAAGAGGCGTTACGCTAGCTTAAACCAAACCGTCGAGAAATTTGTTGCCAATGAATCA

[0041] GGTGCTGTGACAGCTGTGAAAACACCAGTTGGAGAATATGAGGCTGATTTAGTTATTTTATGTGTTGGATTTAA

[0042] ACCAAATACTGATTTGTTGAAGGATAAAGTAGAGATGTTGCCAAATGGTGCCATCGTAGTGGATGAATATATG

[0043] AGAACAAGCGATGAAGCGATTTTTGCTGCTGGCGATAGTTGCGCGGTTCATTATAATCCAACTGGAGGCTCTG

[0044] CGTATATTCCGTTAGCTACAAATGCAGTTAGAATGGGAGCTTTAGTTGGGAAAAATATTGTTTCTCCCAACAGTT

[0045] AAATATCGTGGCACGCAAGCAACTTCTGGTTTATATTTTTGGTTTTAATATAGTTCAACCGGATTGACTGAA

[0046] AATAGCGCTCCTCATTTTGGCGTAGAGGTTCGTTCAGTAGTTGTAGAAGATAATTATCGTCCAGAGTTTATGCC

[0047] GCAACAGAGAAAGTAACGATGAAATTAGTTTGAAGTAGGAACGAATCGGATTGTTGGAGGTCAAATCAT

[0048] GTCAAAATGATGTGACACAATCTGCCAATACGTTATCTTTATGTGTCAAATAAAATGACGATTGAGGATT

[0049] TGGCTTATGTAGATTTCTTCTTCCAACCTCACTTTGATCGTCCTTGGAACTATTTAAATATTTTAGCGCAAGCAGC

[0050] TGTTGAGCAAGAGCGTAAACTAGCAAAATAA

[0051] SE ID Nr. 2:

[0052] MKVVVVGCTHAGTAAVKTILNEHPDASVVYERNDNVSFLSCGIALYVGGVVKDPAGLFYSSPEELASMGAKINM E

[0053] HNVKNIDNENKVVVIENLKTGETFEESYDKLVMTTGSWPIIPPIDGINSENILLCKNYNQANEIIKESKNAKKIVIVGG

[0054] GYIGIELVEAFAESGKQVTLVDGLDRILNKYLDAEFTSVLEHDLQERGVTLALNQTVEKFVANESGAVTAVKTPVGEY

[0055] EADLVILCVGFKPNTDLLKDKVEMLPNGAIVVDEYM RTSDEAIFAAGDSCAVHYNPTGGSAYIPLATNAVRMGALV

[0056] GKNIVSPTVKYRGTQATSGLYLFGFNIGSTGLTENSAPHFGVEVRSVVVEDNYRPEFMPTTEKVTMKLVYEVGTNRI

[0057] VGGQIMSKYDVTQSANTLSLCVQNKMTIEDLAYVDFFFQPHFDRPWNYLNILAQAAVEQERKLAK

[0058] SEQ ID Nr. 3:

[0059] ATGAGCAAAATTGTTATCGTGGGTGCAAATCATGCAGGCACCGCAGCAATTAATACCATTCTGGATAATTATG

[0060] GCAGCGAAAATGAAGTGGTTGTGTTTGATCAGAATAGCAACATTAGCTTTCTGGGTTGTGGTATGGCACTGTG

[0061] GATTGGTAAACAAATTAGCGGTCCGCAGGGTCTGTTTTATGCAGATAAAGAAAGCCTGGAAGCAAAAGGTGC

[0062] CAAAATCTATATGGAAAGTCCGGTTACCGCCATTGATTATGATGCAAAACGTGTTACCGCACTGGTTAATGGTC

[0063] AAGAACATGTTGAAAGCTACGAGAAACTGATTCTGGCAACCGGTAGCACCCCGATTCTGCCTCCGATTAAAGG

[0064] TGCAGCCATTAAAGAAGGTAGTCGCGATTTTGAAGCAACCCTGAAAAATCTGCAGTTCGTGAAACTGTATCAG

[0065] AATGCCGAAGATGTGATTAACAAACTGCAGGATAAAAGCCAGAATCTGAATCGTATTGCAGTTGTTGGTGCAG

[0066] GTTATATTGGTGTTGAACTGGCAGAAGCATTTAAACGTCTGGGTAAAGAAGTGATTCTGATTGACGTTGTTGAT

[0067] ACCTGTCTGGCAGGTTATTATGATCAGGATCTGAGCGAAATGATGCGTCAGAATCTGGAAGATCATGGTATCG

[0068] AACTGGCATTTGGTGAAACCGTTAAAGCAATTGAAGGTGATGGTAAAGTGGAACGTATTGTTACCGATAAAGC

[0069] AAGCCATGATGTGGATATGGTTATTCTGGCAGTTGGTTTTCGTCCGAATACAGCACTGGGTAATGCAAAACTG

[0070] AAAACCTTTCGTAATGGTGCCTTTCTGGTGGATAAAAAACAAGAAACCAGCATCCCGGATGTTTATGCAATTGG

[0071] TGATTGTGCAACCGTGTATGATAATGCCATTAACGACACCAACTATATTGCACTGGCAAGCAATGCACTGCGTA

[0072] GCGGTATTGTTGCAGGTCATAATGCAGCCGGTCATAAACTGGAAAGTCTGGGTGTTCAGGGTAGCAATGGTAT TTCAATTTTTGGCCTGAATATGGTTAGCACCGGTCTGACCCAAGAAAAAGCCAAACGTTTTGGTTATAATCCGG

[0073] AAGTTACCGCCTTTACCGATTTTCAGAAAGCCAGCTTTATCGAGCATGATAACTATCCGGTTACGCTGAAAATT

[0074] GTGTATGACAAAGATAGCCGTCTGGTTCTGGGTGCACAGATGGCCAGCAAAGAAGATATGAGCATGGGTATT

[0075] CACATGTTTAGCCTGGCCATTCAAGAGAAAGTTACCATTGAACGTCTGGCCCTGCTGGATTATTTCTTTCTGCCG

[0076] CATTTAATCAGCCGTACAACTATATGACCAAAGCAGCACTGAAAGCCAAATAA

[0077] SE ID Nr. 4:

[0078] MSKIVIVGANHAGTAAINTILDNYGSENEVVVFDQNSNISFLGCGMALWIGKQISGPQGLFYADKESLEAKGAKIY

[0079] MESPVTAIDYDAKRVTALVNGQEHVESYEKLILATGSTPILPPIKGAAIKEGSRDFEATLKNLQFVKLYQNAEDVINKL

[0080] QDKSQNLNRIAVVGAGYIGVELAEAFKRLGKEVILIDVVDTCLAGYYDQDLSEMMRQNLEDHGIELAFGETVKAIEG

[0081] DGKVERIVTDKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSIPDVYAIGDCATVYDNAINDTNYI

[0082] ALASNALRSGIVAGHNAAGHKLESLGVQGSNGISIFGLNMVSTGLTQEKAKRFGYNPEVTAFTDFQKASFIEHDNYP

[0083] VTLKIVYDKDSRLVLGAQMASKEDMSMGIHMFSLAIQEKVTIERLALLDYFFLPHFNQPYNYMTKAALKAK

[0084] SEQ ID No. 5:

[0085] ATGAAGGTTATTATTGTTGGTTGTACACATGCGGGAACAATTACTGCCACTCAAATTTTACAGAATCATCCAGA

[0086] AACAGAAGTCACAATTTATGAACGGAATGATAACGTTTCATTCCTCTCATGTGGGATTGCAGTTTACTTGAGCG

[0087] GTGATGTTGGTAATCCAGATGCGATGTTTTATTCAAGCCCTGAACAACTTGCTGCCATGGGTGCAACAGTTCAT

[0088] ATGCAACATAATGTAACTGATATTGACCCTAAGACTAAGACAGTCACAGTGACAGACCTTGTGACAGGCGAAA

[0089] CAAAGACCGACCATTATGATAAGTTAGTTGATACTACTGGTTCTTGGCCAGTAATTCCACCAATTGAAGGTGTA

[0090] GATGGCCCTCATGTTTATTTGTGCAAGAATTACCATCATGCTAAAGAATTATTTAACGTTGCTAAAGATGCGCA

[0091] ACGAATTGTTGTGATTGGTGGAGGTTATATTGGGGTTGAATTAGTAGAAGCTTACACTCGTCAAAATAAGGAC

[0092] GTTACATTGATTGATGGGTCTCCACGGATGCTTCATAAATACTTTGACCGCGAGTATACGGATCGAATTCAACA

[0093] GGAATTTGTAGATCACGGCGCCCACTTTGCTTTTGACCAACGCGTAACTGGATTTGAAAACCACGAAAATGGT

[0094] GTAACCGTTAAGACGGATAAGGGCAACTACGAGGCAGATATTGCTATCCTCTGTGTTGGCTTCCGTCCTAATAC

[0095] TGATCTCTTAAAAGGTAAAGTTAAGATGCATGATAATGGGGCAATCATTACGAACGAATACATGCAATCATCT

[0096] GATCCAGATATTTACGCTGCCGGAGATTCAACGGCTGTTCACTATAACCCAACTGGCAAGGATGCATACATTCC

[0097] ATTAGCTACTAACGCTATTCGGCAAGGAACAATTGTTGGAACAAATCTCTTTGGTAATACAATGCGCGATATGG

[0098] GAACCCAATCTAGTTCTGGCTTAAACTTATATGGAACAACGATGGTATCATCTGGCTTAACTTTGGAGAATGCC

[0099] AAAGAAGCGGGCTTTGATGCAGCTGCGGTGACAGTTGAAGACAACTACCGTCCAGAATTTATGCCGACAACAA

[0100] CTCCTGTATTAATGACATTGGTGTGGGATAAGAAGACTCGGCAAATTCTTGGTGGACAGTTTATGAGTAAGCA

[0101] TGATGTTTCTCAATCTGCTAACATTATTTCCTTATGTATCCAGGATAAGCACACGATTGATTATTTAGCATTTGTT

[0102] GATATGCTTTTCCAACCACACTTTGATCGTCCATTTAACTATGTAAATATTCTTGGCCAAGCGCGGTAAAGAA

[0103] ACAAGCTGAATTAGAAAAATAA SEQ ID No. 6:

[0104] MKVIIVGCTHAGTITATQILQNHPETEVTIYERNDNVSFLSCGIAVYLSGDVGNPDAMFYSSPEQLAAMGATVHMQ

[0105] HNVTDIDPKTKTTVTVTDLVTGETKTDHYDKLVDTTGSWPVIPPIEGVDGPHVYLCKNYHHAKELFNVAKDAQRIVVI

[0106] GGGYIGVELVEAYTRQNKDVTLIDGSPRMLHKYFDREYTDRIQQEFVDHGAHFAFDQRVTGFENHENGVTVKTDK

[0107] GNYEADIAILCVGFRPNTDLLKGKVKMHDNGAIITNEYMQSSDPDIYAAGDSTAVHYNPTGKDAYIPLATNAIRQGT

[0108] IVGTNLFGNTMRDMGTQSSSGLNLYGTTMVSSGLTLENAKEAGFDAAAVTVEDNYRPEFM PTTTPVLMTLVWDK

[0109] KTRQILGGQFMSKHDVSQSANIISLCIQDKHTIDYLAFVDM LFQPHFDRPFNYVNILGQAAVKKQAELEK

[0110] SEQ ID No. 7:

[0111] ATGAAAGTTGCAGTAATCGGTTGTACCCATGCTGGGACAGCTGCCGTTAAAACTATCTTAACAGAAACGATG

[0112] ATGTAGAAGTTGTTGTTTTGAACGCAATGATAATATCTCCTTCTTATCTTGTGGGATTGCCCTTTACGTAGGTG

[0113] GCGTGGTCAAAGACGTCAATGGCCTTTTCTATTCTGATCCAAGTGAGTTAGAATCTCTAGGCGCAACAGTTTAT

[0114] ATGAAACACAATGTGCTATCGTTTGACGAAAACACTAAAGTTATCCAAGTAGAAAATATGGAGACTGGCGAAC

[0115] ATTTCCAAGAATCATATGACAAGTTAGTCATTGCCACTGGTTCTTGGCCGATTATTCCTGATTTACCAGGACTAG

[0116] ATCTAGAAAATGTCATGCTATGTAAAAACTTTAAGCATGCCCAAGAATTAATCCAAACTAAGCAAGATAAAAAA

[0117] CGAGTTGCTGTTATTGGTGCTGGTTATATTGGTATCGAGTTAGTTGAAGCCTTTGCTGAAGACGGTAAAGAAG

[0118] TTGTTTTAATCGATGGTGCTGACCGCGTCCTACCTAAGTATCTCGACCAGGAAATGACTGACTTATTAGAAGCA

[0119] AGCTTGGTAGACCATGGCGTACAAATGCAATTAGGGGAATTTGTAGAATCATTCCTAGCCGACGATGAAGGTA

[0120] AGGTACGTGCCGTTAAAACGTCTAAAGGTGAATATGAATGTGATATGGCAGTCCTTTGTGTCGGCTTCCAACCT

[0121] AACACAGAATTATATAAGGGCAAGTTAGAAACTATGCCGAATGGTGCGATTATTGTAGATGACTATATGCACA

[0122] CCTCTCACCCAGATATCTATGCCTGTGGTGACTCATGTGCTGTCAACTATAATCCTAACGATGGCCATGCTTATA

[0123] TCCCGCTTGCCACTAATGCTGTCCGTATGGGTAGCTTAGTTGGTAAAAATATTAAAGCAGACCGCGTCAAATAC

[0124] CGGGGCACCCAGTCAACATCTGGTCTAAAATTATTCGGCTGGAATATTGGCTCAACTGGTGTTACCGACAATTC

[0125] TGCTAGCAGCTTCAACCTTGAAACTCGTAGCGTCTATGTTGAAGATAACTACCGCCCTGAATTCATGCCTACTAC

[0126] AGAAAAAGTTTATGAAGTTAGTCTATGAAGTAGGCACAAATCGAGTTGGCGGTCAGCTTATGTCTAAG

[0127] TATGATATTACCCAATCAGCCAACACCCTATCACTAGCTATCCAAACCAAGCAAACAATTTGAAGATTTAGCCTAT

[0128] GTGGACTTCTTCTTCCAACCACACTTTGACCGCCCTTGGAACTATTTAAATATTTTAGCCCAAGCAGCACTAAGT

[0129] CAAGAAAGAATTAGCTCAAAACTAA

[0130] SE ID Nr. 8:

[0131] MKVAVIGCTHAGTAAVKTILTENDDVEVVVFERNDNISFLSCGIALYVGGVVKDVNGLFYSDPSELESLGATVYM KH

[0132] NVLSFDENTKVIQVENMETGEHFQESYDKLVIATGSWPIIPDLPGLDLENVMLCKNFKHAQELIQTKQDKKRVAVIG

[0133] AGYIGIELVEAFAEDGKEVVLIDGADRVLPKYLDQEMTDLLEASLVDHGVQMQLGEFVESFLADDEGKVRAVKTSK

[0134] GEYECDMAVLCVGFQPNTELYKGKLETMPNGAIIVDDYMHTSHPDIACGDSCAVNYNPNDGHAYIPLATNAVR MGSLVGKNIKADRVKYRGTQSTSGLKLFGWNIGSTGVTDNSASSFNLETRSVEDNYRPEFMPTTEKVYMKLVYE

[0135] VGTNRVVGGQLMSKYDITQSANTLSLAIQTKQTIEDLAYVDFFFQPHFDRPWNYLNILAQAALSQEEELAQN

[0136] SE ID Nr. 9:

[0137] ATGAAAGTAGTAGTAATTGGCTGTACACATGCAGGTACTGCAGCTGTAAAAAGTATTTTAAAAAATCATCCAG

[0138] AAGCAGAAGTGACAGTATATGAAAGAAATGACAATATTTCTTTCTTATCTTGCGGTATCGCACTTTATGTCGGC

[0139] GGAGTAGTAAAGGACCCAGCAGGCTTGTTCTACTCAAATCCTGAAGAATTATCTTCATTAGGAGCAACAGTTA

[0140] AAATGGAACACGATGTAACAGACGTCGATACAGAAAATAAAACAGTAACGGCAAAAGATCTGAAAACAGGAG

[0141] AAACAGAAACGGTTTCTTGATAAATTAGTGATGACAACTGGTTCATGGCCTATTATCCCACCGATTAGTGGA

[0142] ATCGATGCGAAAAATGTACTCCTATGCAAAAACTACAATCAAGCAAATGAAATTATTGCACAAGCGAAAGAAG

[0143] CAAAACGTGTAGTAATTGTTGGTGGTGGATATATCGGTATTGAATTAGTAGAAGCTTTTGTCGAATCTGGAAA

[0144] ACAAGTGACATTGATTGATGGATTAGATCGCATCTTGAATAAATATCTAGATAAACCATTCACAGATATCTTAG

[0145] AAAAAGAATTGACAGACCGCGGTGTTACTCTTGCGCTAGGCGAAAATGTTACTGAATTTATCACAGATGAAGA

[0146] AGGAAAAGTGAAACAAGTAGCTACACCAACAGATACATTTGATACAGATATGGTCATTTTATGTGTGGGCTTC

[0147] CGTCCAAATACAAAACTGATCGAAGAAAAAGTAGATACACTGCCAAACGGGGCAATCAAAGTGAATGAATAC

[0148] ATGCAGACAAGGTGATCCAGACATTTTTGCAGCTGGAGACTCTGCAGTAGTCAACTACAATCCAAGTGGCACAC

[0149] AAAACTATATTCCTTTGGCAACAATTGCCGTTCGTCAAGGTTTACTTGTGGGAAATAACTTGACAGATCATAAA

[0150] ATGGCGTATCGTGGTACTCAAGGAACTTCAGGACTTTACCTATTTGGCTGGACGATCGGTTCGACAGGTGTGA

[0151] CCAAAGAAAGCGCAACTTTGAACGATCTAGAAGTCCAAGCGACTGTATTTGAAGATAATTATCGTCCAGAGTT

[0152] CATGCCAACTACTGAAAAAGTAATGATGGAGCTAGTCTATGAAAAAGGAACGAATCGTATCGTAGGTGCTCAG

[0153] TTCATGTCCAAATACGACATTACACAATCAGCAAATACCATGTCTTTAGCTGTTCAAAATAAAATGACTGTAGA

[0154] AGATCTCGCACTTTCAGATTTCTTCTTCCAACCACATTTTGACCGCCCATGGAATTACTTGAACCTACTTGCACAA

[0155] GCAGCGCTAGGCGAAATCGAAACTGCACAATAA

[0156] SEQ ID No. 10:

[0157] MKVWIGCTHAGTAAVKSILKNHPEAEVTVYERNDNISFLSCGIALYVGGVVKDPAGLFYSNPEELSSLGATVKMEH

[0158] DVTDVDTENKTTVTAKDLKTGETETVSYDKLVMTTGSWPIIPPISGIDAKNVLLCKNYNQANEIIAQAKEAKRVVIVGG

[0159] GYIGIELVEAFVESGKQVTLIDGLDRILNKYLDKPFTDILEKELTDRGVTLALGENVTEFITDEEGKVKQVATPTDTFDT

[0160] DMVILCVGFRPNTKLIEEKVDTLPNGAIKVNEYMQTSDPDIFAAGDSAVVNYNPSGTQNYIPLATNAVRQGLLVGN

[0161] NLTDHKMAYRGTQGTSGLYLFGWTIGSTGVTKESATNLNDLEVQATTVFEDNYRPEFMPTTEKVMM ELVYEKGTNRI

[0162] VGAQFMSKYDITQSANTMSLAVQNKMTVEDLALSDFFFQPHFDRPWNYLNLLAQAALGEIETAQ

[0163] SEQ ID No. 11:

[0164] ATGAAGGTTGCAGTAATCGGTGGAACTCACGCAGGTGTCTTTTCAGCTAAGCAAATTGTGGCAGAAGTACCGG

[0165] GCGCAGAAGTGCATGTATTTGAAAAGACGATACGGTATCATACCTATCATGTGGAACGGCATTATGGATTGG GGATAATGTTTCATCACCAGATCGTATGTTTTATGAATCACCTGAATCAATGAAAGAACAAGGGATTCATATGC

