Hydroxytyrosol manufacturing method

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 considerable attention in recent years due to its beneficial health effects. HTS is considered an active ingredient in the Mediterranean diet. The European Food Safety Authority (EFSA) has approved 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. Studies have also shown that HTS possesses antibacterial properties in vitro against respiratory and gastrointestinal pathogens, including some strains of Vibrio, Salmonella, and Staphylococcus, and that the doses used are likely to compete with those of antibiotics, such as ampicillin. This substance is also thought to have neuroprotective, antiproliferative, and proapoptotic effects. These properties make HTS an interesting and popular substance for use in pharmaceuticals, dietary supplements, functional foods, and other cosmetic products.

[0003] To date, HTS available on the market is mainly derived from olives, olive leaves, or wastewater from olive oil production, and is supplied in the form of extracts: the proportion 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 around 4.5%.

[0004] In addition to 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, and is 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 for producing HTS are known, for example, by hydroxylation of tyrosol in the presence of atmospheric oxygen according to the following scheme: [ka]

[0008] For example, EP3234164B1 describes a method for enzymatically converting tyrosol to HTS by a tyrosinase enzyme from Ralstonia solanacearum or a functional derivative thereof in a reaction mixture with ascorbic acid, wherein the functional derivative is an engineered variant of the tyrosinase enzyme from R. solanacearum having one to five 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] EP 3234164 B1 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 concentrations of ascorbic acid sodium salt up to 0.4 M. In biotransformation, 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 was used to convert 150 mM tyrosol to HTS in the presence of 300 mM sodium ascorbic acid (a two-fold molar excess over the 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 for complete conversion was approximately 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. The HTS is then separated in a multi-step process using 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 support to isolate the higher purity HTS, followed by a separate column chromatography procedure using the method steps described above.

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

[0013] The method disclosed in CN101624607B is not well suited for industrial applications. The amount of tyrosol reactant used is relatively small, even at a maximum. The 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 enzyme must first be purified using a complex method, and the amount of tyrosol reactant used is much lower than for non-immobilized enzyme batches. Furthermore, this method requires the HTS to be processed in five stages: nanofiltration, chromatography, ethyl acetate extraction of the eluate, distillation of the extract, and another column chromatography operation. Due to these cost factors, 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 to 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 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, hereinafter also referred to as a "protective substance." This term is used because 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 oxidases (hereinafter also referred to as proteins (iii)): (1) It has oxidase activity, i.e., it can convert tyrosol in the presence of atmospheric oxygen at a concentration of >0.2 M, and during this reaction it can tolerate 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] An amino acid sequence annotated as polyphenol oxidase / catechol oxidase and homologous to the sequence set forth in SEQ ID NO:2 has at least 86%, preferably at least 90%, and more preferably at least 94% sequence identity with SEQ ID NO:2 over the entire sequence range from amino acid 1 to amino acid 543. The changes in the homologous amino acid sequence are selected from the group consisting of insertion, addition, deletion, and substitution of one or more amino acids. Homologs of SEQ ID NO:2 are selected from the enzyme class identified by the KEGG database number EC1.10.3.1 (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, a method for producing an HTS in which the amino acid sequence homologous to SEQ ID NO:2 is SEQ ID NO:3 is preferred. The sequence of RscK60 oxidase is 47 amino acids long, 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, as envisaged on the basis of the so-called degenerate genetic code, which encode proteins having an amino acid sequence corresponding to SEQ ID NO: 2 or corresponding to SEQ ID NO: 2 or variants homologous to SEQ ID NO: 2, and which have oxidase activity.

[0022] The production 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 HO, as a protective substance to maximize the yield of HTS. As shown in Table 3 of Example 3, tyrosol was completely converted within a short time in a reaction mixture without sodium D-erythorbate, but the HTS yield was low at 40%. Thus, HTS was clearly decomposed in the reaction mixture. In contrast, in a parallel reaction mixture in the presence of sodium D-erythorbate x HO, the tyrosol used was completely converted to HTS. Thus, sodium D-erythorbate as a protective substance inhibited the decomposition of HTS.

