Method for producing hydroxytyrosol

Abstract

The present invention relates to a method for producing hydroxytyrosol (HTS) by enzymatically reacting tyrosol to produce HTS, wherein the reaction starting materials comprise i) tyrosol, ii) a compound selected from the group consisting of erythorbic acid and erythorbate salts, and iii) an oxidase having an amino acid sequence selected from the group consisting of SEQ ID NO:2, amino acid sequences homologous to SEQ ID NO:2, and the HTS is isolated from the reaction starting materials.

Description

[Technical field]

[0001] The present invention relates to a method for producing hydroxytyrosol (HTS) by enzymatic conversion of tyrosol to HTS, characterized in that a reaction mixture comprises i) tyrosol, ii) a compound selected from the group consisting of erythorbic acid and erythorbate salts, and iii) an oxidase having an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and amino acid sequences homologous to SEQ ID NO: 2, and HTS is isolated from the reaction mixture. [Background technology]

[0002] Hydroxytyrosol (HTS; 3,4-dihydroxyphenylethanol; CAS number 10597-60-1) is an effective antioxidant that has attracted great interest in recent years due to its beneficial effects on health. HTS is considered to be an active component in the Mediterranean diet. The European Food Safety Authority (EFSA) has allowed health claims for olive-derived polyphenols and recommends a daily intake of at least 5 mg of HTS. Anti-inflammatory effects of HTS have also been reported. There are also studies showing that HTS has antibacterial properties in vitro against pathogens of the respiratory tract and gastrointestinal tract, such as some strains of the Vibrio, Salmonella and Staphylococcus genera, and that the doses used are likely to compete with those of antibiotics, e.g. ampicillin. The substance is further believed to have neuroprotective, antiproliferative and proapoptotic properties. These properties mean that HTS is a very interesting and popular substance for use in medicines, nutraceuticals, functional foods or other cosmetic products.

[0003] To date, HTS available on the market comes mainly from olives, olive leaves or wastewaters obtained during the production of olive oil, and is supplied in the form of extracts: the percentage of HTS in these products is usually very low. Examples of these are HIDROX®, which has an HTS content of less than 12%, and OPEXTAN™, which has an HTS content of about 4.5%.

[0004] As well as isolating natural HTS from olives, methods for producing this substance synthetically have also been described.

[0005] For example, TN SN03042A1, or the corresponding publication Allouche et al. (2004), J. Agric. Food Chem. 52: 267-273, describes the extraction and purification of HTS from olive oil wastewater.

[0006] A chemical method for producing HTS is disclosed, for example, in EP2774909B1, which shows a compound represented by the general formula (1): [ka] The reactants are reacted in the presence of an aluminum compound and an aqueous solution of a hydroxycarboxylic acid at pH<3, and the formed HTS is isolated by extraction.

[0007] Furthermore, biotechnological methods are known for producing HTS, for example, HTS can be obtained by hydroxylation of tyrosol in the presence of atmospheric oxygen according to the following scheme: [ka]

[0008] For example, EP3234164B1 describes a method for the enzymatic conversion of tyrosol to HTS by a tyrosinase enzyme from Ralstonia solanacearum or a functional derivative thereof in a reaction mixture with ascorbic acid, said functional derivative being an engineered variant of the tyrosinase enzyme from R. solanacearum having from 1 to 5 amino acid changes compared to the wild-type tyrosinase enzyme, the or each change being selected from an amino acid insertion, addition, deletion and substitution.

[0009] EP3234164B1 discloses a tyrosinase whose activity is not inhibited by 27.6 g / L (199.7 mM) tyrosol substrate, 30.8 g / L (199.7 mM) HTS, and sodium salt of ascorbic acid at concentrations up to 0.4 M. In biotransformation, 150 mM tyrosol could be converted to HTS using an engineered mutant of R. solanacearum tyrosinase produced by recombinant means in E. coli at shake flask scale (laboratory scale) and used in the form of a cell-free lysate in the presence of 300 mM sodium salt of ascorbic acid (2-fold molar excess over tyrosol reactant). At an enzyme dosage of 2 mg / L per mM tyrosol of tyrosol substrate (i.e. a very high dosage of 300 mg enzyme per 150 mM tyrosol), the reaction time to complete conversion was about 6 hours.

[0010] Therefore, the method disclosed in EP3234164B1 is not sufficient for industrial application. The productivity of tyrosinase is disclosed only on a laboratory scale. The amount of tyrosol reactant used is relatively small at 150 mM, and it was necessary to prepare a cell-free lysate of tyrosinase-producing cells.

[0011] CN101624607B discloses a method for converting 25 g / L (180 mM) tyrosol (molar ratio of tyrosol to ascorbic acid: 1:1.57) into HTS by oxidase in the presence of 50 g / L (284 mM) ascorbic acid, which is separated in a multi-step process by nanofiltration, column chromatography, extraction and distillation. Under these conditions, HTS was obtained with a purity of 88.3%. Higher purity HTS can only be obtained by complex immobilization of oxidase on a carrier to isolate higher purity HTS, and another column chromatography operation using the steps of the method described above.

[0012] Those skilled in the art would recognize that the method of CN101624607B has some gaps in the disclosure. The source of the oxidase enzyme is not fully disclosed. Similarly, there is no information on the method of producing the enzyme, which is important for economic viability. Furthermore, the method of determining the purity of the product is not detailed.

[0013] The method disclosed in CN101624607B is not well suited for industrial applications. The amount of tyrosol reactant used is relatively small at most. 180 mM (25 g tyrosol per kg batch), and 1.57-fold molar excess of ascorbic acid are very high. For biotransformation using immobilized enzymes, the enzymes must first be purified in a complicated manner, and the amount of tyrosol reactant used is much less than that of the batch using non-immobilized enzymes. In addition, this method requires the HTS to be processed in five stages, namely nanofiltration, chromatography, ethyl acetate extraction of the eluate, distillation of the extract and another column chromatography operation. Due to these cost determinants, the method disclosed in CN101624607B needs improvement to improve the economic viability of biotechnological production of HTS.

[0014] Li et al. (2018), ACS Synth. Biol. 7: 647-654, describe a route for the biosynthetic production of HTS that is not based on the biotransformation of tyrosol. Summary of the Invention [Problem to be solved by the invention]

[0015] The object of the present invention is to provide a process which is improved over the prior art, is more economically viable and allows for the production of high purity hydroxytyrosol in a simple manner. [Means for solving the problem]

[0016] The above mentioned objects are achieved by a method for producing hydroxytyrosol (HTS) by enzymatic conversion of tyrosol to HTS, wherein a reaction mixture comprises i) tyrosol, ii) a compound selected from the group consisting of erythorbic acid and erythorbate salts, and iii) an oxidase having an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and amino acid sequences homologous to SEQ ID NO: 2, and wherein HTS is isolated from the reaction mixture.

[0017] The erythorbic acid used in the context of the present invention is generally preferably D-erythorbic acid. Erythorbate is a salt of erythorbic acid, preferably the sodium salt, D-sodium erythorbate, more preferably D-sodium erythorbate x H2O. Erythorbic acid or its salt is an auxiliary in the reaction, which is also referred to below by the term "protective substance". The reason for this term is that in the presence of a protective substance, HTS is the end product of oxidation from tyrosol, i.e. HTS is "protected", whereas in the absence of a protective substance, HTS is further oxidized. A protective substance is defined as a substance that contributes to the stabilization of the product.

[0018] The following are characteristics of the oxidase (hereinafter also referred to as protein (iii)): (1) It has oxidase activity, i.e., it is capable of converting tyrosol in the presence of atmospheric oxygen at a concentration of >0.2 M, and during this reaction it is also capable of tolerating high concentrations of erythorbic acid or erythorbate of >0.4 M; and (2) Having the amino acid sequence set forth in SEQ ID NO:2 (RscK60 oxidase) or having an amino acid sequence homologous to SEQ ID NO:2, which is a protein identified by accession number CCF97399.1 in the NCBI protein database.

[0019] The amino acid sequence annotated as polyphenol oxidase / catechol oxidase and homologous to the sequence shown in SEQ ID NO:2 has a sequence identity of at least 86%, preferably at least 90%, more preferably at least 94% with SEQ ID NO:2 over the entire sequence range from amino acid 1 to amino acid 543, and each change in the homologous amino acid sequence is selected from the insertion, addition, deletion and substitution of one or more amino acids. The homolog of SEQ ID NO:2 is selected from the enzyme class identified by the number EC1.10.3.1 in the KEGG database (catechol oxidase, diphenol oxidase, o-diphenolase, polyphenol oxidase, pyrocatechol oxidase, dopa oxidase, catecholase, o-diphenol:oxygen oxidoreductase, o-diphenol oxidoreductase). The homologous amino acid sequence is preferably SEQ ID NO:3, hereinafter also referred to as RscK60-del oxidase. That is, the method for producing HTS is characterized in that the amino acid sequence homologous to SEQ ID NO:2 is SEQ ID NO:3. The sequence of RscK60 oxidase is 47 amino acids longer, so SEQ ID NO:3 shares 91.3% identity with SEQ ID NO:2.

[0020] Sequence identity is defined as the percentage of the homologous amino acid sequence that is identical to amino acid positions 1 to 543 of the amino acid sequence annotated as polyphenol oxidase / catechol oxidase in SEQ ID NO:2, and each change in the homologous amino acid sequence is selected from an insertion, addition, deletion and substitution of one or more amino acids.