[0166] ATATGAATACTGAAGTGACGGCCGTTGATTTACAAGCAAAGTCACTTGAAATCAAGTCATTGGCTGATGATTCA

[0167] ACGCGTCAAGATACATTTGATAAGATTGTGATTACAACAGGATCAAAGACGCTAGTACCGCCAATTCCAGGTA

[0168] TTGACAGTGAAAAGATTTATATGAGTAAGAGCTGGAATGATGCGAAGCGTTTGAAGGAACGCACAGCTGATA

[0169] TCAAGAGTGCCATCGTAATCGGAGCTGGTTATATTGGTGCCGAATTAGCAGAACAATTGTCAATTAATGATAA

[0170] AGAAGTGACAATGATCGATGCAGCTGAACGTGTTCTTGCGAATAATGTCTCACCAAAGTTATCTGGCATTGTG

[0171] GCTCAAAAGTATGAAGATCATGGTGTGAAGTTGGTGATGAATGAAAAGGTTGTTGGCTTTGAAGACACAGCT

[0172] GATGGCATTATCGTTAAAACTGATGCCGGAAGTTACGAAGCTGAATTAGCGATTTTGGGAATTGGTTTCCGTCC

[0173] TAACACTGACCTAGTGGCTGATCAATTAGACACGATGGATAATGGGGCAATTAAGACCAACGAATATATGCAA

[0174] ACTTCAGTTGAAGATGTTTATGCTGCTGGTGATTCAACAGCTGTCTTCTATAACCCAACGCAAGCGTATGATTAT

[0175] ATTCCGCTAGCAACTAATGCGGTCCGTCAAGGAATGTTAGTTGGACGTAATATTGAAAAGCCAACAATTGCTTA

[0176] CCAAGGAACACAAGCAAGTTCAGCAGTTGAATTGTATGAAACTGCAATTGGAACAACTGGAATTACAAGTGTT

[0177] GAAGGTGCAACGGCCAAGGGCATTGATGCCGACTCAATCTCAATTGAAGAAGATTACCGTCCTGACTTTATGC

[0178] TATCAACGACTGAAGTTGTTGCTTCATTGGTTTGGGAAAAGGGAACTCGTTCGTTATTGGAGCTGAATTCTTG

[0179] TCAGCGCATGACGTAACGCAAGCGGCCAATGTTATCTCATTAGCAATTGAAAACAAGATGACAATTGATCAAC

[0180] TGGCCATGAGTGACTTCTTCTTCCAACCTAACTTTAGCCAACCGATTAACTATATTTCTTCAGTAGCTATGGCTG

[0181] CTGTTGCCAAAGTTTAA

[0182] SE ID Nr. 12:

[0183] MKVAVIGGTHAGVFSAKQIVAEVPGAEVHVFEKNDTVSYLSCGTALWIGDNVSSPDRM FYESPESMKEQGIHMH

[0184] MNTEVTAVDLQAKSLEIKSLADDSTRQDTFDKIVITTGSKTLVPPIPGIDSEKIYMSKSWNDAKRLKERTADIKSAIVIG

[0185] AGYIGAELAEQLSINDKEVTMIDAAERVLANNVSPKLSGIVAQKYEDHGVKLVMNEKVVGFEDTADGIIVKTDAGSY

[0186] EAELAILGIGFRPNTDLVADQLDTMDNGAIKTNEYMQTSVEDVYAAGDSTAVFYNPTQAYDYIPLATNAVRQGML

[0187] VGRNIEKPTIAYQGTQASSAVELYETAIGTTGITSVEGATAKGIDADSISIEEDYRPDFMLSTTEVVASLVWEKGTRRII

[0188] GAEFLSAHDVTQAANVISLAIENKMTIDQLAMSDFFFQPNFSQPINYISSVAMAAVAKV

[0189] SEQ ID No. 13:

[0190] ATGAAAGTTGCAATTATTGGTAGTACTCATGCAGGTACATTTTCAGCGATTCAAGTTAAACAACAACATCCAGA

[0191] TGCTGAAGTAACGGTATTCGAACAACATACAACCGTCTCGTTTTTATCATGTGGTATCGCATTGTGGTTGGGTG

[0192] ACCATGTTTCTGACGAACAACGTATGTTCTATGAAACACCAGCGACGATGACAGATAAAGGTATCATTATGAA

[0193] GATGCAACATGAAGTGGTTGCCGCGAACCTAGCTGAAAAATCTGTGCAGGTTAAAGACTTATTAACTGGAGAA

[0194] ATATCTTCAGAAATATTTGACAAAATTGTTATCACGACTGGATCAAAACCGTTACTTCCTAAGATTAGTGGCATC

[0195] GACAGTGACCGTATCTATATGGTTAAGTCATGGGAAGATGCTCAGAAAATAAAAGCTGTTGCTAGTGATGTAA

[0196] AACTGCGATTGTGATTGGTGCTGGCTATATTGGTGCTGAAATTGCCGAGCAGGTTTTCGGTGACAGACAAACA

[0197] GGTGACTTTAGTTGATGGCTTTGACCGTGTATTGCCGAAAAACTTTAGCCCAGTCATTTCAAATCGTTTGGCGC AATCATTTCAAGAACATGGTGTGAAGCTGGCTCTCAATCAGATGGTGACCTCATTCGAGGATTTGCCTGATGGC

[0198] GGCATTCGAGTCAATACGGATAAAGGTAGTTATGAAGCTGATATTGCTGTCTTAGGGATTGGATTTTTGCCTAA

[0199] TACACAGTTGTTTGCCGGGCAGATTGATATGCTTGATAATGGGGCTATCATTACTAACAAATATATGGAAACCA

[0200] GCATGCCTGATGTTTATGCTGCTGGAGATGCAACGACTGTTTATTACAATCCAACACACGAATATGATTACTTA

[0201] CCACTAGCTACAAATGCGATTCGTCAAGGCATGTTAGTTGGTAAGAATATTGATGGTCATCATTTACCTTATAG

[0202] TGGTACTCAGGGTACGTCAGCCGTTGAATTATATGGATTTGCCATGTCAGCAACTGGTTTGAATAAGGCAACT

[0203] GCAGAACAACGTGGTTTGGAAGTTGAGGAAACAATCTACGAAGAGAATTATCGGCCTGAATTTATGCTATCAA

[0204] CCACACCAGTATTATCAACCTTAACTTGGGAAAAGAAAACACGGCGTATCGTTGGTGCAGCGTTTATGTCAAAA

[0205] CATGATATTTCCCAAGCTGCTAACGTTGTTTCTTTGGCAATTCAAAATAATATGACGATTGATGATTTGGCAATG

[0206] GCTGACTTCTTTTTCCAACCTAATTTTACAACCGATAATTTGTTGGTGCAACAGCTTTGCAAGCCGTCAAT

[0207] GATAGTGATGATTAG

[0208] SE ID Nr. 14:

[0209] MKVAIIGSTHAGTFSAIQVKQQHPDAEVTVFEQHTTVSFLSCGIALWLGDHVSDEQRM FYETPATMTDKGIIMKM

[0210] QHEVVAANLAEKSVQVKDLLTGEISSEIFDKIVITTGSKPLLPKISGIDSDRIYMVKSWEDAQKIKAVASDVKRAIVIGA

[0211] GYIGAEIAEQFSVTDKQVTLVDGFDRVLPKNFSPVISNRLAQSFQEHGVKLALNQMVTSFEDLPDGGIRVNTDKGSY

[0212] EADIAVLGIGFLPNTQLFAGQIDMLLDNGAIITNKYMETSMPDVYAAGDATTVYYNPTHEYDYLPLATNAIRQGMLV

[0213] GKNIDGHHLPYSGTQGTSAVELYGFAMSATGLNKATAEQRGLEVEETIEENYRPEFMLSTTPPLSTLTWEKTRRI

[0214] VGAAFMSKHDISQAANVVSLAIQNNMTIDDLAMADFFFQPNFTQPINFVGATALQAVNDSDDD

[0215] SEQ ID No. 15:

[0216] ATGAAGATTCTTGTCATTGGTGCTACCCATGCCGGTACATTTGCAACCCAGCAGATTCTAACCGACCATCCAGA

[0217] TGCAGAGGTTACTGTCTACGAACGCAATAACAACCTGTCCTTCCTCTCGTGCGGCATTGCTTTGTGGGTTGGTG

[0218] ATCATGTCAGTGACCCGGATAAAATGTTCTATTCCAGTCCCGAAGCACTCGCTAAACTCGGTGCTAATATGCAA

[0219] ATGGAACATGACGTGCTCAATATTGATCCAGCAACTAAAACAGTTGAAGTCAAGGATCTAAAAACCGGAACCG

[0220] TTACTACCGATACTTATGACAAATTAGTCTACACAACCGGATCGACGCCAATCATTCCAAATATTCCCGGTATTC

[0221] ACGATTCAAACGTCTACTTATGCAAAAATTGGTCCGACGCCAAGACGCTAAAAGATCTGGCCCCGTCCATTAAA

[0222] AGCGCCATTGTCATCGGTGCAGGCTACATCGGTGCAGAATTAGCCGAACAATTTGCGTTAACCGACAAAGAAG

[0223] TCACGTTAATCGATGGACTTCCACGGGTTTTGGCGAAAAACTTTGACGCCACTATCACGGATCGCGTTGAAAAA

[0224] CTGTACACCGATCACGGGGTTCACTTGGCACTCAATGAGATGGTTACCGAGTTCGCACAAGCTGATCAGGGTA

[0225] TCAAGGTTACAACCAATAAAGGCGACTATACCGCGGATATTGCAATTTTATGTACCGGCTTCCGTCCGAACACG

[0226] GATCTGCTAAAGGACCATCTGGACACCCTGCCTAATGGCGCTGTCATAACAAATGCATATATGCAGACCAGTG

[0227] ACCCCGACATTTTCGCTGCTGGTGATACCGCTACCGTCCACTATAATCCGACTGGCAAAAATGACTACATCCCG

[0228] CTTGCGACCAACGCAGTCCGTCAGGGCATTCTTGTTGGTAAAAATATCATGACCCCCACGGAAAAATACCTGG

[0229] GAACACAATCTAGCTCGGCTGTTGAACTTTTTGATCACGCCATTGCGGCAAGCGGCCTAACGGTGGAAGGCGC TCACGCACGTGGACTTGAGCTTGATAGTGTCACGATCGAACAGGATTATCGTCCCGATTTCATGTTAACCACAA

[0230] CGCCGGTGCTCTGCAGCCTGACATGGGATCCCAAGACGCACGAAGTTAAAGGAGGTGCCTTTTTCCCAAGCA

[0231] CGATATCAGCCAAAGGGCTAATGTCATTTCGCTAGCGATCCAGACCCACATGACGATCGAAACACTTGCGATG

[0232] GTTGACATGCTCTTCCAACCTAACTTCGATCAGCCGATTAACTGGGTAAACGCCGTAGCTATGGCGGCAGTTGC

[0233] CAAGGCTAAAAAGAAAACGACAACACCGGTAGCCTAA

[0234] SE ID Nr. 16:

[0235] MKILVIGATHAGTFATQQILTDHPDAEVTVYERNNNLSFLSCGIALWVGDHVSDPDKM FYSSPEALAKLGANMQM

[0236] EHDVLNIDPATKTVVKDLKTGTVTTDTYDKLVYTTGSTPIIPNIPGIHDSNVYLCKNWSDAKTLKDLAPSIKSAIVIGA

[0237] GYIGAELAEQFALTDKEVTLIDGLPRVLAKNFDATITDRVEKLYTDHGVHLALNEMVTEFAQADQGIKVTTNKGDYT

[0238] ADIAILCTGFRPNTDLLKDHLDTLPNGAVITNAYMQTSDPDIFAAGDTATVHYNPTGKNDYIPLATNAVRQGILVGK

[0239] NIMTPTEKYLGTQSSSAVELFDHAIAASGLTVEGAHARGLELDSVTIEQDYRPDFMLTTTPVLCSLTWDPKTHEVKG

[0240] GAFFSKHDISQSANVISLAIQTHMTIETLAMVDM LFQPNFDQPINWVNAVAMAAVAKAKKKTTTPVA

[0241] SEQ ID No. 17:

[0242] ATGAAAGTAGTCGTTGTAGGATGTACGCACGCTGGAACATCAGCAGTAAAAACAATTCTTAATGAGCATCCAA

[0243] ATACTGAAGTAACAGTTTTTGAACGAATGATAATGTGTCATTCTTATCATGCGGAATCGCATTGTATGTTGGT

[0244] GGCGTTGTGAAAGATCCAGCAGGTTTATTCTATTCAAATCCTGAAGAATTAACTGAAATGGGCGCAACTGTGC

[0245] ACATGGAACACAATGTTACAAATATCGATACTGTTGCTAAAAAAGTTACTGTAGAAAATATGCAAACTGGTGA

[0246] AGTCTTTGAAGAATCATACGACAAATTAGTGAATACAACAGGTTCATGGCCGATTGTTCCACCAATCTCTGGTA

[0247] TTGAGTCTAAAAACATTTTATTATGTAAAAACTACAACCAAGCTAATGAAATCATTCGCCAAGCAAAAGACAAA

[0248] CAAAAAGTTGTTATTGTTGGTGGAGGTTACATTGGTATTGAATTAGTGGAAGCGTTTGCTGAATCTGGAAAAG

[0249] ATGTTACGTTGATTGATGGATTAGACCGTATTTTAAACAAATACCTAGATCCAGAATTCACAGATATCTTAGAA

[0250] CACGATTTGCAAGAACGTGGAATCAAATTAGCACTAAACCAAACTGTAAATGGCTTTGAAGCGAATGAAAATG

[0251] GAGAAGTAACTAAAGTTGTCACTTCTGAAAATTCATTTGAAACTGAAATGGTTATCATGTGTGTTGGTTTCCGT

[0252] CCAAACAATGAATTGTTAAAAGACAAAGTAGATATGTTGCCAAATGGCGCTATTATTGTTGATGAATACATGA

[0253] GAACAAGCGATAAAGATATTTACGCTGCTGGAGACAGCTGTGCAGTTCATTACAATCCAAATGGTGGATCTGC

[0254] ATATATTCCATTAGCAACTAACGCTGTTCGCATGGGAACTTTAGTTGGTAAAAACATTGTTGAACCAAGTGTTA

[0255] AAATACCGTGGAACACAATCTACATCTGGATTGTACTTGTTCGGTTTCAACATCGGTTCTACAGGAGTAAACGTA

[0256] AACAGTGCTTCTCATTTCGGATTAGATGTTCGTTCAGTTGTAGTTGAAGACTACTACCGTCCAGAATTCATGCCG

[0257] ACAAATGAAAAAGTATTGATGAAAATTAGTTTATGAAGTTGGAACAAACCGCATCGTTGGTGGACAAGTAATGT

[0258] CTAAATATGACATCACTCAATCAGCTAATACATTGTCATTAGCTGTACAAAACAAAATGACTATTCGAAGATTTG

[0259] GCATATGTTGACTTCTTCTTCCAACCAGTATTCGATCGTCCTTGGAACTACTTGAACTTGCTTGCACAAGCAGCA

[0260] GTAGAACAAGAAAGAAAAATCGCAACAGGTACAGAAGTAACTGTTTAA SEQ. ID No. 18:

[0261] MKVVVVGCTHAGTSAVKTILNEHPNTEVTVFERNDNVSFLSCGIALYVGGVVKDPAGLFYSNPEELTEMGATVHM EHNVTNIDTVAKKVTVENMQTGEVFEESYDKLVNTTGSWPIVPPISGIESKNILLCKNQANEIQQVKVIKVQVK GGGYIGIELVEAFAESGKDVTLIDGLDRLNKYLDPEFTDILEHDLQERGIKLALNQTVNGFEANENGEVTKVVTSENS FETEMVIMCVGFRPPNNELLKDKVDMLPNGAIIVDEYMRTSDKDIYAAGDSCAVHYNPNGGSAYIPLATNAVRMGT LVGKNIVEPSVKYRGTQSTSGLYLFGFNIGSTGVNVNSASHFGLDVRSVVVEDYRPEFMPTNEKVLMKLVEEVGTN RIVGGQVMSKYDITQSANTLSLAVQNKMTIEDLAYVDFFFQPVFDRPWNYLNLLAQAVEQERKIATTV

[0262] The NAD(P)H oxidase leads to the oxidation of NAD(P)H to NAD(P) +comprises or preferably consists of an amino acid sequence having an identity to SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16 or SEQ ID No. 18 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and in particular 100%. The NAD(P)H oxidase particularly preferably comprises or consists of the amino acid sequence SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16 or SEQ ID No. 18.

[0263] Alternatively, the NAD(P)H oxidase for the oxidation of NAD(P)H to NAD(P) +preferably an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15 or SEQ ID No. 17 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid encoding NAD(P)H oxidase comprises or consists of the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15 or SEQ ID No. 17.

[0264] As used herein, stringent conditions refer to conditions under which so-called specific hybrids, but not non-specific hybrids, are formed. For example, the stringent conditions include hybridization in 6xSSC (sodium chloride / sodium citrate) at 45 °C and then washing with 0.2 to 1xSSC, 0.1% SDS at 50 to 65 °C; or such conditions may include hybridization in 1xSSC at 65 to 70 °C and then washing with 0.3xSSC at 65 to 70 °C. The hybridization may be carried out by conventionally known methods, such as those described by J. Sambrook et al. in Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989). Another aspect of the present invention relates to the use of an NAD(P)H oxidase for the oxidation of NAD(P)H to NAD(P) +, in particular in the production of galactaric acid from galacturonic acid, which comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ. ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16 or SEQ ID No. 18 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid exhibiting at least 80% identity to SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15 or SEQ ID No. 17, and iii) an amino acid sequence encoded by a nucleic acid that, under stringent conditions, is attached to a complementary strand of a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15 or SEQ ID No. 17 binds.

[0265] The particularly preferred concentration of galacturonic acid is between 50 and 200 g / l.

[0266] The particularly preferred temperature range for the method according to the invention is between 20 and 40 °C.

[0267] The particularly preferred pH range is between 5 and 9.

[0268] D-Galacturonic acid can be produced, for example, by hydrolysis of pectin, which can be obtained from sugar beet pulp, potato peels, apple pomace or citrus peels, with the latter being preferred.

[0269] The separation of the enzymes can be achieved, for example, by centrifugation or ultrafiltration.

[0270] In a further preferred variant of the process according to the invention, the enzymes are present in a suspension, in the homogenate and / or in the lysate of the corresponding cells producing them, with lysates being particularly preferred.

[0271] In this context, suspension refers to a suspension of resting cells. These are harvested after cultivation (separated from the nutrient medium) and suspended in a suitable buffer system. Unlike fermentative processes, which also utilize whole cells, the resting cells, due to the removal of carbon sources and nutrients, can no longer grow and serve only for substrate conversion (Lin & Tao, 2017). Homogenate, in this context, refers to a physically and / or chemically treated suspension (e.g., treated with pressure, lysozyme, or ultrasound) in which the cell components are released from the cells. A lysate is obtained when the insoluble cell components of the homogenate are removed, for example, by filtration or centrifugation (see Enzyme Production & Lysate Preparation for details).

[0272] In another variant, the enzymes can also be modified at the N-terminus with a water-soluble polymer such as polyethylene glycol, immobilized in or on a solid matrix, or be part of a fusion protein.

[0273] materials

[0274] D-Galacturonic acid monohydrate, galactaric acid, D-glucose, and D-xylose were produced by Sigma-Aldrich; pectinase from Aspergillus niger was produced by TCI; methanol, acetonitrile, L-rhamnose monohydrate, and D-galactose, NAD + , NADH disodium salt, NADP + -Disodium salt and NADPH tetrasodium salt were sourced from PanReac AppliChem (ITW Reagents), L-arabinose, IPTG (isopropyl-β-D-thiogalactopyranoside), potassium dihydrogen phosphate, dipotassium hydrogen phosphate and sodium dodecyl sulfate (SDS) were sourced from Carl Roth and triethanolamine (TEA) was sourced from Chem-Lab NV.