[0023] These results first demonstrate 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 previously described, is that the addition of sodium D-erythorbate x HO provides 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, discloses 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, limiting the amounts of both the protective substance and the tyrosinase reactant used, thereby limiting the economic viability of the production method. The maximum amount of ascorbic acid used was a concentration of 0.4M. Furthermore, in EP3234164B1, the molar ratio of tyrosol to ascorbic acid was 1:2. In CN101624607B, the molar ratio of tyrosol to ascorbic acid was 1:1.57. This means that both production methods use a relatively high amount of ascorbic acid relative to the tyrosol reactant, which negatively impacts the economic viability of the production method.

[0026] It has now surprisingly been found that erythorbate can be used in the production method of the present invention at concentrations above 0.8 M, i.e., much higher than known concentrations 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 production method.

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

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

[0029] As shown in Table 7, this biotransformation yielded 433 mM (66.7 g / L) of HTS from the amount of tyrosol used of 434 mM (60 g / L), corresponding to a molar yield of 99.7%. Table 8 shows that the amount of tyrosol used of 724 mM (100 g / L) yielded 707 mM (109 g / L) of HTS, corresponding to a molar yield of 97.6%. This constitutes a clear improvement over prior art documents such as EP 3234164 B1 and CN 101624607 B, in which only 175 mM and 180 mM of tyrosol, respectively, were converted to HTS.

[0030] The goal of 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. HTS yields of biotransformation of >80%, preferably >90%, more preferably >95%, and especially preferably 100%, relative to the molar amount of tyrosol used, are preferred. Yields are determined by quantitative HPLC of tyrosol and HTS, as described in Example 2.

[0031] The process of the present 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. The oxygen can be introduced passively, for example, by shaking or stirring in an incubation shaker (laboratory scale). The 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. The combination of passive and active introduction of oxygen 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 even 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 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 L-DOPA substrate (3,4-dihydroxy-L-phenylalanine, CAS No. 59-92-7) to dopachrome chromophore (CAS No. 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, oxidase activity can also be detected and quantified by HPLC testing. The test batch contains, per 10 mL batch volume, 0.4 mg / mL protein (or the corresponding amount of cell suspension, isolated cells, cell homogenate, or cell-free enzyme extract), 5.1 mM tyrosol, and 0 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 10% (v / v) H3PO4 solution is immediately added to stop the reaction. After the solid components are separated, for example by centrifugation, the supernatant is used to measure 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] Enzyme activity can be measured directly in the culture broth without re-isolation of the cells (cell suspension) or after re-isolation of the cells (isolated cells). Furthermore, enzyme activity can be measured in a cell homogenate after cell digestion, in which case the cell homogenate can be produced directly from the culture broth or after re-isolation of the cells. Furthermore, enzyme activity can also be measured in a cell extract by removing particulate cellular components from the homogenate, for example, by centrifugation. Finally, enzymes can be isolated from the cell extract by methods known per se, for example, by column chromatography, and used as purified proteins for measuring enzyme activity.

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

[0040] In the genetic information for the Ralstonia solanacearum strain K60 (GenBank CAGT01000120.1) published by Remenant et al. (2012), J. Bact. 194: 2742-274, 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 NCBI protein database accession number CCF97399.1 (SEQ ID NO: 2). The function assigned to this protein 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 and therefore remains uncharacterized. 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 present 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 >0.4 M.