[0021] The present invention encompasses all engineered variants of the DNA sequence of SEQ ID NO: 1, which are envisaged on the basis of the so-called degenerate genetic code, encoding a protein having an amino acid sequence corresponding to SEQ ID NO: 2 or a variant which corresponds to SEQ ID NO: 2 or is homologous to SEQ ID NO: 2 and which have oxidase activity.

[0022] The preparation method of the present invention is further characterized by including erythorbic acid or one of its inexpensive salts, such as sodium D-erythorbate or its monohydrate, sodium D-erythorbate x H2O, as a protective substance to maximize the yield of HTS. As shown in Table 3 of Example 3, tyrosol was completely converted in a short time in the reaction mixture without sodium D-erythorbate, but the HTS yield was low at 40%. Thus, HTS was obviously decomposed in the reaction mixture. In contrast, in the parallel reaction mixture in the presence of sodium D-erythorbate x H2O, the tyrosol used was completely converted to HTS. Thus, sodium D-erythorbate as a protective substance inhibited the decomposition of HTS.

[0023] These results firstly show that RscK60-del oxidase can be efficiently produced as a recombinant enzyme in E. coli and is suitable for direct use in the form of resuspended cells without cell digestion for the biotransformation of tyrosol to HTS, which is one of the key factors for the economic viability of the production method.A further unexpected result of this experiment, which has not been described before, was that the addition of sodium D-erythorbate × HO represents an effective means of maximizing the yield in the biotransformation of tyrosol to HTS by RscK60-del oxidase.

[0024] The amount of erythorbic acid or erythorbate salt used in the process of the present invention depends firstly on the dosage of tyrosol reactant in the reaction mixture and secondly on the tolerance of the enzyme to erythorbic acid or erythorbic acid.

[0025] Prior art, such as EP3234164B1 and CN101624607B, disclose methods for producing HTS using ascorbic acid as a protective substance to maximize the yield of HTS. However, ascorbic acid is also known in the prior art as an inhibitor of tyrosinase, and the amount of both the protective substance and the tyrosinase reactant used is limited, which limits the economic viability of the production method. The maximum amount of ascorbic acid used was at a concentration of 0.4M. Furthermore, in EP3234164B1, the molar ratio of tyrosol to ascorbic acid was 1:2. In CN101624607B, the ratio of tyrosol to ascorbic acid was 1:1.57. This means that in both production methods, the amount of ascorbic acid used relative to the tyrosol reactant is relatively high, which negatively affects the economic viability of the production method.

[0026] It has now surprisingly been found that erythorbate can be used in the process of the invention at concentrations of more than 0.8 M, i.e. much higher than known for ascorbic acid or its salts, without inhibiting the activity of the oxidase, as shown in Examples 4 to 7. Compared to the prior art, this allows for the conversion of much higher concentrations of tyrosol to HTS, which is an important factor for the economic viability of the process.

[0027] Therefore, the method for producing an HTS according to the invention is preferably characterized in that the reaction mixture contains erythorbic acid or an erythorbate salt at a concentration of at least 0.4M, more preferably at least 0.6M, particularly preferably at least 0.8M.

[0028] As shown in Table 6, the tyrosol used could be practically completely converted to HTS with a molar yield of 96.5%. Compared to the prior art such as EP2774909B1, the inventive production method using RscK60 oxidase or a corresponding homologue and erythorbic acid or erythorbate as a protective substance represents an unexpected improvement, since the use of the protective substance in a 1:1 molar ratio to tyrosol is sufficient to completely convert >200 mM tyrosol, whereas according to the prior art, a 2-fold molar excess of sodium ascorbate was required to convert 150 mM tyrosol. Thus, the combination of erythorbate or erythorbic acid with RscK60 oxidase or a corresponding homologue is much more suitable than ascorbic acid and modified tyrosinase mutants to produce HTS from tyrosol in high yields in biotransformation.

[0029] As shown in Table 7, this biotransformation resulted in 433 mM (66.7 g / L) of HTS from the amount of tyrosol used of 434 mM (60 g / L), which corresponds to a molar yield of 99.7%. Table 8 shows that from the amount of tyrosol used of 724 mM (100 g / L), 707 mM (109 g / L) of HTS was obtained, which corresponds to a molar yield of 97.6%. This constitutes a clear improvement over prior art documents such as EP3234164B1 and CN101624607B, in which only 175 mM and 180 mM of tyrosol, respectively, were converted to HTS.

[0030] The aim of the biotransformation is maximum conversion of the tyrosol reactant to the HTS product, i.e. maximum yield of HTS relative to the amount of tyrosol used. A HTS yield of biotransformation of >80%, preferably >90%, more preferably >95%, particularly preferably 100% relative to the molar amount of tyrosol used is preferred. The yield is determined by quantitative HPLC of tyrosol and HTS as described in Example 2.

[0031] The process of the invention is further characterized in that it requires the supply of oxygen to the reaction mixture in the form of atmospheric oxygen, compressed air or pure oxygen. Here, oxygen can be introduced passively, for example by shaking or stirring in an incubation shaker (laboratory scale). Oxygen can also be introduced by active introduction of compressed air or oxygen via a sparging tube or by a combination of passive and active introduction. Introduction of oxygen by a combination of passive and active introduction is preferred.

[0032] Furthermore, the production method of the present invention is carried out under specified conditions of pH and temperature. The pH range of the reaction mixture is preferably 5.0 to 8.5, more preferably 5.5 to 8.0, and more preferably 6.0 to 7.5. The preferred temperature range is 20°C to 60°C, more preferably 25°C to 50°C, and particularly preferably 30°C to 40°C.

[0033] The reaction time for complete conversion of the tyrosol reactant to the HTS product depends on the amount of reactants used and the amount of enzyme used, and is 6 hours or less, preferably 25 hours or less, more preferably 50 hours or less, and especially preferably 80 hours or less.

[0034] The scale of the prepared reaction mixture is at least 0.5L, preferably at least 50L, more preferably at least 500L, and particularly preferably at least 5000L.

[0035] There are a variety of tests available to measure the molar oxidase activity of a protein.

[0036] For example, the L-DOPA test disclosed in Behbahani et al. (1993), Microchemical J. 47: 251-260 can be used, in which the oxidation of the L-DOPA substrate (3,4-dihydroxy-L-phenylalanine, CAS number 59-92-7) to the dopachrome chromophore (CAS number 3571-34-4) is monitored at a wavelength of 475 nm. For this purpose, as described in Example 2 (L-DOPA test) of the present invention, an amount of enzyme solution (cell suspension, isolated cells, cell homogenate or cell-free enzyme extract) containing 4 mg of protein is mixed with an amount of KPi buffer (50 mM potassium phosphate, 1 mM EDTA, pH 6.5) containing 10 mM L-DOPA. The test batch is incubated at 37°C and 140 rpm. After 0, 30, 60 and 120 minutes, aliquots of the test batch are taken, the solid components are separated, for example by centrifugation, and the absorbance of the supernatant is measured spectrophotometrically at 475 nm.

[0037] As described in Example 2, the oxidase activity can also be detected and quantified in an HPLC test. The test batch contains, for every 10 mL batch volume, 0.4 mg / mL protein (or a corresponding amount of cell suspension, isolated cells, cell homogenate or cell-free enzyme extract), 5.1 mM tyrosol and 0 mM or 10 mM sodium D-erythorbate in KPiE buffer (50 mM potassium phosphate, 10 mM EDTA, pH 6.5). The test batch is incubated at 30° C. and 140 rpm. After 0, 1, 2 and 4 hours, aliquots of the test batch are taken and in each case a H3PO4 solution at a concentration of 10% (v / v) is immediately added to stop the reaction. After the solid components have been separated, for example by centrifugation, the supernatant is used for the measurement of tyrosol and HTS by correspondingly calibrated HPLC (as known to those skilled in the art or as described in more detail in Example 2).

[0038] The enzyme activity can be measured directly in the culture broth without reisolation of the cells (cell suspension) or after reisolation of the cells (isolated cells). Furthermore, the enzyme activity can be measured in a cell homogenate after cell digestion, which can be produced directly from the culture broth or after reisolation of the cells. Furthermore, the measurement of the enzyme activity can also be carried out in a cell extract, for example by removing particulate cell components from the homogenate by centrifugation. Finally, the enzymes can be isolated from the cell extract by methods known per se, for example by column chromatography, and used as purified proteins for the measurement of the enzyme activity.

[0039] The enzyme activity is measured from the reisolated cells or from a cell homogenate, more preferably directly from the culture broth or from the reisolated cells, particularly preferably directly from the culture broth.

[0040] In the genetic information of the Ralstonia solanacearum strain K60 published by Remenant et al. (2012), J. Bact. 194: 2742-274 (GenBank CAGT01000120.1), the cds is identified by SEQ ID NO: 1, which is accessible via the GenBank Locus Tag RSK60_20060005, nt 3460-5091, and encodes a protein with the accession number CCF97399.1 (SEQ ID NO: 2) in the NCBI protein database. The function assigned to the protein therein was that of a "putative polyphenol oxidase, catechol oxidase". However, the enzymatic function of the protein resulting from this annotation has not been experimentally tested so far and therefore has not been characterized so far. Based on the annotation of polyphenol oxidase / catechol oxidase, the cds having the DNA sequence of SEQ ID NO: 1 was identified as rscK60-cds, and the protein having the protein sequence of SEQ ID NO: 2 in the present invention was identified as RscK60 oxidase.