[0275] Production of enzymes & production of lysates

[0276] General information on the expression of recombinant enzymes in E. coli

[0277] For recombinant enzyme production in an Escherichia co / / strain, the gene to be expressed was first amplified in PCR using genomic DNA or its synthetically adapted equivalent (adapted to the codon usage of E. coli) as a template, along with specific oligonucleotides carrying additional recognition sequences for restriction endonucleases. The resulting DNA fragment was then isolated from the reaction mixture. After nucleic acid digestion with the restriction enzymes Sphl and Hindll, the gene fragment encoding the target enzyme was ligated into the Sphl-Hindll backbone of the expression vector pQE70-Kan. The ligation product was transformed into chemically competent E. co / / cells (ToplOF), and the resulting colonies were used for plasmid isolation and restriction analysis.

[0278] The result of the cloning step was verified by restriction enzyme digestion and DNA sequencing. The resulting construct carries the target gene under the IPTG-inducible T5 promoter.

[0279] For the overexpression of the enzyme in E. coli, the resulting expression plasmid was transformed into competent RB791 expression cells. After 24 h incubation at 37 °C, the resulting colonies were inoculated into LB medium for expression assays. The following day, expression cultures with an optical density (OD550) of 0.02 were inoculated and shaken at 37 °C until an OD550 of 0.3 was reached. Subsequently, the temperature was lowered to 25 °C, and the cultures were induced with 0.1 mM IPTG upon reaching an OD550 of 0.5. After 22 h, the cultures were harvested (separated from the medium by centrifugation as a cell pellet) and analyzed for the expression of the recombinant enzyme using SDS-gel electrophoresis and activity determination (for use in a use test or optical zymatic assay).

[0280] Production of cell lysates using Sonifier digestion

[0281] To prepare a cell suspension, the cell pellet produced according to the above procedure was weighed into a suitable container and mixed with buffer and lysozyme (final concentration 0.5 mg / ml) (e.g., triethanolamine (TEA) - HCl) and dissolved with stirring. The biomass fraction is typically 20% by mass, the remainder being the buffer.

[0282] A Branson Sonifier 450 was used for cell disruption. The suspension was treated three times with 15 ultrasonic pulses each (device settings: Timer = 15; Duty Cycle = 50; Output Control = 3-5).

[0283] The resulting homogenate was centrifuged for 10 min at 4 °C and 16000 rpm (Eppendorf centrifuge 5417R) to separate the insoluble cell fragments and obtain the lysate.

[0284] Table 1. Enzyme classes and donor organisms for the enzymes used in the examples.

[0285] Analytical methods

[0286] High Performance Anion Exchange Chromatography

[0287] A Dionex ICS6000 system with an AS-AP autosampler was used to quantify D-galacturonic acid / D-galacturonate and galactaric acid / galactarate by HPAEC (High Performance Anion Exchange Chromatography). Measurements were performed using conductivity detection (CD) coupled to an electrolytically regenerated Dionex AERS 500 suppressor in external water mode. A Dionex lonPac ASll-HC-4pm column with a suitable guard column and a NaOH gradient was used for analyte separation. The mobile phase was additionally pretreated with a Dionex ATC (Anion Trap Column). A Dionex ICS6000 system with an AS-AP autosampler was also used to quantify L-arabinose, D-glucose, D-galactose, L-rhamnose, and D-xylose by HPAEC (High Performance Anion Exchange Chromatography). A Dionex CarboPac PA20-fast-4pm column with a corresponding guard column and a NaOH gradient was used to separate the analytes.The analytes were detected using a pulsed amperometric detector (PAD, gold electrode) and the "Carbo, Quad" waveform.

[0288] Determination of enzyme activities (optical-enzymatic assay)

[0289] Enzyme activities in the lysates were determined using a Shimadzu UV-1900 spectrophotometer. The formation or consumption of NAD(P)H was monitored at a wavelength of 340 nm by measuring changes in absorption. The measurements were performed with 0.2 mM cofactor (NAD(P)H). +or NAD(P)H). For this purpose, 20 µl of a 10 mM stock solution of the cofactor were placed in a cuvette (Greiner bio-one semi-micro cuvette made of polystyrene), and the desired pH was adjusted with 100 mM TEA-HCl buffer (870 µl). 10 µl of lysate (diluted or undiluted) and 100 µl of substrate solution were added to the cuvette, and the measurement was started immediately thereafter. The measurements were carried out at a standard temperature of 25 °C. The extinction coefficient of NAD(P)H at 340 nm (E = 6220 L mol) was used. 1 cm 1 The enzyme activity of the lysate can be determined in U / ml (based on the volume of the lysate) or U / g (based on the biomass used for production). 1 U represents 1 pmol of substrate conversion per minute (1 U = 1 pmol / min = l.67-10 _8 cat).

[0290] The following examples describe preferred variants of the process according to the invention in more detail. The lysates used in these examples were produced according to the processes described above.

[0291] Example 1

[0292] Selective oxidation of D-galacturonate to galactarate in the presence of sugars

[0293] The following components were mixed in two 2 ml glass vials (Vials I and II): 93.1 pl deionized water, 125 pl of a 1 M TEA buffer (pH 8), 250 pl of a sugar mixture* (99 g / l L-arabinose, 100 g / l D-galacturonic acid, 100 g / l D-glucose, 24 g / l D-galactose, 11 g / l L-rhamnose, 10 g / l D-xylose; adjusted to pH 8), and 5 pl of a 10 mM NAD + -Solution.

[0294] To start the reaction, 2 U of NADH oxidase I lysate and 5 U of UDH I lysate were added to Vial I, and 1.1 U of UDH II lysate (same biomass as UDH I) was added to Vial II. The mixtures were incubated for a total of 20 h with continuous shaking (Eppendorf thermomixer; 30 °C).

[0295] For analysis, 50 µl of a sample was mixed with 200 µl of methanol and incubated in an Eppendorf Thermomixer at 60 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 µl of deionized water, vortexed, incubated for a further 10 min at 60 °C, and then centrifuged for 5 min at max. g. The supernatant was diluted 1:400 with ultrapure water and measured by HPAEC (conductivity detection). For sugar determination by HPAEC (PAD), the supernatant was diluted 1:1600.

[0296] In this way, >99% of the D-galacturonate (50 g / l) was converted to galactarate in both reactions (Vial I: 56.0 g / l; Vial II: 55.0 g / l). No change in sugar concentration was observed, as the UDHs used here selectively oxidize D-galacturonate.

[0297] *The sugar mixture used here corresponds in its composition to a completely hydrolyzed sugar beet pulp (Micard et al., 1996; Ward et al., 2015).

[0298] Example 2

[0299] Conversion of citrus pectin to galactaric acid

[0300] The reaction was carried out in a Multifors benchtop bioreactor (Infors AG). A glass reactor vessel with an attached stirrer, pH electrode, and C2 sensor was used. pH control was achieved by adding 5M NaOH or 5M H2SO4.