[0042] Sequence comparison was also performed on tyrosinase from R. solanacearum strain GMI1000, which is 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), because it can also be used for the bioconversion of tyrosol to HTS, or engineered variants of this enzyme. Comparison of the amino acid sequence of the RscK60 oxidase (SEQ ID NO: 2), annotated as a polyphenol oxidase / catechol oxidase, with that of the tyrosinase from R. solanacearum GMI1000 (Genbank accession number NP_518458) in EP3234164B1 revealed clear differences. The sequence of the RscK60 oxidase from R. solanacearum K60, annotated as a polyphenol oxidase / catechol oxidase, 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 approximately 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: -Oxidases for production purposes can be produced by fermentation at a scale of 1 L or more. ->Tyrosol at a concentration of 0.2M is converted into 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 manufacturing method of the present invention compared to 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 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 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 on a 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 culture medium and maximize the activity of the protein / enzyme. The terms "laboratory scale" and "industrial scale" simply depend on 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 into an expression vector, which means that the gene construct contains all the information to express the protein from the cloned cds.

[0049] Those skilled in the art are aware of various methods for generating suitable genetic constructs. The expression vector pKKj, disclosed in EP 2670837 A1, is preferred. pKKj contains the known tac promoter, which allows expression of a coding gene sequence operably linked to the tac promoter to be induced by the addition of IPTG inducer (isopropyl-β-thiogalactoside). The genetic constructs of the present invention are pRscK60 (FIG. 1) and pRscK60-del (FIG. 2).

[0050] The corresponding gene construct is then transformed into a microorganism (production host) suitable for protein production by known methods to create a strain for producing the corresponding oxidase. The production host is preferably selected from the Escherichia coli species, and particularly preferably a microorganism derived from the E. coli K12 JM105 strain (commercially available from DSMZ German Collection of Microorganisms and Cell Cultures GmbH under strain number DSM 3949). The production strain is more preferably E. coli JM105 x 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, which may 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 may be tested for oxidase activity.

[0052] In a fermentation method 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, followed by the oxidase. Herein, the formation of biomass and oxidase may be correlated in time or separated from each other in time, with biomass formed in a first fermentation step and enzyme production initiated in a second step by an inducer of gene expression. A preferred fermentation method is disclosed in Example 4, in which the formation of biomass and oxidase is separated in time and oxidase production 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 liter, particularly preferably more than 10 liters, particularly preferably more than 1000 liters, even more preferably more than 5000 liters.

[0054] The terms "culture," "growth," and "fermentation," as well as, for example, the terms "culture medium," "growth medium," and "fermentation medium," are used interchangeably 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 oxidase (iii) and cell culture medium.

[0055] Culture media are well known to those skilled in the art from practical microbial cultures and 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. A 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 yeast extract, proteose peptone, or corn steep liquor, the latter in liquid form or in a dry form called CSD.

[0058] Cultivation can be carried out in the so-called batch mode, in which 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 culture can also be carried out in a so-called fed-batch mode, where, after the initial stage of growth in the batch mode, additional nutrients are added to compensate for the consumption of 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 separately as individual feedstocks. Furthermore, inducers can also be added to the feedstock. The feedstock can be fed continuously or in portions (discontinuously), or a combination of continuous and discontinuous feeding. Cultivation in a 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 carbon source content in the fermenter during the production phase does not exceed 10 g / L, with a maximum concentration of preferably 2 g / L, more preferably 0.5 g / L, and 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] Additional medium additives may include salts of the elements phosphorus, chlorine, sodium, magnesium, nitrogen, potassium, calcium, and iron, as well as trace amounts (i.e., μM concentrations) of salts of the elements molybdenum, boron, cobalt, manganese, zinc, copper, and nickel. Additionally, 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 culture is carried out under pH and temperature conditions that promote 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 growing 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 of at least 10% (v / v), preferably at least 20% (v / v), more preferably at least 30% (v / v) is established. 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: the amount of compressed air supplied to the fermentation batch, reported as liters of compressed air per liter of fermentation volume per minute). Compressed air is preferably introduced at 0.2 vvm to 8 vvm, more preferably 0.4 to 6 vvm, and 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 absorbance at 600 nm / mL (OD) of the fermenter broth, was determined by known photometric methods. 600 OD of 10 / mL is determined by measuring the absorbance at 600 nm of the fermenter broth (OD / mL). 600 to, more preferably, an OD of 30 / mL 600 From OD 50 / mL, particularly preferably 600 It is preferable to add IPTG from the

