WO2010012871A1

WO2010012871A1 - Use of a hydroxyphenylacetate hydroxylase (hpah) for producing hydroxytyrosol

Info

Publication number: WO2010012871A1
Application number: PCT/FR2008/001134
Authority: WO
Inventors: Pierre-Pol Liebgott; Jean Lorquin
Original assignee: Institut De Recherche Pour Le Developpement
Priority date: 2008-07-30
Filing date: 2008-07-30
Publication date: 2010-02-04

Abstract

The invention relates (i) to the use of a HPA hydroxylase for catalysing the bioconversion of tyrosol into hydroxytyrosol, (ii) to a method for the in vitro preparation of hydroxytyrosol, which comprises at least one step of converting tyrosol into hydroxytyrosol by an HPAH, (iii) to a method for the in vitro preparation of hydroxytyrosol, using a microbial strain expressing an enzyme having an HPAH activity and not expressing any enzyme degrading tyrosol and/or hydroxytyrosol, (iv) to a bacterial strain transformed so as to express an HPAH without degrading tyrosol or hydroxytyrosol, (v) to the hydroxytyrosol obtained by the above methods, and (vi) to a portion or the entirety of the microbial culture that can be obtained by said in vivo method and that contains hydroxytyrosol.

Description

Use of Hydroxyphenylacetate Hydroxylase (HPAH) to produce

Phydroxytyrosol

The invention relates to the field of enzymatic and microbiological production of hydroxytyrosol (2- (3,4-dihydroxyphenyl) ethanol) by the conversion of tyrosol (2- (4-hydroxyphenyl) ethanol) catalyzed by an enzyme with hydroxyphenylacetate hydroxylase (HPA hydroxylase or HPAH) activity (see Figure 1).

Among the many natural sources of antioxidants, olive oil and especially co-products resulting from its production are the natural sources of the most interesting active ingredients to fight the oxidation of fatty substances (Servili et al, 1999 Visioli et al, 2002). Indeed, recent work has demonstrated the role of some antioxidant compounds present in the olive on the good resistance to the radical oxidation of virgin olive oils. These are mainly oleuropein, tyrosol, caffeic acid and especially hydroxytyrosol (Bisignano et al, 1999, Bonoli et al, 2003).

Hydroxytyrosol is a single phenolic compound having a 1-position side chain of the alcohol (Cl) ring, as well as two hydroxyl substitutions positioned at the C3 and C4 positions. It is therefore an ortwo-diphenol, family of compounds generally known to have interesting antioxidant properties. Hydroxytyrosol is a natural antioxidant found in olive, which is found mainly in the by-products of the olive industry.

Its consumption would explain the beneficial effect of the Cretan diet on human health. This compound in fact comes from the hydrolysis of oleuropein carried out during the olive oil production process (Capasso et al, 1999, Casalino et al, 2002, Tuck et al, 2001, Amiot et al, 1986). ). Many studies carried out on hydroxytyrosol have shown the interest of this compound as an antioxidant; it is considered promising for improving human health because of a broad spectrum of action (Table I) and probably owes its good activity to its high stability.

The antioxidant activity of hydroxytyrosol also gives it preservative properties of fatty substances; the protective activity against the oxidation of fatty substances was measured by the RANCIMAT technique (Rossignol-Castera and Bosque, 1994), and was compared with other reference compounds; the results of the experiment are illustrated in Figure 2 and show that hydroxytyrosol has a strong antioxidant activity. Only propyl gallate (E310) and octyl gallate (E31 1) exhibit superior antioxidant activity; however, they are toxic synthetic antioxidants from a certain concentration and can cause allergies. Hydroxytyrosol is also known for its beneficial properties on human health (Table I). It is therefore considered as a very good supplement or food additive and as a useful active agent to enhance cellular protection against oxidative stress and pathologies that it generates.

Action on health References

Anti-inflammatory Manna el al, 1997, Maiuπ (Ji al, 2005

. . .. . . . . Kohyarna e / a /, 1997, from Ia Puerta el al, 1999, Petroni

Inhibition of 5- and! 2-hpoxygenase j _{| g9} _

Inhibition of platelet aggregation Petroni m al, 1995

Inhibition of TXB2 * Pet.om a al, 1995

Hypocholesterolemic FKi et al, 2007

_,. _{,, ^} . . . . Aruoma el al, 1998, Bonanome et al, 2000, D'Angelo

Low Density Protection Lipopiotein _{el g / 2QQ]}

Prevention of cell damage by metabolites Viaoii m al, 1998, Espm et al, 2001, Visioii and Gaiii, oxygen reagents (Free radicals) 2001, Schaffer et al, 2007

Hypotensive Visioh et al, 2002

Anti-carcinogenic Visioh and Galli, 1998, D'Angelo elal, 2005

Protection of DNA oxidation Aruoma m al, 1998, Deiana et al, 1999

. .. ,. Bisignano el al, 1999, Capasso ef û /, 1995, Furnen el

Antimicrobial al. , 2.0.0.4.

Inhibition of HIV integrase 1 Lee-Huang eι al, 2007

* TXB2 = thromboxane B2

Table I. Biological properties of hydroxytyrosol.

At present, and despite important industrial needs, in particular cosmetics and agro-food, there is no method for the production of hydroxytyrosol on an industrial scale. Only small-scale synthesis methods have been described; however, these processes are not economically viable because they lead to excessively high production costs.