[0041] Surprisingly, both the protein encoded by SEQ ID NO:1, as well as SEQ ID NO:2 (RscK60 oxidase) and amino acid sequences homologous to SEQ ID NO:2, have the oxidase activity of the invention, i.e. are able to convert tyrosol to HTS in the presence of atmospheric oxygen without being inhibited by erythorbic acid or erythorbate at concentrations >0.4M.

[0042] Sequence comparison was performed because tyrosinase from R. solanacearum strain GMI1000 disclosed in EP3234164B1B1 (Salanoubat et al. 2002, Nature 415: 497-502; Hernandez-Romero et al. 2006, FEBS J. 273: 257-270; Molloy et al. 2013, Biotechnol. and Bioengineering 110: 1849-1857) or engineered mutants of this enzyme can also be used for the biotransformation of tyrosol to HTS. Clear differences were found when comparing the amino acid sequence of the RscK60 oxidase annotated as a polyphenol oxidase / catechol oxidase (SEQ ID NO: 2) with that of the tyrosinase from R. solanacearum GMI1000 (Genbank accession number NP_518458) of EP3234164B1. The sequence of the RscK60 oxidase annotated as a polyphenol oxidase / catechol oxidase from R. solanacearum K60 is 47 amino acids longer than the sequence of the tyrosinase from R. solanacearum GMI1000 and differs by an additional 34 amino acids, resulting in only about 85% identity.

[0043] A protein having the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence homologous to SEQ ID NO: 2 is particularly suitable for the production of HTS, especially on an industrial scale, for the following reasons: -The oxidase of interest can be produced by fermentation at a scale of 1 L or more. -> Tyrosol at a concentration of 0.2M is converted to HTS In contrast to ascorbic acid or ascorbate, the protective substances, i.e. erythorbic acid or erythorbate, can be used at concentrations >0.4M to avoid degradation of the HTS. The molar ratio of tyrosol reactant to erythorbic acid or erythorbate in the mixture is only 1:1 to 1:1.2, which has the effect of saving costs in the production process of the present invention compared with the prior art, where the molar ratio of tyrosol to ascorbic acid or ascorbate is 1:1.57 to 1:2. The -oxidase can be used in the form of the fermenter broth without further processing, eliminating the need for costly re-isolation and processing of fermenter cells.

[0044] In summary, the provision of the oxidases of the present invention allows for a more economically viable biotechnological production of HTS.

[0045] The protein (iii) having oxidase activity, the amino acid sequence of SEQ ID NO:2 or an amino acid sequence homologous to SEQ ID NO:2, can be produced by fermentation or by chemical synthesis of the amino acid sequence.

[0046] The method for producing HTS by enzymatic conversion of tyrosol to HTS is preferably characterized in that the oxidase (iii) is produced by recombinant means by fermentation in E. coli, thereby producing a fermentation broth.

[0047] Fermentation is a process for the production of cell cultures at laboratory or industrial scale by growing a microbial production strain containing a genetic construct for the expression of an oxidase enzyme under defined conditions of medium, temperature, pH, oxygen supply and medium mixing to maximize the cell concentration produced in the cell medium and maximize the activity of the protein / enzyme. The terms "laboratory scale" or "industrial scale" simply differ according to the size of the culture. For example, a batch volume of less than 1000 mL is referred to as laboratory scale (e.g., described as shake flask culture), whereas a batch volume of more than 1000 mL is referred to as industrial scale.

[0048] For fermentation, a gene construct is first made by cloning the cds of RscK60 oxidase (SEQ ID NO:1), the cds of the engineered mutant RscK60-del (nt 142 to 1632 of SEQ ID NO:1), the cds encoding a protein homologous to SEQ ID NO:2, or the cds of the protein to be tested in an expression vector, which means that the gene construct contains all the information to express the protein from the cloned cds.

[0049] A person skilled in the art knows various methods to generate suitable genetic constructs. The expression vector pKKj disclosed in EP2670837A1 is preferred. pKKj contains the known tac promoter, whereby the expression of a coding gene sequence operably linked to the tac promoter can be induced by adding IPTG inducer (isopropyl-β-thiogalactoside). The genetic constructs of the present invention are pRscK60 (Figure 1) and pRscK60-del (Figure 2).

[0050] The corresponding gene construct is then transformed by known methods into a microorganism (production host) suitable for protein production, thereby creating a strain for producing the corresponding oxidase. The production host is preferably selected from the species Escherichia coli, particularly preferably a microorganism from the strain E. coli K12 JM105 (commercially available under strain number DSM3949 from DSMZ German Collection of Microorganisms and Cell Cultures GmbH). The production strain is more preferably E. coli JM105×pRscK60-del.

[0051] The RscK60 oxidase, or an oxidase homologous to SEQ ID NO:2, is expressed by culturing the production strain in a medium. The culturing can be on a laboratory scale by shake flask culture (known to those skilled in the art and described in Example 3) or on an industrial scale by fermentation (known to those skilled in the art and described in Example 4), and an aliquot of the resulting culture broth can be examined for oxidase activity.

[0052] In the fermentation process for producing the inventive oxidase of SEQ ID NO: 2 or a sequence homologous to SEQ ID NO: 2, first the biomass of the production strain is formed and then the oxidase is formed. Herein, the formation of the biomass and the oxidase may be correlated in time or may be separated from each other in time, such that the biomass is formed in a first fermentation step and then the enzyme production is initiated in a second step by an inducer of gene expression. A fermentation process as disclosed in Example 4 is preferred, in which the formation of the biomass and the oxidase is separated in time and the production of the oxidase is initiated by an inducer. A preferred inducer is IPTG (isopropyl-β-thiogalactoside).

[0053] The process for producing HTS by enzymatic conversion of tyrosol to HTS is preferably characterized in that the oxidase (iii) is produced by fermentation on an industrial scale, more preferably by fermentation with a fermentation volume of more than 1 L, particularly preferably more than 10 L, particularly preferably more than 1000 L, even more preferably more than 5000 L.

[0054] The terms "culture", "growth" and "fermentation", as well as, for example, the terms "culture medium", "growth medium" and "fermentation medium", are used synonymously in the context of the present invention. The term "culture broth" or "fermentation broth" refers to the end product of fermentation, which comprises cells containing the oxidase (iii) and cell culture medium.

[0055] Culture media are well known to those skilled in the art from practical microbial cultures: they usually consist of a carbon source (C-source), a nitrogen source (N-source) and additives such as vitamins, salts, trace elements, etc. that optimize cell growth and oxidase production.

[0056] The C source is one that can be utilized by the production strain for the formation of biomass. The preferred C source is glucose.

[0057] The N source is one that can be utilized by the production strain for the formation of biomass. Preferred N sources are gaseous ammonia or ammonia in aqueous solution as NH4OH or their salts, such as ammonium sulfate or ammonium chloride. N sources also include complex amino acid mixtures, preferably including yeast extract, proteose peptone or corn steep liquor, the latter in liquid form or in dry form called CSD.

[0058] Cultivation can be carried out in the so-called batch mode, where the medium is inoculated with a starter culture of the production strain, after which cell growth proceeds without further supply of nutrients.

[0059] As shown in Example 4, the cultivation can also be carried out in the so-called fed-batch mode, where after the initial stage of growth in the batch mode, further nutrients are supplied to compensate for the consumption of the nutrients. The feedstock consists of a C source, an N source, one or more vitamins or trace elements, including Cu(II) ions, important for production, or a combination of the above. The components of the feedstock can be metered together as a mixture or metered separately as individual feedstocks. Furthermore, inducers can also be added to the feedstock. The feedstock can be fed continuously or in portions (discontinuously) or by a combination of continuous and discontinuous feeding. Cultivation in the fed-batch mode is preferred.

[0060] The preferred source of C in the feedstock is glucose.

[0061] The C source is preferably metered into the culture so that the content of carbon source in the fermenter in the production phase does not exceed 10 g / L. The maximum concentration is preferably 2 g / L, more preferably 0.5 g / L, particularly preferably 0.1 g / L.

[0062] The preferred source of N in the feedstock is ammonia in gaseous form or in an aqueous solution such as NH4OH.

[0063] Further medium additives added may be salts of the elements phosphorus, chlorine, sodium, magnesium, nitrogen, potassium, calcium, iron, and trace amounts (i.e. μM concentrations) of the elements molybdenum, boron, cobalt, manganese, zinc, copper and nickel. In addition, organic acids (e.g. acetate, citrate), amino acids (e.g. isoleucine) and vitamins (e.g. vitamin B1, vitamin B6) may also be added to the medium.

[0064] The cultivation is carried out under pH and temperature conditions that promote the growth and gene expression of the production strain. A useful pH range is pH 5 to pH 9. A pH range of pH 5.5 to pH 8 is preferred. A pH range of pH 6.0 to pH 7.5 is particularly preferred.

[0065] The preferred temperature range for the growth of the production strain is 20° C. to 40° C. A temperature range of 25° C. to 37° C. is particularly preferred, with 28° C. to 34° C. being especially preferred.

[0066] The production strain can optionally be grown without oxygen (anaerobic culture) or with oxygen (aerobic culture), with aerobic culture being preferred.

[0067] In the case of aerobic cultivation, a saturation of the oxygen content is established of at least 10% (v / v), preferably at least 20% (v / v), more preferably at least 30% (v / v). The oxygen saturation in the medium is automatically regulated according to the prior art by a combination of gas supply and agitation speed.