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

[0073] The culture batch obtained by the above-described method contains RscK60 oxidase or a corresponding homologue. The oxidase can be further used in the production method of the present invention directly as a fermenter broth, as a cell suspension after cell reisolation, or as a cell homogenate after cell digestion without further workup. It can also be further used as a cell-free enzyme extract or purified enzyme either directly from the fermenter broth or after reisolation. In the production method of the present invention, the oxidase is preferably used in the fermentation broth without further workup, as a cell suspension after cell reisolation, or as a cell homogenate after cell digestion without further workup. In the production method of the present invention, the oxidase is particularly preferably used in the fermentation broth without further workup, or as a cell suspension after cell reisolation without further workup. In the production method of the present invention, the oxidase is particularly preferably used in the fermentation broth without further workup.

[0074] 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 primarily by a growth medium that promotes good cell growth and further by additional additives that selectively stimulate enzyme production. A known method for stimulating enzyme production is based on a gene construct with an inducible promoter, such as that 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 a gene operably linked to the tac promoter can be significantly enhanced by the addition of the IPTG inducer (isopropyl-β-thiogalactoside).

[0075] Another way to increase enzyme activity is to add a cofactor for enzyme activity. Since only the gene sequence of the RscK60 gene of the present invention was known, and no experimental studies of its enzyme activity were known, various ways to enhance enzyme activity were investigated. The annotation of the RscK60 gene as a polyphenol oxidase / catechol oxidase suggested that its enzyme activity was metal-dependent. For example, the effect of adding Cu(II) ions to the medium on enzyme production was investigated. Surprisingly, it was found that increasing the Cu(II) ion concentration significantly increased enzyme activity (Example 3). The RscK60 oxidase herein 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 dependence 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, or coding sequence) is a region of DNA or RNA that begins with a start codon and ends with a stop codon and encodes a sequence of amino acids for a protein. ORFs are also called coding regions; 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 for producing biologically active RNA. A gene includes a section of DNA that is transcribed to produce a single-stranded RNA copy, as well as expression signals that control this copying. 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 additional genetic elements (e.g., promoter, terminator, selection marker, origin of replication). The genetic elements of a genetic construct are initially inherited extrachromosomally during cell growth, resulting in the production of the protein encoded by the gene.

[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 produced 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 operational 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 (extractant), thereby transferring the reaction product to the extractant. After separation of the reaction mixture and extractant (phase separation), the product can be isolated by removing the extractant.

[0084] In genetics and bioinformatics, annotation 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) within that sequence, including the encoded protein.

[0085] In a preferred embodiment, the method for producing HTS by enzymatic conversion of tyrosol to HTS is characterized in that the fermentation for oxidase production 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, and 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 (CuSO4 x 5H2O) in anhydrous or pentahydrate form.

[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), supplementing the medium with Cu(II) ions in the form of CuSO4 x 5H2O increased the enzymatic activity of the oxidase by more than 10-fold. Thus, supplementing the medium with Cu(II) ions constitutes an efficient method for optimizing the production of enzymes suitable for the manufacture of HTS, which has not yet been described in the prior art.

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

[0090] To maximize economic efficiency, tyrosol is converted to the maximum concentration during biotransformation to produce HTS. 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, and particularly preferably more than 700 mM.

[0091] In the HTS manufacturing method, When the molar amount of tyrosol is 1, Erythorbic Acid or Erythorbic Acid salt of The molar amount is 1 .50 or less is preferable is 10.2 or less, especially preferred is 1 The following are particularly preferred: is 0 .5 or less do.

[0092] Furthermore, in the method for producing HTS, it is preferred that the volume fraction of the fermentation broth from the fermentation for producing 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 will vary depending on the dose of tyrosol, which is determined by measuring the amount of tyrosol and HTS by HPLC (see Example 2, HPLC Testing for an explanation).