The only natural source of hydroxytyrosol is at present olive and by-products of olive oil (Bezançon et al., 2000). Olive oil contains only a small amount of hydroxytyrosol, with contents ranging from 0.01 to 1 mg / 100 g of oil. This is explained by the high solubility of hydroxytyrosol in water (approximately 5 g / 100 ml); as a result, it is strongly present in olive-growing waters commonly called 'margines' (hydroxytyrosol is about 100 times more important than in olive oil), and is the majority of cases rejected in the wild. A first route of production of hydroxytyrosol relies on its purification from these liquid olive oil by-products (vegetable waters). The majority of this type of process often leads to the production of an impure product given the great diversity and the large amount of polyphenols listed in vegetable waters (2.5-3% by weight, about a hundred different compounds). Labat et al., 2000). In addition, obtaining a purified fraction containing hydroxytyrosol from this waste involves several chromatographic steps using large amounts of solvents (Capasso et al., 1992, 1994 and 1999, Visioli et al, 1998). Therefore, for several years studies have multiplied in an attempt to find a cheaper method of purification of hydroxytyrosol, these attempts have been shown overall without much success.

Other production routes based on chemical methods include the synthesis of hydroxytyrosol by chemical reduction of 3,4-dihydroxyphenylacetic acid (Bai et al., 1998, Tuck et al, 2000), and by catalytic conversion of tyrosol to hydroxytyrosol by a mixture of methylrhénium trioxide (MTO) and hydrogen peroxide (Patent Application EP 1 623 960).

Two enzymatic production routes of hydroxytyrosol have also been described: - production of hydroxytyrosol by hydrolysis of oleuropein in the presence of β-glucosidase (Briante et al., 2001). The β-glucosidase used in the process of Briante et al. is produced by a recombinant Escherichia coli strain, this β-glucosidase comes from the archaea Sulfolobus solfataricus which is a hyperthermophilic bacterium. This process has several disadvantages: first, the enzyme and oleuropein must be previously purified from the bacterial culture and from olive leaves respectively, and secondly, the final extract is constituted a mixture of hydroxytyrosol and two forms of elenolic acid; an enzymatic synthesis by conversion of tyrosol in the presence of a tyrosinase extracted from a fungus (Espin et al 2001). This second enzymatic method also has limitations: on the one hand, the process requires the prior purification of tyrosinase with several steps that further increase production costs; on the other hand, the process requires the presence of ascorbic acid to inhibit the tyrosinase activity cresolase and thus avoid the formation of quinones; an additional final purification step must therefore be implemented to remove ascorbic acid from the reaction mixture.

Another type of production was made from whole cells grown on tyrosol. The producing bacteria are Serratia marscecens (Allouche and Sayadi, 2005), Pseudomonas aeruginosa (Allouche et al, 2004, Bouallagui and Sayadi, 2006), Pseudomonas putida F6 (Brooks et al, 2006) and Halomonas sp. HTB24 (Liebgott et al, 2007). The enzymatic system responsible for the bioconversion of tyrosol to hydroxytyrosol has never been identified in the various bacterial species mentioned above. These microbiological processes also have limitations:

- P. aeruginosa is a Class 2 pathogenic bacterium, which causes great reluctance to use the hydroxytyrosol it produces;

for all these strains, the production of hydroxytyrosol remains low. Indeed, there is a degradation of hydroxytyrosol 3,4-dihydroxyphenylacetate (3,4-DHPA), the latter subsequently degrading succinate and pyruvate.

In summary, the extraction from natural raw materials gives small amounts of hydroxytyrosol, it presents the risk of contamination by the extraction solvents and results, because of too many steps, a price too high. Chemical synthesis, on the other hand, is rather limited; it uses substrates or catalysts that are toxic and difficult to obtain. Finally, for the moment, the microbiological processes described use, for some, pathogenic bacteria with the disadvantages that are known and have a yield that is too low to be economically attractive.

In the course of their work, the inventors have now demonstrated that the enzyme involved in the bioconversion of tyrosol to hydroxytyrosol is a protein with hydroxyphenylacetate hydroxylase (also called HPAH or HPA hydroxylase), HPA 3-monooxygenase activity. This is surprising because tyrosol is not the natural substrate for this enzyme; the natural substrate of the enzyme is 4-hydroxyphenylacetic acid (4-HPA). Subsequently, the term HPA refers to 4-HPA or 3-HPA, preferably 4-HPA.

Thus, the objects of the present invention relate to (i) the use of HPAH to catalyze the bioconversion of tyrosol to hydroxytyrosol, (ii) an in vitro hydroxytyrosol synthesis process comprising at least one conversion step of tyrosol to hydroxytyrosol by HPAH, (iii) an in vivo hydroxytyrosol synthesis method using a microbial strain expressing an enzyme with HPAH activity and not expressing a tyrosol and / or hydroxytyrosol degrading enzyme, (iv) a bacterial strain transformed in such a way that it optimally expresses an HPAH and that it does not degrade tyrosol or hydroxytyrosol, (v) the hydroxytyrosol obtained by the processes above and (vi) all or part of the microbial culture obtainable by said process in vivo and containing hydroxytyrosol. According to a first of its objects, the present invention thus relates to the use of an enzyme with hydroxyphenylacetate hydroxylase activity, free or immobilized, to catalyze the conversion of tyrosol to hydroxytyrosol, and to a process for the in vitro preparation of hydroxytyrosol comprising at least one conversion step by conversion of tyrosol to hydroxytyrosol catalyzed by an enzyme with hydroxyphenylacetate hydroxylase activity.