[0068] The oxygen supply is ensured by the introduction of compressed air or pure oxygen. Aerobic cultivation with the introduction of compressed air is preferred. The useful range of compressed air supply in aerobic cultivation is 0.05 vvm to 10 vvm (vvm: compressed air supply to the fermentation batch, reported as liters of compressed air per liter of fermentation volume per minute). It is preferred that compressed air is introduced at 0.2 vvm to 8 vvm, more preferably 0.4 to 6 vvm, particularly preferably 0.8 to 5 vvm.

[0069] The maximum stirring speed is 2500 rpm, preferably 2000 rpm, more preferably 1800 rpm.

[0070] Protein production is induced by the addition of IPTG. It is preferred to add IPTG at a concentration of at least 0.1 mM, more preferably at least 0.2 mM, and particularly preferably at least 0.4 mM. The inducer may be added all at once, in multiple portions, or continuously. It is preferred to add the IPTG inducer all at once.

[0071] IPTG may be added directly at the beginning of the fermentation or when the cell density in the fermenter reaches a certain threshold. 600 The cell density, referred to as the number of cells in the fermenter broth, was determined by photometry in a known manner, as the absorbance at 600 nm per mL (OD 600 OD of 10 / mL is determined by measuring the absorbance at 600 nm of the fermenter broth. 600 to, more preferably, an OD of 30 / mL 600 From OD of 50 / mL is particularly preferred. 600 It is preferable to add IPTG from

[0072] The culture time is 10 to 100 hours. The culture time is preferably 20 to 70 hours. The culture time is particularly preferably 25 to 50 hours.

[0073] The culture batch obtained by the above-mentioned method comprises RscK60 oxidase or a corresponding homologue. The oxidase can be further used in the inventive method directly as fermenter broth, as a cell suspension after reisolation of the cells, as a cell homogenate after digestion of the cells without further work-up, either directly from the fermenter broth or after reisolation, as a cell-free enzyme extract or as an enzyme purified therefrom. In the inventive method, the oxidase is preferably used in the inventive method as fermentation broth without further work-up, as a cell suspension after reisolation of the cells or as a cell homogenate after digestion of the cells. In the inventive method, the oxidase is particularly preferably used in the inventive method directly as fermentation broth without further work-up, or as a cell suspension after reisolation ....

[0074] The cultivation can be carried out on a laboratory scale by shake flask cultivation (as described in Example 3) or on an industrial scale by fermentation (as described in Example 4) with the aim of generating a cell culture with maximum enzymatic activity based on the conversion of tyrosol to HTS. Maximum enzymatic activity is achieved firstly by a growth medium that promotes good cell growth and then by further additives that selectively stimulate enzyme production. A known method of stimulating enzyme production is based on gene constructs with inducible promoters, such as those present in the expression vector pKKj used in Example 1. The use of the expression vector pKKj is disclosed, for example, in EP 2 670 837 A1. pKKj features the known tac promoter. Expression of genes operably linked to the tac promoter can be significantly enhanced by the addition of the IPTG inducer (isopropyl-β-thiogalactoside).

[0075] A further means of increasing the enzyme activity is to add a cofactor for the enzyme activity. Since only the gene sequence of the RscK60 gene of the present invention was known, and no experimental studies of the enzyme activity were known, various means of enhancing the enzyme activity were investigated. The annotation of the RscK60 gene as polyphenol oxidase / catechol oxidase suggested a metal dependency of the enzyme activity. For example, the effect of adding Cu(II) ions to the medium on the enzyme production was investigated. Surprisingly, it was found that the enzyme activity was significantly increased by increasing the concentration of Cu(II) ions (Example 3). In this specification, the RscK60 oxidase differs from the prior art in that, for example, in the production of the enzyme in EP3234164B1, Cu(II) ions were not used to increase the enzyme yield, even though its dependency on copper was known in the technical literature (Hernandez-Romero et al. (2006), FEBS J. 273: 257-270; Molloy et al. (2013), Biotechnol. and Bioengineering 110: 1849-1857).

[0076] An open reading frame (ORF, cds, coding sequence) is a region of DNA or RNA that begins with a start codon and ends with a stop codon and codes for a sequence of amino acids in a protein. ORFs are also called coding regions, and stop codons are not translated into amino acids.

[0077] CDs are flanked by non-coding regions. A gene is a segment of DNA that contains all the basic information to produce biologically active RNA. A gene includes a DNA section that is transcribed to produce a single-stranded RNA copy, and expression signals involved in controlling this copying process. Expression signals include, for example, at least one promoter, a transcription start site, a translation start site, and a ribosome binding site. In addition, terminators and one or more operators can also be used as expression signals.

[0078] In the context of the present invention, a genetic construct refers to a circular DNA molecule (plasmid, expression vector) in which the cds of a gene are linked to further genetic elements (e.g., promoter, terminator, selection marker, origin of replication). The genetic elements of the genetic construct are initially responsible for their extrachromosomal inheritance during cell growth, and the protein encoded by the gene is produced.

[0079] The abbreviation WT (Wt) stands for wild type. A wild-type gene refers to the form of a gene that naturally evolves and is present in the wild-type genome. The DNA sequence of the Wt gene is publicly available in databases such as NCBI.

[0080] Engineered variants / functional derivatives / genetically generated variants of an enzyme define enzyme variants that arise through mutation, i.e. as a result of changes in the nucleotide sequence from the DNA of the Wt gene, resulting in an enzyme with a modified protein sequence, which may contain any desired alterations by insertion, addition, deletion and substitution of amino acids, as long as the original enzyme function is preserved.

[0081] The method for producing HTS in the context of the present invention is preferably a biotransformation process and more preferably consists of the following operating steps: in a first step, a recombinant oxidase enzyme is produced by fermentation, in a second step, the resulting fermentation broth is reacted directly in a reaction mixture with the tyrosol reactant (starting material) and further auxiliaries without further work-up, and in a third step, the HTS product is isolated from the reaction mixture by extraction with a solvent followed by distillative removal of the solvent without further work-up steps.

[0082] Biotransformation is defined as the enzyme-catalyzed conversion of reactants to products.

[0083] Extraction is defined as the process of mixing a reaction mixture with an insoluble liquid (the extractant), thereby transferring the reaction product to the extractant. After separation of the reaction mixture and the extractant (phase separation), the product can be isolated by removing the extractant.

[0084] Annotation in genetics and bioinformatics refers to the assignment of function derived from either experimental results or computer-aided prediction. Annotation of a DNA sequence specifically describes the protein-coding regions (cds) that contain the encoded proteins within that sequence.

[0085] In a preferred embodiment, the method for producing HTS by enzymatic conversion of tyrosol to HTS is characterized in that the fermentation for the production of oxidase is carried out in the presence of Cu(II) ions at a concentration of at least 0.02 mM, more preferably at least 0.1 mM, particularly preferably at least 0.2 mM, particularly preferably at least 0.5 mM. In this specification, Cu(II) ions can be provided by any known Cu(II) salt, such as copper(II) sulfate, copper(II) chloride, copper(II) acetate or copper(II) nitrate, preferably copper(II) sulfate in anhydrous or pentahydrate form (CuSO4×5H2O).

[0086] Increasing the content of Cu(II) ions in the medium has the advantage that an improved yield of enzyme activity can be achieved.

[0087] As summarized in Table 2 (Example 3), supplementation of the medium with Cu(II) ions in the form of CuSO4×5H2O increased the enzymatic activity of the oxidase by more than 10-fold. Thus, supplementation of the medium with Cu(II) ions constitutes an efficient method, not yet described in the prior art, for optimizing the production of enzymes suitable for the manufacture of HTS.

[0088] Furthermore, it is preferred that the fermentation broth from the fermentation for the production of the oxidase is used directly in the process for producing the HTS without further work-up.

[0089] As shown in Table 4 of Example 4, RscK60-del oxidase can be produced for industrial use by recombinant means in E. coli and can be used directly as fermentation broth without further cell isolation or digestion in the biotransformation of tyrosol to HTS. When the cell dosage in the fermenter is high enough, tyrosol was quantitatively converted to the product HTS at a concentration of 180 mM, which is very high compared to the conventional technology (Table 5). Unexpectedly, compared to the conventional technology using ascorbic acid as a protectant, the dosage of the D-erythorbate sodium x H2O protectant at a molar ratio of 1:1 was sufficient to inhibit the degradation of HTS.

[0090] In order to maximize the economic efficiency, tyrosol is converted in the biotransformation to produce HTS at a maximum concentration. In a preferred embodiment, the method for producing HTS is characterized in that tyrosol is used at a concentration of more than 200 mM, more preferably more than 400 mM, particularly preferably more than 700 mM.

[0091] In the process for producing HTS, it is preferred that the molar ratio of tyrosol to the amount of erythorbic acid or erythorbic acid used is 1:1.50 or less, more preferably 1:1.2 or less, particularly preferably 1:1 or less, and especially preferably 1:0.5 or less.

[0092] Furthermore, in the method for producing an HTS, it is preferred that the volume fraction of fermentation broth from the fermentation to produce the oxidase in the reaction mixture is 90% or less, more preferably 50% or less.

[0093] The process for producing HTS is characterized by reacting the reaction mixture for a period of time until at least 90% of the tyrosol used is converted to HTS.

[0094] The specific time required varies with the dose of tyrosol, which is determined by measuring the amount of tyrosol and HTS by HPLC (see Example 2, HPLC Testing for illustration).