[0095] For example, 70 g / L of tyrosol was converted to about 95% within 24 hours. Preferably, the tyrosol used is converted to HTS to a degree of at least 80% within 24 hours.

[0096] In the method for producing HTS of the present invention, it is preferred that HTS be 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 HTS from the reaction mixture, the reaction mixture is mixed with an immiscible solvent without any intermediate steps, and after phase separation, the solvent phase containing the product is separated. A suitable solvent for the extraction of HTS is known from EP 2 774 909 B1. A preferred solvent for extraction is ethyl acetate (CAS number 141-78-6). Extraction, preferably with ethyl acetate, can be repeated as many times as necessary until the HTS is completely removed from the reaction mixture. Alternatively, extraction, preferably with ethyl acetate, can be carried out continuously by known methods, such as countercurrent extraction. To achieve 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 proteins present in the mixture.

[0098] The process for preparing HTS is preferably characterized by isolating 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 reactant and HTS product, including spectrophotometry, NMR, gas chromatography, HPLC, mass spectrometry, gravimetry, elemental analysis, or a combination of these analytical methods.

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

[0103] The production method of the present invention may further comprise an inexpensive and simple step, namely, using the 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 explanation of the drawings]

[0104] [Figure 1] FIG. 1 shows the 4.5 kb vector pRscK60 prepared in Example 1. [Figure 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 [Example]

[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), which encodes a putative polyphenol oxidase / catechol oxidase, is disclosed in the NCBI (National Center for Biotechnology Information) nucleotide database as Locus Tag CAGT01000120.1, nt 3460-5091 (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, encoding the protein of amino acid sequence SEQ ID NO: 2, hereinafter referred to as RscK60 oxidase; -rscK60-del-cds: nt 142 to nt 1632 of SEQ ID NO: 1, encoding the protein of amino acid sequence SEQ ID NO: 3, hereinafter referred to as RscK60-del oxidase. RscK60-del oxidase is a protein in which 47 amino acids are truncated at the N-terminus compared to RscK60 oxidase.

[0110] The DNA fragment rscK60-cds was isolated as a 1.6 kb fragment by PCR (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 (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) starting from 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) starting from 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, which had been previously digested with EcoRI and HindIII, to generate 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 fermentors (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 the suspension (culture broth, fermenter broth) by centrifugation of the suspension (10 min, 15,000 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, the cell pellet from a 100 mL culture was suspended in 20 mL of 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 the cell suspension was digested in a 1.5 mL tube containing glass beads ("Lysis Matrix B") prepared by the manufacturer (3 × 20 seconds at a shaking frequency of 6000 rpm, with 30 second intervals 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 enzyme 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 No. 59-92-7) to dopachrome chromophore (CAS No. 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 of KPi buffer, 10 mM L-DOPA (Sigma-Aldrich) and 4 mL of test sample (cell suspension, isolated cells, cell homogenate or cell-free enzyme extract).

[0124] When testing samples from shake flask cultures, cells were first concentrated by centrifuging 25 mL of the shake flask mixture to isolate a cell pellet (10 min, 15,000 rpm, Sorvall RC5C centrifuge with SS34 rotor), which was 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 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.

[0126] The reaction was initiated by the addition of each test sample. The test batch was incubated at 37°C and 140 rpm in an Infors shaker. 1 mL aliquots were taken at 0, 30, 60, and 120 minutes and immediately centrifuged at 13,000 rpm for 5 minutes (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 specific oxidase 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] Determination of Tyrosol and HTS (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 and 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 × HO (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 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 on a shaker (Infors) at 30 °C and 140 rpm. 1 mL samples were taken at 0, 1, 2, and 4 hours and immediately mixed with 0.1 mL of concentrated H3PO4 in each case to stop the reaction. After centrifugation (13,000 rpm for 5 minutes in a Heraeus™ Fresco™ 21 centrifuge, Thermo Scientific™), 1 mL of supernatant was dispensed for tyrosol and HTS determination by HPLC.