Hydroxylases, also known as monooxygenases, are the enzymes that catalyze the initial hydroxylation reactions of non-monohydroxylated phenolic compounds. There are different kinds of bacterial monooxygenases comprising metalloprotein (for example tyrosinase) or flavoprotein type enzymes (for example A-hydroxyphenylacetate 3-monooxygenase). The latter, which carry out on the one hand the reduction of a flavin and then the actual hydroxylation of the phenolic compound, thus contain two functions: a reductase function and an oxygenase function. These functions are either carried by a single polypeptide chain (single-component Flavoprotein-monooxygenase), or by several independent chains (multi-component enzymes, which can have from 2 to 6 components), the most widespread are those with double component (Flavoprotein monooxygenase double-component).

Are useful for converting tyrosol to hydroxytyrosol according to the present invention, any enzyme with HPA hydroxylase activity that can occur:

1. in the form of a single protein with a reductase activity component and a hydroxylase activity component; or

2. in the form of two enzymes, one being a hydroxylase and the other being any reductase capable of generating FADH ₂ formation.

The enzyme with HPAH activity can be extracted from a microorganism that expresses it. For example, the following bacteria express HPAH: Escherichia coli (Prieto et al., 1996), Micrococcus luteus (Sparnins and Chapman, 1976), Acinetobacter baumanii (Thotsaporn et al., 2004), Pseudomonas putida (Arunachalam et al, 1992) , Psendomonas aeruginosa (Zeng and Jin, 2003), Halomonas sp. HTB24 (see Example 1 below), Serratia marscecens (Trias et al, 1989) and, more generally, any bacteria capable of degrading 3- or 4-HPA to form 3,4-dihydroxyphenylacetic acid (DHPA).

The enzyme with HPAH activity can also be produced by a genetically modified microorganism by introducing an expression system allowing and / or optimizing the production of said enzyme. According to a particular embodiment of the invention, the enzyme with HPAH activity is hydroxyphenylacetate 3-monooxygenase (EC 1.14.13.3 also known as HPA 3-monooxygenase) which is described for catalyzing the reaction shown schematically in FIG.

This enzyme is known to be in particular expressed by Escherichia coli in which the role in the catabolism of HPA has been studied by Cooper and Skinner (1980).

According to another particular embodiment of the invention, the enzymatic activity comes from cell fractions of microorganisms containing the enzyme HPAH activity, these fractions being obtained from any microbial strain naturally expressing an HPAH. In the particular case of HPA 3-monooxygenase, the strains that can be selected include Escherichia coli, Micrococcus luteus, Acinetobacter baumanii, Pseudomonas putida, Pseudomonas aeruginosa and Halomonas sp. HTB24.

In the context of the in vitro hydroxytyrosol production process, the enzymatic reaction producing the hydroxytyrosol is advantageously carried out in an aqueous reaction medium buffered at a pH of between 6 and 9 with an enzyme concentration of between 1 and 200 mg. / ml and NAD (P) H at a concentration of between 1 and 100 μM, especially 1 to 50 μM; the expression NAD (P) H denotes either NADH or NADPH; these cofactors can be produced by cell extraction and purification.

The reaction starts with the introduction into the reaction medium of the tyrosol whose concentration is adjusted to the enzymatic activity of the reaction medium; it can be between 5 and 250 mM, and stirring of the various reagents (NAD (P) H, tyrosol) and ends after exhaustion of the tyrosol in the culture medium.

The enzyme may be immobilized on a support such as a matrix of calcium alginate or the like, or else free in the reaction medium, for example from the cell fraction containing it. Insofar as the enzyme is not consumed during the reaction, it is preferable to immobilize it on a support by techniques known to those skilled in the art in order to recover it after the reaction.

According to another of its objects, the present invention relates to a process for the in vivo preparation of hydroxytyrosol, characterized in that it uses a microorganism expressing an enzyme with hydroxyphenylacetate hydroxylase activity and which does not express a tyrosol degrading enzyme. and / or hydroxytyrosol.

By microorganism, also referred to hereinafter strain or microbial strain is meant in particular and without limitation a bacterium, a yeast or a filamentous fungus. In particular, the microorganism does not express an enzyme with an aryl dehydrogenase activity oxidizing tyrosol or hydroxytyrosol, which notably excludes Pseudomonas aeruginosa, Serratia marcescens and Halomonas sp. and Pseudomonas putida.

Aryl dehydrogenases are enzymes that catalyze the conversion of an alcohol to an aldehyde, an aldehyde to an acid, or both at once, i.e., an alcohol to an acid.

In particular, they oxidize tyrosol or hydroxytyrosol, respectively, to 4-HPA and 3,4-DHPA (Liebgott et al, 2007, Hirano et al, 2005, MacKintosh and Fewson, 1988). Indeed, in the processes for producing hydroxytyrosol from different bacteria described in the literature, the major problem remains that of the degradation of hydroxytyrosol in the culture medium, by oxidation to 3,4-DHPA. Thus, the use of a microorganism that does not express aryl dehydrogenase makes it possible to obtain a better production of hydroxytyrosol.

The microorganisms that can be used according to this process can be selected by testing their enzymatic activity.

Concretely, this test can be carried out by a first phase of culturing a microbial strain in a minimum culture medium comprising tyrosol; the selected strain is one that does not degrade tyrosol and therefore has no aryl dehydrogenase activity.

Subsequently, in a second phase, 1ΗPA is added to the culture and measurement of the amount of tyrosol, hydroxytyrosol, HPA and DHPA in the medium is performed every hour.