[0095] For example, 70 g / L of tyrosol was converted to an extent of 95% within 24 hours. It is preferred that the tyrosol used is converted to an HTS to an extent of at least 80% within 24 hours.

[0096] In the method for producing HTS of the present invention, it is preferred that HTS is produced from tyrosol in a molar yield of preferably at least 70%, more preferably at least 90%, and particularly preferably at least 95%.

[0097] To isolate the HTS from the reaction mixture, the reaction mixture is mixed with an immiscible solvent without intermediate steps and, after phase separation, the solvent phase containing the product is separated. Solvents suitable for the extraction of the HTS are known from EP 2 774 909 B1. A preferred solvent for extraction is ethyl acetate (CAS number 141-78-6). The extraction, preferably with ethyl acetate, can be repeated as many times as necessary until the HTS is completely removed from the reaction mixture. Alternatively, the extraction, preferably with ethyl acetate, can be carried out continuously in known manner, for example by countercurrent extraction. In order to achieve a better phase separation, the mixture can be pretreated before extraction, for example by acidification with sulfuric acid, heating or a combination of these two measures, in order to denature the proteins present in the mixture.

[0098] The process for preparing HTS is preferably characterized by isolating the HTS from the reaction mixture by extraction with ethyl acetate, which is then more preferably separated by distillation.

[0099] The extraction is preferably carried out at neutral pH, ie pH 6.5 to 7.5.

[0100] After distilling off the solvent (ethyl acetate), HTS is obtained in high yield and purity. The molar yield based on the amount of tyrosol used is preferably >60%, more preferably >70%, particularly preferably >80%. The method for producing HTS is preferably characterized in that HTS is isolated from the reaction mixture with a purity of at least 80%, more preferably at least 90%, particularly preferably at least 93%.

[0101] A variety of analytical methods are available for identifying, quantifying and determining the purity of the tyrosol reactants and HTS products, including spectrophotometric, NMR, gas chromatographic, HPLC, mass spectrometry, gravimetric, elemental analysis, or a combination of these analytical methods.

[0102] In summary, as shown by the examples, the inventive process for producing HTS using the RscK60-del oxidase or a homologous oxidase and erythorbic acid or erythorbate as components of this novel process has unexpected improvements over the prior art such as EP2774909B1 and CN101624607B. Even small amounts of erythorbic acid or erythorbate used are sufficient to completely convert a significant amount of tyrosol (in the examples, it was sufficient to completely convert >400 mM tyrosol when the molar ratio of tyrosol to erythorbic acid was 1:1 to 1:1.2), whereas according to the prior art, a 2-fold and 1.57-fold molar excess of sodium ascorbate or ascorbic acid over tyrosol was required to convert 150 mM and 180 mM tyrosol, respectively. As a result, the inventive process is efficient and inexpensive. This allows the production of HTS with unexpectedly high yields and high purity.

[0103] The production method of the present invention may further comprise an inexpensive and simple step, namely, using cell culture broth from the fermentation of a strain producing the RscK60-del oxidase or the corresponding homologue of said oxidase in the biotransformation without further work-up, and isolating the product by direct extraction from the reaction mixture. [Brief description of the drawings]

[0104] [Figure 1] FIG. 1 shows the 4.5 kb vector pRscK60 prepared in Example 1. [Diagram 2] FIG. 2 shows the 4.4 kb vector pRscK60-del prepared in Example 1.

[0105] Abbreviations used in this application: cds coding DNA sequence (see above for definition) nt nucleotide HTS Hydroxytyrosol HPLC High Performance Liquid Chromatography EXAMPLES

[0106] The present invention is illustrated by the following examples, but is not limited thereto.

[0107] Example 1: Construction of expression vectors pRscK60 and pRscK60-del Genomic DNA from Ralstonia solanacearum strain K60 (commercially available from DSMZ German Collection of Microorganisms and Cell Cultures GmbH under strain number DSM9544) was used to isolate the cds of the RscK60 gene.

[0108] The coding sequence of the isolated RscK60 (hereinafter referred to as rscK60-cds, SEQ ID NO: 1), encoding a putative polyphenol oxidase / catechol oxidase, is disclosed in the NCBI (National Center for Biotechnology Information) nucleotide database as Locus Tag CAGT01000120.1, nt3460-5091 (SEQ ID NO: 1) and encodes a protein with Genbank accession number CCF97399.1 (SEQ ID NO: 2), hereinafter referred to as RscK60 oxidase.

[0109] The vectors pRscK60 and pRscK60-del were generated using the following DNA fragments from the putative polyphenol oxidase / catechol oxidase cds: -rsck60-cds: nt 1 to nt 1632 of SEQ ID NO: 1, which encodes the protein of amino acid sequence SEQ ID NO: 2, hereinafter referred to as RscK60 oxidase; - rscK60-del-cds, which encodes a protein of amino acid sequence SEQ ID NO: 3, hereinafter referred to as RscK60-del oxidase: nt 142 to nt 1632 of SEQ ID NO: 1. RscK60-del oxidase is a protein in which the N-terminus is truncated by 47 amino acids compared to RscK60 oxidase.

[0110] The DNA fragment rscK60-cds was isolated as a 1.6 kb fragment by PCR reaction ("Phusion™ High-Fidelity" DNA polymerase, Thermo Scientific™). For this purpose, genomic DNA from R. solanacearum strain K60 and primers rsck60-1f (SEQ ID NO: 4) and rsck60-2r (SEQ ID NO: 5) were used.

[0111] The DNA fragment rscK60-del-cds was isolated as a 1.5 kb fragment by PCR reaction ("Phusion™ High-Fidelity" DNA polymerase, Thermo Scientific™). For this purpose, genomic DNA from R. solanacearum strain K60 and primers rsck60-3f (SEQ ID NO: 6) and rsck60-2r (SEQ ID NO: 5) were used.

[0112] Primer rsck60-1f contains an EcoRI cleavage site flanked by 23 nucleotides (nt) (nt 1 to 23 of SEQ ID NO: 1) beginning at the start of the cds of RscK60 oxidase.

[0113] Primer rsck60-2r contains a HindIII cleavage site flanked by 24 nucleotides (nt) (nt 1609 to 1632 of SEQ ID NO: 1, reverse complement) starting from the 3′ region of the cds of RscK60 oxidase.

[0114] Primer rsck60-3f contains an EcoRI cleavage site flanked by 24 nucleotides (nt) (nt 142 to 165 of SEQ ID NO: 1) beginning in the 5′ region of the cds of RscK60 oxidase.

[0115] The PCR products were digested with EcoRI (present in primers rsck60-1f and rsck60-3f) and HindIII (present in primer rsck60-2r) and cloned into the pKKj expression vector previously digested with EcoRI and HindIII, generating the 4.5 kb expression vector pRscK60 (Figure 1) and the 4.4 kb expression vector pRscK60-del (Figure 2).

[0116] The expression vector pKKj disclosed in EP2670837A1 is a derivative of the expression vector pKK223-3. The DNA sequence of pKK223-3 is disclosed in the GenBank gene database under accession number M77749.1. Approximately 1.7 kb was removed from the 4.6 kb plasmid (bp 262 to 1947 of the DNA sequence disclosed in M77749.1) to generate the 2.9 kb expression vector pKKj.

[0117] Example 2: Analytical Testing Cell suspensions and isolated cells: Cells cultured in shake flasks (Example 3) or fermenters (Example 4) were either used directly as cell suspensions (culture broth, fermentation broth) without further isolation for analytical testing, or the cells were isolated.

[0118] Cells were isolated from suspensions (culture broth, fermenter broth) by centrifugation of the suspension (10 min, 15000 rpm, Sorvall RC5C centrifuge with SS34 rotor). The resulting cell pellet was washed once with 0.9% (w / v) NaCl. For further use as isolated cells, cell pellets from 100 mL cultures were suspended in 20 mL KPi buffer (50 mM potassium phosphate, 1 mM EDTA, pH 6.5).

[0119] Preparation of cell homogenate: To prepare the cell homogenate, a FastPrep-24™ 5G cell homogenizer from MP Biomedicals was used. 2×1 mL of cell suspension was digested in a 1.5 mL tube containing glass beads ("Lysis Matrix B") preformed by the manufacturer (3×20 s at a shaking frequency of 6000 rpm with an interval of 30 s in each case).

[0120] Preparation of enzyme extract: Cell-free enzyme extracts were prepared from the cell homogenates by centrifugation (10 min, 15000 rpm, Sorvall RC5C centrifuge with SS34 rotor) and isolation of the resulting supernatant.

[0121] Determining the protein content of a sample: The protein content of cell suspensions, isolated cells, cell homogenates or enzymatic extracts was measured in a Qubit 3.0 fluorometer from Thermo Fisher Scientific using the "Qubit® Protein Assay Kit" according to the manufacturer's instructions.

[0122] Measurement of oxidase enzyme activity (L-DOPA test): Oxidase enzyme activity was measured using a photometric assay in which the oxidation of L-DOPA enzyme substrate (3,4-dihydroxy-L-phenylalanine, CAS number 59-92-7) to dopachrome chromophore (CAS number 3571-34-4) was monitored at a wavelength of 475 nm (Behbahani et al. (1993), Microchemical J. 47: 251-260). Enzyme assays were performed using cell suspensions (e.g., to monitor the progress of production in fermenters), isolated cells, cell homogenates or cell-free enzyme extracts.

[0123] Test batches contained the following in a 100 mL Erlenmeyer flask with a batch volume of 8 mL: 4 mL KPi buffer, 10 mM L-DOPA (from Sigma-Aldrich) and 4 mL of test sample (cell suspension, isolated cells, cell homogenate or cell-free enzyme extract).