[0133] HPLC analysis of tyrosol and HTS: Quantification of tyrosol and HTS was performed using HPLC methods calibrated for tyrosol and HTS, respectively. Calibration reference materials, 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 to a wavelength of 274 nm. A Phenomenex Luna C18(2) column, 250 mm long, 4.6 mm internal diameter, and 5 μm particle size, was conditioned in a column oven at 30 °C. Eluent A: 5 mL of H3PO4 in 1 L of 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 the reaction conditions. The yield is reported as a volumetric yield (mM or g / L) in absolute amount of product based on volume, or as a relative yield of product in percent (also called percent 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 plasmid DNA isolation, the expression vectors pRscK60 and pRscK60-del from Example 1 were each transformed into NEB® 10-β (New England Biolabs), a commercially available E. coli strain used for cloning purposes. Cell cultures were prepared 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) at 37°C, 120 rpm, on an Infors tray shaker. Plasmid DNA was isolated from the cells using a plasmid DNA isolation kit (QIAprep® Spin Miniprep Kit, 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 a known method. 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 the pKKj vector (E. coli JM105×pKKj), from which transformants were generated in the same manner.

[0138] Precultures were prepared 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 / mL ampicillin) (growth overnight at 37°C and 120 rpm in an Infors tray shaker).

[0139] Two 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 x 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, Sigma-Aldrich) was added and the mixture was shaken overnight at 30° C. and 140 rpm.

[0140] SM3 medium contains 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); and 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] Subsequently, cells from shake flask cultures were used to verify enzyme activity by HPLC and photometric L-DOPA assays.

[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 in each case suspended in 5 mL of KPiE buffer. In each case, 5 mL of isolated and resuspended cells were used in the HPLC study described in Example 2.

[0144] Two HPLC experiments were performed. The first batch contained, in a 10 mL batch volume, 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), 4.9 mL of KPiE buffer, and 0.1 mL of 1 M sodium D-erythorbate × HO dissolved in KPiE buffer (see HPLC experiment in Example 2). The second batch, also in a 10 mL batch volume, contained 5 mL of JM105×pRscK60 cells isolated and resuspended from the shake flask culture described above, 7 mg of tyrosol, 4.9 mL of KPiE buffer, and 0.1 mL of 1 M sodium D-erythorbate × HO dissolved in KPiE buffer. The batches were incubated on a shaker (Informs) at 30°C and 140 rpm. 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 assay. Cell homogenates were generated from the two strains (cell digestion as described in Example 2) and used in an L-DOPA assay using L-DOPA as the enzyme substrate. The specific enzyme activity measured in the L-DOPA assay using 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] Changes in Cu(II) concentration in the medium: In preliminary experiments to optimize RscK60-del enzyme activity in shake flask cultures, one of the parameters varied was the Cu(II) concentration in the medium. Supplementing SM3 medium with Cu(II) ions proved advantageous. SM3 medium supplemented with a trace element solution containing 0.25 mg / L CuSO4 × 5H2O (final concentration in the medium: 1 μM) essentially contained only a low content of Cu(II) ions.

[0148] The effect of Cu(II) ions on the enzymatic activity of RscK60-del oxidase was examined 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 ranging from 0 to 0.5 mM, as described above. Cells from each batch were used as cell homogenates for the determination of activity by the 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 ) were isolated by centrifugation and resuspended in 10 mL of KPiE buffer. Five mL of each isolated and resuspended cells was used in the HPLC study described in Example 2.

[0151] Two comparative biotransformation experiments 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 10 mL batch volume. The second batch also contained 5 mL of isolated and resuspended JM105×pRscK60-del cells, 7 mg of tyrosol, 4.9 mL of KPiE buffer, and 0.1 mL of 1 M sodium D-erythorbate×HO dissolved in KPiE buffer in a 10 mL batch volume (see HPLC experiment in Example 2). The batches were incubated at 30°C and 140 rpm on a shaker (Infors). Samples from this experiment were taken at 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 fermentor (working volume 2 L) from Sartorius BBI Systems GmbH.