During the implementation of this test, we observe four cases:

(1) tyrosol is converted into hydroxytyrosol and the latter does not disappear over time. The strain tested is therefore selected for the process;

(2) the tyrosol is oxidized to HPA before being hydroxylated to hydroxytyrosol, before the addition of HPA in the culture medium. The strain therefore has aryl dehydrogenases which degrade tyrosol and is not retained for the process;

(3) tyrosol and HPA are not converted, respectively, into hydroxytyrosol and 3,4-DHPA, so the strain does not have an enzyme with HPAH activity. The strain is not selected;

(4) tyrosol and HPA are hydroxylated, respectively, to hydroxytyrosol and 3,4-DHPA, but the hydroxytyrosol disappears over time in the culture medium. In this case, the tyrosol is not oxidized to HPA while the hydroxytyrosol is oxidized to DHPA; strain therefore has an enzymatic aryl dehydrogenase activity which degrades hydroxytyrosol. The strain is not selected.

The microorganism can also be genetically engineered to express HPA-3-monooxygenase and to repress aryl dehydrogenase. It is thus possible to select a strain naturally expressing the previously mentioned HPA-3-monooxygenase, in particular Escherichia coli, Micrococcus luteus, Acinetobacter baumanii, Halomonas sp., Pseudomonas putida and Pseudomonas aeniginosa, and modify them to suppress the expression of aryl dehydrogenases capable of oxidizing tyrosol and / or hydroxytyrosol.

Said in vivo method advantageously comprises at least one step of culturing the microorganism in an aqueous culture medium at a pH of between 6 and 8 with the main carbon source of the tyrosol and optionally of the HPA.

According to a particular embodiment, the culture medium contains per liter: between 0.35 and 0.85 g of KH ₂ PO ₄ , preferably 0.6 g; between 0.35 and 0.85 g of K ₂ HPO ₄ , preferably 0.6 g; between 0.25 and 0.75 g of NaCl, preferably 0.5 g; between 0.75 and 1.25 g of NH ₄ Cl, preferably 1 g. The pH is adjusted to 7.0 with 10M KOH solution. The cultures are carried out in the presence of peptide derivatives such as yeast extract or peptone at concentrations of 0.5 to 2 g / l. Tyrosol is present at a concentration of between 1 and 20 mM. Other sources of carbon such as sugars can also be used at concentrations optimized for adequate enzyme production.

In order to optimize the yield, the in vivo method is carried out by culturing the cells according to the so-called "resting cell" method which consists of inhibiting cell growth using a very dense suspension of cells in the stationary phase (Allouche et al, 2005 ). This method uses microbial cells deficient carbonaceous source which, therefore, no longer multiply and remain in the state of cell maintenance. This alternative aims to prevent the degradation of the reaction product by the growing cells, as well as a synthesis of undesired proteins (Barghini et al, 1998).

These conditions are achieved by pre-culturing cells, centrifuging them under conditions such that they are recovered intact and then incubating them for a very short time in a medium containing tyrosol.

The culture medium described above is suitable for a microbiological culture under stationary conditions ("resting cells"), provided that the carbon source is limited to tyrosol and possibly to HPA.

According to one particular embodiment, the in vivo method uses microbial cells immobilized on a solid support, for example, consisting of alginate beads. calcium, and on which flows the medium containing the tyrosol (Bouallagui and Sayadi, 2006, Brooks and α / ,, 2006).

This technique allows a greater reuse in the long term of the fixed cells. It thus becomes easier to produce molecules with high added value via microorganisms acting as "stable enzyme bags". This makes it possible to obtain processes that are both less expensive, less restrictive and more efficient.

Among the microorganisms expressing an HPAH, there are (i) strains which express the enzyme after induction by its natural substrate, 1ΗPA (inducible expression microorganism of HP AH); and (ii) strains in which the expression of the enzyme is constitutive, that is to say independent of the presence or absence of HPA in the culture medium (microorganism with constitutive expression of HPAH) .

When the in vivo process according to the invention is carried out with an inducible HPAH-inducing microorganism, it is possible to carry out an additional pre-culture step, prior to the culture, in the presence of HPA to induce the synthesis of HPAH. , while the following culture is carried out in the presence of tyrosol to produce hydroxytyrosol. According to one variant of this in vivo method, the inducible expression microorganism of HPAH is the bacterium Escherichia coli, in particular it is the strain W ά'Escherichia coli.

The in vivo method according to the invention then comprises the following steps:

1. preliminary step of growth of the inducible expression microorganism of HPAH in the presence of HPA to induce the genes necessary for the synthesis of HPAH;

2. step of culturing said microorganism in carbon source deficiency medium and in the presence of tyrosol.

It is during step (2) that the conversion of tyrosol to hydroxytyrosol takes place.

The culture medium that can be used in step (1) is that described above without tyrosol and in the presence of HPA at concentrations of between 1 and 20 mM.

Step (2) is then conducted in the presence of tyrosol with a constant addition of HPA, these two products may be present at the following concentrations: between 1 to 20 mM for tyrosol and between 1 and 10 mM, preferably 5 mM, for the HPA which is added every 2 hours. The culture of step (2) is advantageously carried out under stationary growth conditions of the microorganism ("resting cell" conditions).

In the case where the in vivo method according to the invention is implemented with a microorganism with constitutive expression of HPAH, that this constitutive expression is natural or results from a genetic modification, the use of HPA can be avoided. . This An alternative embodiment of the process in vivo is advantageous because it facilitates the extraction of hydroxytyrosol.