[0301] Initially, 3 g of pectinase from Aspergillus niger were dissolved in 200 ml of water and 17.5 ml of a 1 M KPP buffer (pH 6). Gradually, 35 g of citrus pectin* were stirred into the solution. The pH was adjusted by adding 5 M NaOH. The mixture was then transferred to the reactor, mixed with 55 ml of water, and heated to 30 °C while stirring.

[0302] After 45 minutes, 7 ml of a 10 mM NAD+ solution were administered. + -Solution, 4 kU NADH oxidase I lysate and 30 ml UDH I lysate were introduced and compressed air was set.

[0303] During operation, samples were continuously taken from the reactor solution and analyzed as follows: 50 µl of the reactor solution were mixed with 200 µl of methanol and incubated in an Eppendorf Thermomixer at 60 °C with shaking for 20 min. The sample was briefly centrifuged, mixed with 750 µl of deionized water, vortexed, incubated for a further 10 min at 60 °C, and then centrifuged for 5 min at max. g. The supernatant was filtered using an Agilent syringe filter (PTFE, 0.2 pm). The filtrate was diluted 1:800 with ultrapure water and measured by HPAEC (conductivity detection).

[0304] After 27 hours, 306 mg of pectinase from Aspergillus niger (dissolved in 10 ml H2O) was added.

[0305] After 44 h, 70 g / l galactarate (corresponding to 91% conversion of the theoretical amount of D-galacturonic acid in pectin*) and 1.7 g / l D-galacturonate were detected.

[0306] The entire reactor contents were then heated to 70 °C for 30 minutes. The enzymes were removed by centrifugation (10 minutes at 4500 rpm). The yellowish, slightly cloudy supernatant was filtered through a P3 glass frit under vacuum. The filtrate was then acidified with 12 M H₂SO₄ (pH 1.4) and incubated overnight in a refrigerator, resulting in the formation of a colorless precipitate. This was filtered off through a P3 glass frit and dried overnight in a vacuum drying oven at 50 °C. Further fractions were obtained by concentrating and cooling the solution.

[0307] In this way, 13.2 g of galactaric acid could be obtained in high purity (> 95%).

[0308] * Note: According to the specification for "Pectin from Citrus" from Tokyo Chemical Industry (TCI; product number: P0024), the pectin consists of at least 58% D-galacturonic acid. Enzymatic hydrolysis using pectinase from Aspergillus niger in preliminary tests showed that the D-galacturonic acid content is approximately 70%, which was used for the conversion calculation.

[0309] Example 3

[0310] Oxidation of D-galacturonate to galactarate - comparison of two regeneration systems

[0311] The following components were mixed in two 2 ml glass vials (Vial I and II): 142.2 pl of deionized water (Vial I) and 33.4 pl of deionized water (Vial II), 125 pl of a 1 M TEA buffer (pH 8), 200 pl of a D-galacturonic acid solution (250 g / l), and 5 pl of a 10 mM NAD + -solution plus an additional 112 mg sodium pyruvate in Vial II.

[0312] To initiate the reaction, 5 U UDH I-lysate and 1 U NADH oxidase I I-lysate were added to Vial I, and 1 U L-lactate dehydrogenase (EC 1.1.1.27; from rabbit muscle) was added to Vial II. The mixtures were incubated for a total of 20 h with continuous shaking (Eppendorf Thermomixer; 30 °C). For analysis, 25 µl of one mixture was treated with 200 µl of methanol and incubated in an Eppendorf Thermomixer at 60 °C for 15 min. The sample was briefly centrifuged, mixed with 775 µl of deionized water, vortexed, incubated for a further 10 min at 60 °C, and then centrifuged for 5 min at max. g. The supernatant was diluted 1:400 with ultrapure water and measured on the HPAEC (conductivity detection) instrument.

[0313] In this way, 60% of the D-galacturonate (100 g / l) could be converted to galactarate (58.9 g / l) in Vial I and 18% of the D-galacturonate (100 g / l) to galactarate (21.7 g / l) in Vial II.

[0314] This example shows that cofactor regeneration via NADH oxidase leads to higher conversion rates than cofactor regeneration via lactate dehydrogenase (cf. Chang & Feingold (1969)).

[0315] Example 4

[0316] Oxidation of D-galacturonate to galactarate

[0317] The following components were mixed in 18 2 ml glass vials (vials 1 - 18): 45 pl deionized water, 50 pl a 500 mM TEA buffer (pH 7.9), 25 pl a 500 mM D-galacturonic acid solution (final concentration 25 mM), and 25 pl a 100 mM NAD + -solution (final concentration 5 mM).

[0318] To start the reaction, 5 pl of NADH oxidase lysate (see Table 2 below) and 5 pl of UDH lysate (UDH I: Vials 1–9; UDH II: Vials 10–18) were added to all batches. The batches were incubated for a total of 20 h with continuous shaking (Eppendorf thermomixer; 30 °C).

[0319] For analysis, 50 µl of a sample was mixed with 200 µl of methanol and incubated in an Eppendorf Thermomixer at 60 °C for 15 min. The sample was briefly centrifuged, mixed with 750 µl of deionized water, vortexed, incubated for a further 10 min at 60 °C, and then centrifuged for 5 min at max. g. The supernatant was diluted 1:80 with ultrapure water and measured using HPAEC (conductivity detection).

[0320] In this way, D-galacturonate (25 mM) could be completely converted to galactarate in all approaches.

[0321] Table 2.

[0322] In contrast to Example 4 of EP 3460067 Al, it is not necessary to add NADH oxidase during the reaction. Surprisingly, the NADH oxidases used lead to complete oxidation of the D-galacturonic acid.

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Claims

Patent claims 1. Method for the preparation of galactaric acid by reacting galacturonic acid in an aqueous solution in vitro with a dehydrogenase containing NAD(P) + as a cofactor to form reduced cofactor NAD(P)H, wherein the reduced cofactor NAD(P)H is oxidized with an NAD(P)H oxidase, characterized in that the NAD(P)H oxidase is used to oxidize NAD(P)H to NAD(P) +an amino acid sequence that is selected from the group consisting of: i) an amino acid sequence that has an identity to SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ. ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16 or SEQ ID No. 18 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid exhibiting at least 80% identity to SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15 or SEQ ID No. 17, and iii) an amino acid sequence encoded by a nucleic acid that, under stringent conditions, is attached to a complementary strand of a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15 or SEQ ID No. 17 binds.

2. Method according to claim 1, characterized in that a uronic acid / uronate dehydrogenase is used as the dehydrogenase.

3. Method according to one of claims 1 or 2, characterized in that D-galacturonic acid is used as the galacturonic acid.

4. Use of an NAD(P)H oxidase for the oxidation of NAD(P)H to NAD(P) + , which comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence exhibiting at least 80% identity with SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16 or SEQ ID No. 18, ii) an amino acid sequence encoded by a nucleic acid exhibiting at least 80% identity with SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15 or SEQ ID No. 17, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a complementary strand of a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15 or SEQ ID No. 17.