[0154] Shake flask preculture: Inoculate 2 x 100 mL of LBamp medium in a 1 L baffled Erlenmeyer flask with the JM105xpRscK60-del strain from the agar plate to a cell density of OD 600 The mixture was incubated on an incubation shaker (Infors) at 30°C and 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 the 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 automated metering 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 of fermentation time, a cell density (OD) of 45-60 / mL was achieved. 600 was achieved.

[0156] Production fermentation: A 150 mL fermentation culture was inoculated into 1.35 L of pH 7.0 FM2 medium supplemented with 20 g / L glucose, 0.5 mM CuSO4 × 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%. The oxygen partial pressure (pO2) was controlled by the stirrer speed. Fermentation duration was 30–32 h.

[0157] FM2 medium: 5 g / L (NH4)2SO4; 0.50 g / L NaCl; 0.075 g / L FeSO4 x 7H2O; 1 g / L Nacitrate, 0.30 g / L MgSO4 x 7H2O, 0.015 g / L CaCl2 x 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 fermentation was adjusted to 50%. After O2 saturation fell below the target value, a closed-loop control cascade was initiated to bring 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 decreased from the initial 20 g / L to approximately 5 g / L, a 60% (w / w) glucose solution was continuously fed. The feed rate was then 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 the fermenter is 50-60 / mL OD 600At 7.5 hours of fermentation time, expression of the oxidase RscK60-del was initiated by a single addition of IPTG inducer (final concentration 0.4 mM). Fermentation was stopped 22.5 hours after induction, corresponding to a total fermentation time of 30 hours, and enzyme activity (L-DOPA assay) and analytical-scale tyrosol to HTS conversion (HPLC assay) 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 divided into 50 mL aliquots 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 when 1.6 mL of fermenter broth with a protein concentration of 5 mg / mL was used in the L-DOPA test.

[0163] For 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 for tyrosol and HTS content by HPLC to determine the yield, i.e., the percentage of 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 of tyrosol (final concentration 180 mM) and 389 mg of sodium D-erythorbate × HO (180 mM) were initially charged into a 100 mL Erlenmeyer flask and dissolved in 1 mL of KPi buffer. Without further workup, 9 mL of fermenter broth of RscK60-del oxidase-expressing JM105 × pRscK60-del cells was added to initiate the reaction. The batch volume was 10 mL. The molar ratio of tyrosol to sodium D-erythorbate × HO was 1:1. The actual pH of the reaction mixture measured within the batch was 6.7. The batch was incubated on a shaker (Infors) at 37 °C and 140 rpm. Sampling and HPLC analysis (as described in Example 2) were performed at 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 of tyrosol by JM105×pRscK60-del fermentor cells expressing RscK60-del in the presence of a 1:1 molar ratio of tyrosol to sodium D-erythorbate and sodium D-erythorbate × H2O. 300 mg of tyrosol (final concentration 220 mM) and 475.4 mg of sodium D-erythorbate x HO (220 mM) were initially charged into a 100 mL Erlenmeyer flask and dissolved in 5 mL of KPiE buffer. Without further workup, 5 mL of fermenter broth of E. coli K12 JM105 x pRscK60-del strain from Example 4 was added to initiate the reaction. The batch volume was 10 mL. The molar ratio of tyrosol to sodium D-erythorbate x HO was 1:1. The actual pH of the reaction mixture measured within the batch was 6.7. The batch was incubated on a shaker (Infors) at 30 °C and 140 rpm. Sampling and HPLC analysis (Example 2) were performed at 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 of tyrosol by RscK60-del fermentor cells in the presence of sodium D-erythorbate × HO (molar ratio of tyrosol to sodium D-erythorbate 1:1.2). 600 mg of tyrosol (final concentration 434 mM) and 1126 mg of sodium D-erythorbate x HO (521 mM) were initially charged into a 100 mL Erlenmeyer flask and suspended in 5 mL of KPiE buffer. While sodium D-erythorbate x HO showed good solubility under these conditions, 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 initiate the reaction. The batch volume was 10 mL. The molar ratio of tyrosol to sodium D-erythorbate x HO was 1:1.2. The batch was incubated on a shaker (Infors) at 30 °C and 140 rpm. Sampling and HPLC analysis (Example 2) were performed at 0, 3, and 24 hours. The reaction progress is shown in Table 7.