According to another of its objects, the present invention also relates to a microorganism genetically modified to express an enzyme with hydroxyphenylacetate hydroxylase activity and not to express an enzyme with aryl dehydrogenase activity.

The construction of such a self-induced strain by insertion of a constitutive promoter can be carried out as follows:

introduce a constitutive promoter upstream of the genes encoding HPA monooxygenase, or

introducing a plasmid containing the HPA monooxygenase gene regulated by a strong promoter.

According to a particular embodiment, it is a modified Escherichia coli strain comprising a heterologous constitutive promoter of expression of HPA-3 monooxygenase.

The present invention also relates to hydroxytyrosol obtained by one of the in vitro or in vivo methods described above. To do this, it is necessary to purify the hydroxytyrosol according to methods known to those skilled in the art. In particular, it may be successively (i) a liquid-liquid extraction of the culture medium with ethyl acetate followed by dry evaporation of the organic phase and redissolution in a methanol-water mixture ( 10: 90, v / v), then (ii) a solid phase extraction on a media type grafted silica Ci ₈ (e.g., LiChroprep RP-18 Merck, 1.09303.0100 reference), hydroxytyrosol being then eluted with pure methanol which is evaporated to dryness. Hydroxytyrosol is finally weighed.

Finally, the invention relates to a hydroxytyrosol-rich fraction originating from all or part of the microbial culture that may be derived from the in vivo process described above. To avoid the excessive accumulation of hydroxytyrosol in the medium and the risk that it polymerizes, it is preferable to extract the hydroxytyrosol gradually from the culture medium. Another variant to avoid this polymerization consists in stabilizing the hydroxytyrosol by derivation of one of the hydroxyl functions of the aromatic ring and then extracting it later.

Preferably, since the hydroxytyrosol is present in the culture medium at the end of the process, the hydroxytyrosol-rich fraction may be the culture medium after removal of the cells, for example by filtration or centrifugation. The invention is now described in more detail with reference to the following figures:

Figure 1 is a diagram showing the bioconversion of tyrosol to hydroxytyrosol by enzymatic catalysis with HPAH.

Figure 2 is a histogram which represents the induction time (expressed in hours) obtained during the accelerated oxidation test for various anti-oxidant compounds. The measurement is carried out by the RANCIMAT technique, here at 98 ° C on refined sunflower oil. The dose of antioxidant is fixed at 400 ppm in this experiment (after Rossignol-Castera and Bosque, 1994).

Figure 3 is a diagram showing the conversion reaction of 3- or 4-HPA to 3,4-dihydroxyphenylacetate catalyzed by HPA 3-monooxygenase.

Figure 4 comprises three graphs A, B and C representing the growth curve of the species Halomonas under the different culture conditions according to Example 1 (part LA.).

Figure 5 is a graph showing the purification (DEAE-Sepharose) of HPA 3-monooxygenase performed in Example 1.

Figure 6 is a graph showing the production of hydroxytyrosol (HTyr) by various aerobic bacteria in resting-cell condition. (A), Pseudomonas aeruginosa; (B), Halomonas sp. strain HTB24; (D), H. neptunia; (+), ^C, H. alkaliantarctica; (Δ), Serratia marcescens; (O), Micrococcus luteus; (•), Escherichia coli strain W.

In Figure 7 are shown: in A) the gas phase chromato gram (GC) showing the two compounds, tyrosol and hydroxytyrosol, both derived by trimethylsilyl (TMS); in B) the mass spectrum of the hydroxytyrosol derived by the TMS and realized in electronic impact.

Figure 8 is a graph showing the production of hydroxytyrosol (HTyr) by Escherichia coli strain W. The concentration of various metabolites is followed by HPLC: (A), HTyr; (G), 4-HPA; (Δ), Tyrosol; (•), OD at 600 nm

Figure 9 is a graph showing HPLC analysis of the culture taken at 6 h according to Example 3 (1, hydroxytyrosol; 2, tyrosol).

Figure 10 is a graph showing HPLC analysis of the culture taken at 10 h according to Example 3 (I ₃ hydroxytyrosol; 2, tyrosol).

Fig. 11 is a graph showing HPLC analysis of the culture taken at 10 h according to Example 3 (1, hydroxytyrosol; 2, tyrosol; 3, HPA).

Fig. 12 is a graph showing HPLC analysis of the 24 hour culture according to Example 3 (1, hydroxytyrosol; 2, tyrosol). Example 1 Characterization of the Enzyme Responsible for the Synthesis of Hydroxytyrosol

THE. Culture in ^' Ηalomonas in the presence of tyrosol and / or 4-HPA

It has first been studied behavior ^'Ηalomonas HTB24, a newly isolated halophilic bacterium (Liebgott et al., 2007) and variations in the production of hydroxytyrosol with these bacteria based on different conditions of preculture.

The bacteria are cultured in a culture medium containing 5 mM tyrosol, yeast extract at 1 g / L and 50 g / L NaCl at 30 ^° C. with stirring of 150 rpm, according to the following three conditions:

(A) the bacteria are cultured after being pre-cultured on yeast extract as the only carbon source;

(B) the bacteria are cultured after being pre-cultured on 5 mM tyrosol;

(C) the bacteria are cultured by inoculating a concentrated inoculum from a preculture carried out on 5 mM tyrosol.

The results presented in graphs A, B and C of FIG. 4 show that the appearance of hydroxytyrosol correlates with the presence of 4-HPA in the culture medium. It is assumed that the Halomonas HTB24 strain adapts to the presence of 4-HPA and induces genes encoding 4-HPA-3-monooxygenase.