[0124] When samples from shake flask cultures were tested, 25 mL of the shake flask mixture was first centrifuged to concentrate the cells (10 min, 15,000 rpm, Sorvall RC5C centrifuge with SS34 rotor) to isolate a cell pellet, which was then used to prepare isolated cells, cell homogenates or cell-free enzyme extracts as described above, and the volume of the test sample was adjusted to 4 mL with KPi buffer for further use.

[0125] When samples from fermentations were tested, 1.6 mL of the fermentation batch was used directly as a cell suspension or for the production of isolated cells, cell homogenates or cell-free enzyme extracts as described above, and the volume of the test sample was adjusted to 4 mL with KPi buffer for further use.

[0126] The reaction was started by adding the respective test sample. The test batches were incubated at 37°C and 140 rpm in a shaker (Infors). 1 mL aliquots were taken at 0, 30, 60 and 120 min and immediately centrifuged at 13,000 rpm for 5 min (Heraeus™ Fresco™ 21 centrifuge, Thermo Scientific™) and the absorbance of the supernatant was measured at 475 nm (Genesys™ 10S UV-VIS spectrophotometer, Thermo Scientific™).

[0127] One unit (U) of oxidase activity is defined as the amount of enzyme that produces 1 μmol of dopachrome from L-DOPA in 1 min under test conditions (the extinction coefficient of dopachrome, ε 475nm =0.37×10 4 L×Mol -1 ×cm-1 ).

[0128] The oxidase specific activity was calculated based on the oxidase enzyme activity per mg of total protein in the measurement sample (cell extract, homogenate or cell suspension) (U / mg protein).

[0129] The oxidase (specific) activity measured by the L-DOPA test is hereinafter referred to as the oxidase (specific) activity in the L-DOPA test.

[0130] Tyrosol and HTS Determination (HPLC Test): The biotransformation study of tyrosol to HTS was carried out on an analytical scale.

[0131] Tyrosol solution: 7 mg of tyrosol (Sigma-Aldrich, final concentration in test 5.1 mM) was weighed into a 100 mL Erlenmeyer flask, dissolved in 4.9 mL of KPiE buffer (50 mM potassium phosphate, 10 mM EDTA, pH 6.5) and 0.1 mL of 1 M sodium D-erythorbate x H2O (Sigma-Aldrich, final concentration in test 10 mM) was added. For comparison purposes, in each test, 7 mg of tyrosol was dissolved in 5 mL of KPiE buffer without the addition of sodium D-erythorbate.

[0132] Test batch: 50 mL of cell suspension from shake flask cultures or 10 mL of cells from fermenters cultures were suspended in 5 mL of KPiE buffer and added to the tyrosol solution at the start of the reaction. The test batch (volume 10 mL) was incubated at 30° C. and 140 rpm on a shaker (Infors). Samples of 1 mL each were taken at 0, 1, 2 and 4 h and immediately mixed in each case with 0.1 mL of concentrated H3PO4 to stop the reaction. After centrifugation (13,000 rpm for 5 min, Heraeus™ Fresco™ 21 centrifuge, Thermo Scientific™), 1 mL of each supernatant was dispensed for the determination of tyrosol and HTS by HPLC.

[0133] HPLC Analysis of Tyrosol and HTS: For the quantification of tyrosol and HTS, HPLC methods calibrated for tyrosol and HTS, respectively, were used. The calibration reference substances tyrosol and HTS were provided by Sigma-Aldrich. An Agilent Infinity II HPLC instrument equipped with a diode array detector was used. The detector was set at a wavelength of 274 nm. Furthermore, a Luna C18(2) column from Phenomenex, 250 mm long, 4.6 mm inner diameter, 5 μm particle size, was adjusted to a temperature of 30 °C in the column oven. Eluent A: 5 mL H3PO4 in 1 L H2O. Eluent B: acetonitrile. Separation was performed in gradient mode from 5% to 10% eluent B in 5 min, followed by 10% to 16% eluent B in 15 min, at a flow rate of 1 mL / min. Retention time of tyrosol: 13.9 min; retention time of HTS: 10 min.

[0134] The yield of the reaction in the context of the present invention is defined as the amount of tyrosol (reactant) used that is converted to HTS (product) under reaction conditions. The yield is reported as volumetric yield in absolute amount of product based on volume (mM or g / L) or relative yield of product in percentage (also called percentage yield), i.e., the absolute yield is based on the tyrosol (reactant) used (taking into account the molecular weight of tyrosol (reactant) of 138.2 g / mol and the molecular weight of HTS (product) of 154.2 g / mol).

[0135] Example 3: Expression of RscK60 and RscK60-del oxidase in E. coli by shake flask cultivation For the isolation of plasmid DNA, the expression vectors pRscK60 and pRscK60-del from Example 1 were transformed into the commercially available E. coli strain NEB® 10-β (New England Biolabs), used for cloning purposes. Cell cultures were made for each clone from the transformation by culturing in LBamp medium (10 g / L tryptone, GIBCO™, 5 g / L yeast extract from BD Biosciences, 5 g / L NaCl, 100 mg / L ampicillin) (37° C., 120 rpm, tray shaker from Infors), and plasmid DNA was isolated from the cells using a plasmid DNA isolation kit (QIAprep® Spin Miniprep Kit, from Qiagen) according to the manufacturer's instructions.

[0136] Plasmid DNA of the expression vectors pRscK60 and pRscK60-del was transformed into E. coli K12 JM105 strain by known methods. E. coli JM105 strain is commercially available from DSMZ German Collection of Microorganisms and Cell Cultures GmbH under strain number DSM3949.

[0137] Clones for transformation were selected on Lbamp plates (10 g / L tryptone, GIBCO™, 5 g / L yeast extract from BD Biosciences, 5 g / L NaCl, 15 g / L agar, 100 mg / mL ampicillin) and identified as JM105×pRscK60 and JM105×pRscK60-del, respectively. The control used was E. coli JM105 transformed with pKKj vector (E. coli JM105×pKKj), from which transformants were generated in the same manner.

[0138] Precultures were made for each clone of E. coli strain JM105×pKKj, JM105×pRscK60, and JM105×pRscK60-del in 30 mL of Lbamp medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 100 mg / m L ampicillin) (growth overnight at 37°C and 120 rpm, Infors tray shaker).

[0139] 2 mL of each preculture was used as inoculum for the main culture of 100 mL of SM3 medium (1 L Erlenmeyer flask) supplemented with 15 g / L glucose, 0.5 mM CuSO4×5H2O and 100 mg / L ampicillin. Cell density OD 600 The main culture was shaken at 30°C and 140 rpm until the OD reached 2.0 (OD 600 (Photometric determination of cell density by measuring absorbance at 600 nm). Then, IPTG inducer (isopropyl-β-thiogalactoside, final concentration 0.4 mM, from Sigma-Aldrich) was added and the mixture was shaken overnight at 30° C. and 140 rpm.

[0140] SM3 medium: 12 g / L K2HPO4, 3 g / L KH2PO4, 5 g / L (NH4)2SO4, 0.3 g / L MgSO4 x 7H2O, 0.015 g / L CaCl2 x 2H2O, 0.002 g / L FeSO4 x 7H2O, 1 g / L Nacitrate x 2H2O, 0.1 g / L NaCl; 5 g / L peptone (Oxoid); 2.5 g / L yeast extract (BD Biosciences); 0.005 g / L vitamin B1 (Sigma-Aldrich); 1 mL / L trace element solution.

[0141] Composition of trace element solution: 0.15 g / L Na2MoO4×2H2O, 2.5 g / L H3BO3, 0.7 g / L CoCl2×6H2O, 0.25 g / L CuSO4×5H2O, 1.6 g / L MnC12×4H2O, 0.3 g / L ZnSO4×7H2O.

[0142] The enzyme activity was then verified by HPLC and photometric L-DOPA assays using cells from shake flask cultures.

[0143] Comparison of RscK60 oxidase activity with RscK60-del oxidase activity: For HPLC testing, E. coli JM105×pRscK60 (cell density OD of 6.4 / mL) was used. 600 ) and JM105 × pRscK60-del (cell density OD of 5.9 / mL 600 Cells from 50 mL shake flask cultures of each strain of were isolated by centrifugation and suspended in 5 mL of KPiE buffer in each case. In each case, 5 mL of isolated and resuspended cells were used in the HPLC studies described in Example 2.

[0144] Two HPLC tests were performed. The first batch contained in a batch volume of 10 mL: 5 mL JM105×pRscK60-del cells isolated and resuspended from the shake flask culture described above, 7 mg tyrosol (final concentration in the batch: 5.1 mM), 4.9 mL KPiE buffer, and 0.1 mL 1 M sodium D-erythorbate×H2O dissolved in KPiE buffer (see HPLC test in Example 2). The second batch also contained in a batch volume of 10 mL: 5 mL JM105×pRscK60 cells isolated and resuspended from the shake flask culture described above, 7 mg tyrosol, 4.9 mL KPiE buffer, and 0.1 mL 1 M sodium D-erythorbate×H2O dissolved in KPiE buffer. The batches were incubated at 30° C. and 140 rpm on a shaker (Infors). Samples from this test were taken at 0, 2, 4 and 6 hours and analyzed by HPLC. The results are shown in Table 1.