[0171] [Table 7]

[0172] Test 3: Biotransformation of 100 g / L of tyrosol by RscK60-del fermentor 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 × HO (872 mM) were initially charged into 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 JM105 × pRscK60 cells expressing RscK60-del oxidase from Example 4 were added. Sodium D-erythorbate × HO 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 × HO 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 phases were removed and combined. The combined ethyl acetate phases (volume 40 mL) were analyzed by HPLC for the presence of tyrosol and HTS as described in Example 2, and only HTS was detected. Based on 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, 404.8 mg of HTS was detected, which corresponds to 93.8% of the theoretical yield.

[0176] The solvent of the extract was separated on a rotary evaporator (Buchi Rotavapor R-205) (batch temperature 62 °C, vacuum to 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 HTS production A 1 L jacketed thermostatic glass vessel (Diehm) was connected to a thermostat (Lauda) via a hose connection and the temperature was adjusted to 37 °C. 35 g of tyrosol (final concentration 507 mM) and 65.6 g of sodium D-erythorbate × HO (607 mM) were initially charged to the glass vessel, and without further workup, 250 mL of KP3 buffer (50 mM potassium phosphate, 5 mM EDTA, pH 6.5) and 250 mL of fermenter broth of E. coli K12 JM105 × pRscK60-del strain (Example 4) were added. The molar ratio of tyrosol to sodium D-erythorbate × HO was 1:1.2. The batch volume was 0.5 L. Mixing was performed using a magnetic stirrer. Compressed air was introduced into the batch via a glass tube to supply oxygen. The reaction was initiated 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 material was first incubated at 80°C for 30 minutes with magnetic mixing, then centrifuged at 4000 rpm for 30 minutes (Heraeus Megafuge 1.0 R) to separate particulate matter. The supernatant was extracted three times with ethyl acetate. The first extraction was performed with 1 L of ethyl acetate, and the second and third extractions were each performed with 0.5 L of ethyl acetate. 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 reduced pressure of 270 mbar and a temperature of 60°C, and then at a reduced pressure of 20 mbar and a temperature of 85°C. Removal of the ethyl acetate left 34.1 g of residue, corresponding to the HTS product of the inventive manufacturing method. To determine the purity, 32 mg of the HTS product was weighed, dissolved in 1 mL of HO (concentration 32 mg / mL), and analyzed by HPLC. HPLC analysis gave an HTS content of 30 mg / mL, corresponding 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. Considering 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. Considering a purity of 93.8%, 34.1 g of HTS product would contain 31.9 g of HTS. For the entire process, this corresponded 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: reacting the pre-reaction mixture to obtain a post-reaction mixture; and isolating the HTS from the post-reaction mixture. wherein the pre-reaction mixture comprises i) tyrosol and 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 amino acid sequences homologous to the amino acid sequence shown in SEQ ID NO: 2; Including, The amino acid sequence homologous to the amino acid sequence set forth 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 set forth in SEQ ID NO: 2, and is derived 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 of 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. 3. The method of claim 1 or 2, wherein the oxidase is produced by recombinant means by fermentation in E. coli.

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

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

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

8. The method 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 pre-reaction mixture is at most 90%.

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

10. 10. The process according to any one of claims 1 to 9, wherein the 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 post-reaction mixture by extraction with ethyl acetate and subsequent distillative removal of the ethyl acetate.

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

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