LB. Induction tests of the enzyme responsible for the conversion of tyrosol to hydroxytyrosol under stationary conditions

It is then confirmed that 4-HPA is responsible for the induction of the enzyme catalyzing the conversion of tyrosol to hydroxytyrosol by the implementation of various induction tests on strain HTB24.

During these tests, it was studied the behavior ¹ Halomonas HTB24 depending on different conditions of preculture and culture.

Two successive pre-cultures containing 5 mM of aromatic inducer and in some cases 1 g / L of yeast extract were carried out (column ^a of Table II). Strain HTB24 does not believe in 2-tyrosol and 2-HPA. For the bioconversions reactions, the cells were prepared at a rate of 6 g / L (fresh weight). Beyond this concentration value, the results are too fluctuating, probably because of the lower availability of oxygen.

Then, the strain was cultured in medium containing yeast extract 1 g / L, glucose 20 mM, and various phenolic compounds such as 4-HPA, 3,4-DHPA or 4-hydroxybenzoate (4-HB) (column ^b of Table II). The culture conditions are as follows: the cells were incubated at 30 ^° C. with stirring of 150 rpm for 20 h in the presence of 4-tyrosol or 4-HPA (5 mM each). After 48 h of culture, the medium is centrifuged and the cells placed in stationary condition (resting cells) in the presence of 5 mM 4-HPA or tyrosol.

The aromatic compounds were identified by GC-MS (gas chromatography coupled to a mass spectrometer) and quantified by HPLC (average of three determinations, with standard deviations less than 0.1). CHMS was determined spectrophotometrically at 380 nm (Sparnins et al, 1974, Cooper and Skinner 1980). The percentages of degradation (4-tyrosol or 4-HPA) are indicated in parentheses and were calculated by the areas of peaks obtained in HPLC at their respective maximum wavelength (column ^c of Table II).

Pre-grovvth conditions "Aromatic substrate used for conversion Htyrb (mM)

YE Aronidlio used for induction 4-HPA (% lonverled) '4-tyrosol (% toneded)'

4-HPA 3,4-DHPA ₁ CHMS (100) HTyr (52) 2 6

4-HPA 3,4-DHPA CHMS (IOO) HTyr (56) 2 8

4-HPΛ + glucose -

3-HPA 3,4-DHPA, CHMS (100) HTyr (47) 235

4-tyrosol 3,4-DHPA, CHMS (100) HTyr, 4-HPΛ, 3,4-DHPΛ, CHMS, (100) 1 54

4-tyrosol 3,4-DHPA, CHMS (100) HTyr, 4-HPA, 3,4-DHPA, CHMS, (100) 1 64

4-tyrosol + glucose HPA traces

3-tyrosol 3,4-DHPA, CHMS (100) 1 45

3,4-DHPA [3,4-DHPA, CHMS] traces

4-HB -

4-C - tyrosine [1,4-DHPA, CHMS] traces

4-hydroxyphenylpyruvate [3,4-DHPA CHMS] traces

Table II. Conversion of tyrosol and 4-HPA by cells of the strain Halomonas sp. HTB24 (conditions of "resting cells").

Abbreviations: 4-HPA, 4-hydroxyphenylacetic acid; 4-tyrosol, 2- (4-hydroxyphenyl) ethanol; HTyr, hydroxytyrosol; 3,4-DHPA, 3,4-dihydroxyphenylacetic acid; 4-HB, 4-hydroxybenzoic acid; 4-C, 4-coumaric acid; CHMS, 5-carboxymethyl-2-hydroxymuconic acid semialdehyde.

The results clearly show that the production of hydroxytyrosol is correlated with the presence of 4-HPA in the pre-culture medium.

LC. Isolation of the cell fraction containing the enzymatic activity responsible for the conversion of tyrosol to hydroxytyrosol

To confirm that the enzyme responsible for the conversion of tyrosol to hydroxytyrosol is indeed 4-HPA 3-monooxygenase, the enzymatic activity of the fraction bacterial in which the enzyme should be found (according to the literature, this enzyme is intracellular) is measured.

The purification of the enzyme was carried out in several stages by simultaneously following in the various fractions the hydroxylating activity of tyrosol and that of 4-HPA. The purification steps were carried out according to different chromatographic modes (DEAE-Sepharose, gel permeation, MonoQ). The hydroxylating activity of tyrosol or 4-HPA was determined at 25 ^° C. in a glass tube containing 2 μl of 1 mM FAD, 10 μl of 2 mM NADH, 10 μl of extract containing a reductase component, 50 μl of enzyme (graphical chromato fraction), and 18 μl of 50 mM Tris-HCl buffer, pH 8.0. The reaction is started by the addition of 5 mM tyrosol or 4-HPA. The reaction is stopped by the addition of 5% (v / v) glacial acetic acid. The sample is then centrifuged at 10,000 g for 10 min and the reaction product, hydroxytyrosol or 3,4-DHPA is quantified by HPLC (see protocol detailed in point 1.1.4 of Example 3). ).

It has thus been demonstrated that the hydroxylating activity of 4-HPA and tyrosol coincide systematically (same fraction). Figure 5 represents one of these purification steps (two successive DEAE-Sepharose). Conclusion

These studies confirm that 4-HPA 3-monooxygenase, which normally catalyzes the hydroxylation of 4-HPA to 3,4-DHPA, also allows the hydroxylation of tyrosol to hydroxytyrosol. The enzyme is therefore not specific for 4-HPA. This has been characterized in various bacteria such as Escherichia coli (Prieto et al., 1996), Micrococcus luteus (Sparnins and Chapman, 1976), Pseudomonas aeruginosa (Ballou et al., 2005, Zeng and Jin 2003), Serratia marscecens (Trias et al., 1989), but without any description of its possible property to hydroxylate tyrosol.