[0145] [Table 1]

[0146] Photometric L-DOPA Test: The enzyme activity of the JM105×pRscK60-del strain was determined by comparison with the control strain JM105×pKKj in a photometric L-DOPA test. Cell homogenates were produced for the cells of the two strains (digestion of cells as described in Example 2) and used in an L-DOPA test with L-DOPA as the enzyme substrate. The specific enzyme activity measured in the L-DOPA test with L-DOPA as the enzyme substrate was 1.5 mU / mg for the cell homogenate of the E. coli JM105×pRscK60-del strain and 0 mU / mg for the JM105×pKKj control strain.

[0147] Change in Cu(II) concentration in the medium: In preliminary experiments for the optimization of RscK60-del enzyme activity in shake flask cultures, one of the parameters that was varied was the Cu(II) concentration in the medium. The addition of Cu(II) ions to SM3 medium proved to be advantageous. SM3 medium supplemented with a trace element solution containing 0.25 mg / L CuSO4 × 5H2O (final concentration in the medium 1 μM) contained essentially only a low content of Cu(II) ions.

[0148] The effect of Cu(II) ions on the enzymatic activity of RscK60-del oxidase was tested by shake flask cultivation of E. coli JM105×pRscK60-del strain in SM3 medium supplemented with 15 g / L glucose, 100 mg / L ampicillin, and CuSO4×5H2O at concentrations between 0 mM and 0.5 mM, as described above. Cells from the batches were used as cell homogenates for the determination of activity by L-DOPA test (Table 2).

[0149] [Table 2]

[0150] Effect of sodium D-erythorbate on the stability of HTS: E. coli JM105×pRscK60-del strain (cell density OD of 4.7 / mL) 600Combined cells from 2 x 50 mL shake flask cultures of 100% HO were isolated by centrifugation and resuspended in 10 mL of KPiE buffer. Five mL of each of the isolated and resuspended cells was used in the HPLC study described in Example 2.

[0151] Two comparative biotransformation studies were performed. The first batch contained: 5 mL of JM105×pRscK60-del cells isolated and resuspended from the shake flask culture described above, 7 mg of tyrosol (final concentration in the batch: 5.1 mM), and 5 mL of KPiE buffer in a batch volume of 10 mL. The second batch also contained: 5 mL of JM105×pRscK60-del cells isolated and resuspended, 7 mg of tyrosol, 4.9 mL of KPiE buffer, and 0.1 mL of 1 M sodium D-erythorbate×H2O dissolved in KPiE buffer (see HPLC study in Example 2), also in a batch volume of 10 mL. The batches were incubated at 30° C. and 140 rpm on a shaker (Infors). Samples from this study were taken after 0, 1, and 4 hours and analyzed by HPLC. The results are shown in Table 3.

[0152] [Table 3]

[0153] Example 4: Expression of RscK60-del oxidase in E. coli by fermentation The E. coli JM105×pRscK60-del strain was used for the fermentation, which was carried out in a Biostat B fermenter (working volume 2 L) from Sartorius BBI Systems GmbH.

[0154] Shake flask preculture: Inoculate 2 × 100 mL of LBamp medium in a 1 L Erlenmeyer flask with baffles with the JM105 × pRscK60-del strain from the agar plate and grow to a cell density OD 600 The mixture was incubated at 30°C on an incubation shaker (Infors) at 120 rpm for 7 to 8 minutes until the concentration reached 2 to 4 / mL.

[0155] Pre-fermentation: 1.5 L of FM2 medium supplemented with 40 g / L glucose and 100 mg / L ampicillin was inoculated with 7.5 mL of shake flask preculture. Fermentation conditions were: temperature 30 °C; constant pH 7.0 (automatically corrected with 25% NH4OH and 6.8 N H3PO4 as described below); foam control by automatic dosing of 4% v / v Struktol J673 (Schill & Seilacher) in H2O; stirrer speed 450-1300 rpm; constant ventilation with compressed air sterilized using a sterile filter at 1.7 vvm (vvm: introduction of compressed air into the fermentation batch reported in liters of compressed air per liter of fermentation volume per minute); pO2 ≥ 50%. The oxygen partial pressure pO2 was controlled by the stirrer speed. After 16 h fermentation time, a cell density OD of 45-60 / mL was obtained. 600 was achieved.

[0156] Production fermentation: 150 mL of fermentation culture was inoculated into 1.35 L of pH 7.0 FM2 medium supplemented with 20 g / L glucose, 0.5 mM CuSO4 x 5H2O and 100 mg / L ampicillin. Fermentation conditions were: temperature 30°C; constant pH 7.0 (automatically corrected with 25% NH4OH and 6.8 N H3PO4 as described below); foam control by automatic dosing of 4% v / v Struktol J673 (Schill & Seilacher) in H2O; stirrer speed 450-1300 rpm; constant ventilation at 1.7 vvm; pO2 ≥ 50%. Oxygen partial pressure pO2 was controlled by the stirrer speed. Fermentation time was 30-32 h.

[0157] FM2 medium: 5 g / L (NH4)2SO4; 0.50 g / L NaCl; 0.075 g / L FeSO4×7H2O; 1 g / L Na3 citrate, 0.30 g / L MgSO4×7H2O, 0.015 g / L CaCl2×2H2O, 1.50 g / L KH2PO4, 0.005 g / L Vitamin B1 (Sigma-Aldrich); 5.00 g / L peptone (Oxoid); 2.50 g / L yeast extract (BD Biosciences), 10 mL / L trace element solution (corresponding to that used in Example 2).

[0158] The pH in the fermenter was adjusted to 7.0 at the start by pumping in a 25% NH4OH solution. During the fermentation, the pH value was maintained at 7.0 by automatic correction with 25% NH4OH or 6.8N H3PO4. For inoculation, 150 mL of preculture was pumped into the fermenter vessel. The starting volume was therefore 1.5 L. At the start, the culture was stirred at 350 rpm and sparged with a ventilation rate of 1.7 vvm. Under these starting conditions, the oxygen probe was calibrated to 100% saturation before inoculation.

[0159] The target value of O2 saturation (pO2) during the fermentation was adjusted to 50%. After the O2 saturation fell below the target value, a closed-loop control cascade was initiated to bring the O2 saturation back to the target value. The stirrer speed was continuously increased (up to a maximum of 1500 rpm).

[0160] Fermentation was carried out at a temperature of 30°C. When the glucose content in the fermenter dropped from the initial 20 g / L to approximately 5 g / L, a 60% (w / w) glucose solution was continuously fed. After that, the feeding rate was adjusted so that the glucose concentration in the fermenter did not exceed 2 g / L again. Glucose was measured using a YSI (Yellow Springs, OH, USA) glucose analyzer.

[0161] Target density in fermenter is OD of 50-60 / mL 600At the time point (7.5 h fermentation time), expression of the oxidase RscK60-del was started by a single addition of IPTG inducer (final concentration 0.4 mM). Fermentation was stopped 22.5 h after induction, corresponding to a total fermentation time of 30 h, and the enzyme activity (L-DOPA test) and analytical scale conversion of tyrosol to HTS (HPLC test) in samples of the fermentation batch were quantified as described in Example 2. In both tests, the fermentation broth was used directly without further workup. The remaining fermentation broth was aliquoted in 50 mL portions and stored frozen at -20°C for further experiments.

[0162] Without further treatment, the specific enzyme activity of the fermenter broth using L-DOPA as the enzyme substrate (L-DOPA test) was 28.4 mU / mg protein using 1.6 mL of fermenter broth with a protein concentration of 5 mg / mL in the L-DOPA test.

[0163] In the HPLC test to determine enzyme activity in the presence of sodium D-erythorbate (Example 2), 100 μl of fermenter broth (OD of 63.8 / mL) was added to the HPLC test after 30 hours of fermentation time, so that the actual cell density in the 10 mL test batch was 0.64 / mL. 600 ) was used. Samples from the test were taken after 0, 1, 2 and 4 hours and analyzed by HPLC for tyrosol and HTS content to determine the yield, i.e. the percentage of the tyrosol used that was converted to HTS (% HTS). After 4 hours of incubation, 99.4% of the tyrosol used was converted to HTS (see Table 4).

[0164] [Table 4]

[0165] In a further batch, the suitability of fermentation cells of the E. coli JM105×pRscK60-del strain for the biotransformation of tyrosol to HTS, which is important for the economic viability of the production method of the present invention, was tested in terms of the conversion of tyrosol at the maximum concentration and in the minimum time (space-time yield).

[0166] 249 mg tyrosol (final concentration 180 mM) and 389 mg sodium D-erythorbate x H2O (180 mM) were initially charged into a 100 mL Erlenmeyer flask, dissolved in 1 mL KPi buffer, and without further workup, 9 mL of fermenter broth of RscK60-del oxidase expressing JM105 x pRscK60-del cells was added to start the reaction. The batch volume was 10 mL. The molar ratio of tyrosol to sodium D-erythorbate x H2O was 1:1. The actual pH of the reaction measured in the batch was 6.7. The batch was incubated at 37°C and 140 rpm on a shaker (Infors). Sampling and analysis by HPLC (as described in Example 2) were performed after 0, 2 and 4 hours. The progress of the reaction is shown in Table 5.