Example 2 Comparison of Several Strains Described in the Literature as Producing Hydroxytyrosol and Strains Expressing 4-HPA-3-Monoxygenase

Experiments for the production of hydroxytyrosol under the condition of "resting cells" by various strains described in the literature or known to express 4-HPA-3-monooxygenase have been carried out.

The strains tested are: Pseudomonas aeruginosa DSM 50071 ^τ , Serratia marcescens DSM 30121 ^τ , Escherichia coli strain W DSM 1116 ^T , Halomonas alkaliantarctica DSM 15686 ^T , H. nephmia DSM 15720 ^τ , and Micrococcus luteus DSM 20030, all obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Brunschweig in Germany (DSMZ). Halomonas sp. HTB24 was recently isolated and deposited in the Institut Pasteur collection (France) under the number CIP 109599.

The bacteria were pre-cultured twice in the presence of 5 mM 4-HPA as inducer, then removed and concentrated to resuspended at 6 g / L in pH 7.5 phosphate buffer. Incubation begins immediately after adding 5 mM tyrosol to the medium. The production of hydroxytyrosol is monitored by HPLC (Figure 6).

Beyond 10 h, in all the strains except Escherichia coli W, a decrease of the oxidized hydroxytyrosol in 3,4-DHPA, due to aryl dehydrogenase activities, is observed.

Only Escherichia coli W keeps a constant concentration of hydroxytyrosol as well as unprocessed tyrosol. Additional tests of batch culture in the presence of tyrosol have also been carried out with these same strains. Only ^• Escherichia coli W does not oxidize tyrosol to 4-HPA.

Escherichia coli W may therefore be one of the bacteria to be retained for producing hydroxytyrosol from tyrosol.

EXAMPLE 3 Production of Hydroxytyrosol by Escherichia Coli I. Confirmation of Hydroxytyrosol Production by Escherichia Coli

Ll Materials and Methods

1.1.1 Description of the strain

Escherichia coli strain W (DMS 1116, ATCC 9637, TLC 2024, NCIB 8666) is a non-pathogenic bacterium that is described to produce diaminopimelate decarboxylase (Dewey, 1954), glutamine synthetase (Wu and Yuan, 1968), hydrogenases ( Ackrell et al., 1966), penicillin acylase and cephalosporins by bioconversion (US Pat. No. 3,945,888).

1.1.2 Culture media and stock solutions

The culture medium contains per liter: 0.6 g of KH ₂ PO ₄ , 0.6 g of K ₂ HPO ₄ , 0.5 g of NaCl, 1 g of NH ₄ Cl. The pH is adjusted to 7.0 with 10M KOH solution. The medium is then autoclaved at 120 ° C for 20 min. Routinely, cultures of 25 ml are carried out in 250 ml flasks of Erlenmeyer flasks in the presence of 5 mM of 4-HPA and 1 g / l of yeast extract.

Buffers. For the resting-cell experiments (bioconversion by intact cells under stationary conditions), the buffer used is a 50 mM phosphate buffer at pH 7.0. Mother solutions. The aromatic compounds were prepared in anaerobiosis, sterilized by filtration (Millipore filter, porosity 0.22 μm), and kept in penicillin bottles under N ₂ to ambient temperature and protected from light (vials covered with aluminum foil). Stock solutions of acidic aromatic compounds are prepared at a concentration of 250 mM and neutralized to pH 7 with NaOH (0.4 g per 25 mL). They are added to the culture medium before inoculation at 5 mM (final concentration).

The stock solution of yeast extract [25% (w / v)] (yeast extract, from PANREAC, reference 403687.1210) is prepared and sterilized by autoclaving for 20 min at 120 ^° C.

1.1.3 Pre-crops and crops

The experiments were carried out from 25 ml cultures in 250 ml cotton flasks. E. coli W is previously thawed from the laboratory collection and then inoculated into the medium in the presence of 5 mM 4-HPA. After 10 h at 37 ° C. in INFORS incubators with a stirring of 150 rpm, the pre-cultures are inoculated at a rate of 10% (v / v) of the preceding preculture into the medium containing 5 mM of 4-HPA. . The cultures are finally stirred under the same conditions in the presence of 4 mM of 4-HPA and tyrosol (2 or 3 mM) for several hours with monitoring of the appearance and disappearance of aromatic compounds, as well as growth. measured by absorbance at 600 nm.

1.1.4 Extraction and chemical identification by HPLC and GC-MS

The production of hydroxytyrosol is controlled by HPLC, its structure confirmed by mass spectrometry coupled to a gas chromatograph after extraction and bypass.

Extraction. 1 ml of bacterial culture is removed and centrifuged (8000g, 5 min); 400 .mu.l of supernatant are acidified with 20 .mu.l of glacial acetic acid and then centrifuged again (precipitation of the extracellular proteins). Finally, 10 to 20 μl of the supernatant obtained (called the primary solution) are injected in HPLC. To control the chemical structure of the compounds, the primary solution is extracted twice with ethyl acetate. After dry evaporation, two orientations are possible:

a control by HPLC is carried out again by taking up the dry extract in methanol and injecting 10 μl; or

the dry extract is derived by BSTFA for injection into GC-MS.