[0167] [Table 5]

[0168] Example 5: Preparation of HTS Test 1: Biotransformation of 30 g / L tyrosol by JM105×pRscK60-del fermenter cells expressing RscK60-del in the presence of tyrosol and sodium D-erythorbate at a molar ratio of 1:1 and sodium D-erythorbate × H2O 300 mg tyrosol (final concentration 220 mM) and 475.4 mg sodium D-erythorbate x H2O (220 mM) were initially charged into a 100 mL Erlenmeyer flask, dissolved in 5 mL KPiE buffer, and without further workup, 5 mL of fermenter broth of E. coli K12 JM105 x pRscK60-del strain from Example 4 was added to start the reaction. The batch volume was 10 mL. The molar ratio of tyrosol to sodium D-erythorbate x H2O was 1:1. The pH of the reaction measured in the batch was 6.7. The batch was incubated on a shaker (Infors) at 30°C and 140 rpm. Sampling and analysis by HPLC (Example 2) were performed after 0, 3 and 24 hours. The progress of the reaction is shown in Table 6.

[0169] [Table 6]

[0170] Test 2: Biotransformation of 60 g / L tyrosol by RscK60-del fermenter cells in the presence of sodium D-erythorbate × HO (molar ratio of tyrosol to sodium D-erythorbate 1:1.2) 600 mg tyrosol (final concentration 434 mM) and 1126 mg Sodium D-erythorbate x H2O (521 mM) were initially charged into a 100 mL Erlenmeyer flask and suspended in 5 mL KPiE buffer. Sodium D-erythorbate x H2O showed good solubility under these conditions, whereas tyrosol could only react in suspension. Without further workup, 5 mL of fermenter broth of E. coli K12 JM105 x pRscK60-del strain from Example 4 was added to start the reaction. The batch volume was 10 mL. The molar ratio of tyrosol to Sodium D-erythorbate x H2O was 1:1.2. The batch was incubated at 30 °C and 140 rpm on a shaker (Infors). Sampling and analysis by HPLC (Example 2) were performed after 0, 3 and 24 hours. The progress of the reaction is shown in Table 7.

[0171] [Table 7]

[0172] Test 3: Biotransformation of 100 g / L tyrosol by RscK60-del fermenter cells in the presence of sodium D-erythorbate × HO (molar ratio of tyrosol to sodium D-erythorbate 1:1.2) 1000 mg of tyrosol (final concentration 724 mM) and 1885 mg of sodium D-erythorbate x H2O (872 mM) were initially charged in a 100 mL Erlenmeyer flask and, without further workup, 1 mL of KP2 buffer (500 mM potassium phosphate, 10 mM EDTA, pH 6.5) and 9 mL of fermenter broth of cells JM105 x pRscK60 expressing RscK60-del oxidase from Example 4 were added. Sodium D-erythorbate x H2O showed good solubility under these conditions, whereas tyrosol could only react in suspension. The batch volume was 10 mL. The molar ratio of tyrosol to sodium D-erythorbate x H2O was 1:1.2. The batch was incubated at 37 °C and 140 rpm on a shaker (Infors). Sampling and analysis by HPLC (Example 2) was performed after 3, 6, 24 and 29 hours. The progress of biotransformation over time is shown in Table 8.

[0173] [Table 8]

[0174] Example 6: Extraction of HTS 6.5 mL of the biotransformation product from experiment 2 of Example 5 was extracted four times with 10 mL of ethyl acetate (ethyl ethanoate, CAS number 141-78-6) each time, and after phase separation, the upper ethyl acetate phase was removed and combined. The combined ethyl acetate phase (volume 40 mL) was analyzed by HPLC for the presence of tyrosol and HTS as described in Example 2, and only HTS was detected. Based on the HPLC analysis, the purity of HTS was 97%.

[0175] The molar yield of HTS was calculated. The amount of tyrosol reactant used was 390 mg (6.5 mL of a 60 g / L tyrosol batch), which corresponds to 2.8 mmol of tyrosol (molecular weight of tyrosol: 138.2 g / mol). Assuming a 100% yield, this resulted in 2.8 mmol of HTS, which corresponds to 431.8 mg (molecular weight of HTS: 154.2 g / mol). By HPLC measurement, 404.8 mg of HTS was detected, which corresponds to 93.8% of the theoretical yield.

[0176] The extract solvent was stripped on a rotary evaporator (Buchi Rotavapor R-205) (batch temperature 62° C., vacuum at 500 mbar) and the solvent residue was removed under reduced pressure. The remaining tan oil was weighed. The yield was 400 mg, which was in good agreement with the yield determined by HPLC.

[0177] Example 7: Preparative-scale production of HTS A jacketed 1 L thermostatic glass vessel (Diehm) was connected to a thermostat (Lauda) via a hose connection and the temperature was adjusted to 37°C. The glass vessel was initially charged with 35 g tyrosol (final concentration 507 mM) and 65.6 g sodium D-erythorbate x H2O (607 mM) and, without further workup, 250 mL KP3 buffer (50 mM potassium phosphate, 5 mM EDTA, pH 6.5) and 250 mL fermenter broth of E. coli K12 JM105 x pRscK60-del strain (Example 4) were added. The molar ratio of tyrosol to sodium D-erythorbate x H2O was 1:1.2. The batch volume was 0.5 L. Mixing was performed with a magnetic stirrer. Compressed air was introduced into the batch by a glass tube to supply oxygen. The reaction was started by starting the magnetic stirrer and sparging. Sampling and analysis by HPLC (Example 2) was performed after 0, 3, 6 and 24 hours. At this time, tyrosol was 100% converted. The progress of biotransformation over time is summarized in Table 9.

[0178] [Table 9]

[0179] Based on the amount of 506.5 mM tyrosol used at the start of the reaction, the molar yield of 458 mM HTS was 90.4%.

[0180] To isolate HTS, 0.5 L of biotransformation was first incubated at 80°C for 30 min with magnetic stirring and then centrifuged at 4000 rpm for 30 min to separate particulate matter (Heraeus Megafuge 1.0 R). The supernatant was extracted three times with ethyl acetate. The first extraction was performed with 1 L of ethyl acetate, while the second and third extractions were performed with 0.5 L of ethyl acetate each. The ethyl acetate phases were combined to obtain 2 L of extract.

[0181] Ethyl acetate was distilled in a rotary evaporator (Buchi Rotavapor R-205) and the remaining ethyl acetate was removed first at a vacuum of 270 mbar and a temperature of 60° C., then at a vacuum of 20 mbar and a temperature of 85° C. Removal of the ethyl acetate left a residue of 34.1 g, corresponding to the HTS product of the process of the invention. To determine the purity, 32 mg of the HTS product was weighed, dissolved in 1 mL of H2O (concentration 32 mg / mL) and analyzed by HPLC. The HPLC analysis gave an HTS content of 30 mg / mL, which corresponds to a purity of 93.8% based on the amount of 32 mg of HTS product weighed.

[0182] The yield of HTS was calculated. The amount of tyrosol reactant used was 35 g. Taking into account the difference in molecular weight (138.2 g / mol for tyrosol and 154.2 g / mol for HTS), the maximum yield of HTS was expected to be 39 g. Taking into account the purity of 93.8%, 34.1 g of HTS product would contain 31.9 g of HTS. For the entire process, this corresponds to a yield of 81.7% based on the maximum achievable yield of 39 g of HTS.

Claims

1. 1. A method for producing hydroxytyrosol (HTS) by enzymatic conversion of tyrosol, comprising: The reaction mixture is i) tyrosol; ii) a compound selected from the group consisting of erythorbic acid and erythorbate salts; iii) an oxidase having an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO:2 and an amino acid sequence homologous to the amino acid sequence shown in SEQ ID NO:2; Including, The HTS is isolated from the reaction mixture; The amino acid sequence homologous to the amino acid sequence shown in SEQ ID NO:2 has at least 94% sequence identity over the entire sequence range from amino acid 1 to amino acid 543 of the amino acid sequence shown in SEQ ID NO:2, and is from the enzyme class identified by the number EC1.10.3.1 in the KEGG database; and The above-mentioned production method, wherein the molar amount of the erythorbic acid or erythorbate salt is 1.50 or less when the molar amount of the tyrosol is 1.

2. The method according to claim 1, wherein the amino acid sequence homologous to the amino acid sequence shown in SEQ ID NO:2 is the amino acid sequence shown in SEQ ID NO:

3.

3. The process according to claim 1 or 2, wherein the oxidase is produced by recombinant means by fermentation in E. coli.

4. 4. The process according to claim 3, wherein the fermentation for the production of the oxidase is carried out in the presence of Cu(II) ions at a concentration of at least 0.02 mM.

5. 5. The process according to claim 3 or 4, wherein the fermentation broth from the fermentation for the production of the oxidase is used directly in the process for the production of the HTS without further post-treatment.

6. The process according to any one of claims 1 to 5, wherein tyrosol is used at a concentration of more than 200 mM.

7. The process according to any one of claims 1 to 6, wherein the reaction mixture comprises erythorbic acid or an erythorbate salt at a concentration of at least 0.4 M.

8. The process according to any one of claims 1 to 7, wherein the volume fraction of fermentation broth from the fermentation for the production of oxidase in the reaction mixture is up to 90%.

9. The process according to any one of claims 1 to 8, wherein the reaction mixture is reacted for a period of time until at least 90% of the tyrosol used is converted into HTS.

10. The process according to any one of claims 1 to 9, wherein HTS is produced from tyrosol in a molar yield of at least 70%.

11. The process according to any one of claims 1 to 10, wherein the HTS is isolated from the reaction mixture by extraction with ethyl acetate followed by distillative removal of the ethyl acetate.

12. The method of claim 11 , wherein the HTS is isolated from the reaction mixture with a purity of at least 93%.

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