Identification by HPLC. The HPLC analyzes are carried out on a WATERS system equipped with a membrane degasser, a RHEODYNE 7725i injector (La JoIa, USA), a 1525 binary pump, a thermostatically controlled oven at 30 ° C. and a 2996 diode array detector. The device is controlled by Millenium 32 software version 4.0. The separation is ensured by a SYMETRY Cig column (150 x 4.6 mm, porosity 5 μm, WATERS). The mobile phase, entrained at a flow rate of 0.8 mL / min, is composed of two solvents: acetonitrile (A) and water distilled acidified with 1% acetic acid (B). The total elution time is 55 min. The gradient used is done in three steps: Step I ₅ 5 to 20% A in B for 30 min; Step 2, 20 to 100% A to B for 20 min; Step 3, return to 5% from A to B in 5 min. The identification of the compounds is carried out on the one hand by the determination of retention times and on the other hand by the UV / VIS spectra of each compound eluted, compared to standards.

Derivation and identification by GC-MS. The derivation is carried out on the dry extract with a mixture of BSTFA + 1% TMCS (N, O-bis (trimethylsilyl) trifluoroacetamide + 1% trimethylchlorosilane) from SIGMA-ALDRICH (Ref T6381). To 0.3-1 mg of dry sample are added 100 μL of pyridine and 100 μL of reagent (B STF A + 1% TMCS). The mixture is then placed in an oven for 20 min at 60 ° C., evaporated under nitrogen and the residue taken up in methanol or ethyl acetate. One μL is injected into GC-MS.

The analyzes were carried out with a GC-MS (AGILENT TECHNOLOGIES) apparatus consisting of a 6890N GC System chromato graph, a 5973 Mass Selectiv Detector mass spectrometer equipped with an electron impact ion source and a quadrupole type analyzer, MSD-CHEMSTATION acquisition software. The compounds are separated using a DB-IMS capillary column (30 mx 0.25 mm, from JW Scientific) and whose temperature limits are between -60 ⁰ C and 350 ⁰ C. The mobile phase is helium. The pressure in the column is 10.5 psi and the flow rate is 1 mL / min. The temperature of the injector (Inlet) is 280 ° C. The programming of the temperature gradient is as follows: 1 min at 100 ^° C., then increasing from 100 ° to 260 ^° C. at 4 ° / min, then 10 min at 260 ^° C. The total elution time is 51 min. The mass spectra obtained are compared with those of the NIST and WHILEY libraries contained in the software.

1.2 Results obtained

1.2.1 Escherichia coli produces hydroxytyrosol

Escherichia coli is cultured in the medium described previously (see 1.1.2). It is found that there is synthesis of hydroxytyrosol when the bacterium has been previously precultured in the presence of 4-HPA, then grown in a medium containing 4-HPA and tyrosol, compared to a culture performed under the same conditions but in absence of 4-HPA. The GC-MS analysis clearly shows the synthesis of hydroxytyrosol confirmed by its fragmentation in mass spectrometry and the NIST and WHILEY spectral libraries (FIGS. 7A and 7B). 1.2.2 Bacterial growth in the presence of tyrosol and 4-HPA

In the presence of 1 g / l of yeast extract, 2 mM of tyrosol and 4 mM of 4-HPA in the culture medium, the bacterium first degrades 4-HPA completely in 6 h of time, in forming 3,4-DHPA as an intermediate compound (Figure 8). After 6 h of culture, sampling and HPLC analysis are performed (FIG. 9), showing a very low level of hydroxytyrosol (equivalent to 0.08 mM, peak 1).

After 6 h of culture, the tyrosol is hydroxylated to hydroxytyrosol, a transformation which lasts 2 h of time (FIG. 8). The hydroxytyrosol then keeps a constant concentration of about 0.7 mM, even after 10 h of culture (Figure 10).

In the same way, after 20 hours and beyond, the hydroxytyrosol and the tyrosol remain constant in the culture which is in the stationary phase (absorbance of 2.0). E. coli no longer degrades tyrosol, and has no action on hydroxytyrosol. 4-HPA monooxygenase no longer seems to work; it is believed to be inhibited by the reaction product which is hydroxytyrosol.

The previous experiment is again performed under the same conditions with this time 3 mM tyrosol. The results concerning the production of hydroxytyrosol are identical to the previous observations, which indicate that the tyrosol has not been totally transformed. After 10 h of growth, 4 mM of 4-HPA are again added to the medium. At this point, HPLC sampling and analysis are immediately performed (Figure 11). After 24 h, the analyzes are shown in Figure 12. There was a significant increase in hydroxytyrosol which reached a concentration of 2.2 mM (340 mg / L), as well as a total consumption of 4-HPA .

Thus, the addition of 4-HPA in the culture medium makes it possible to considerably increase the level of hydroxytyrosol.

1.3 Conclusions

If the substrate (tyrosol) and the inducer (4-HPA) are adjusted to the correct concentrations and added to the medium at well-defined times, it is possible to develop a process in which hydroxytyrosol remains the only compound present in the middle. Extraction with ethyl acetate makes it possible to obtain it with a degree of purity approaching 98-99%. Bibliographical references

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Claims

1. Use of an enzyme with free or immobilized hydroxyphenylacetate hydroxylase activity to catalyze the conversion of tyrosol to hydroxytyrosol.

2. Use according to claim 1, characterized in that said enzyme with hydroxyphenylacetate hydroxylase activity is hydroxyphenylacetate 3-monooxygenase.

3. Process for the in vitro preparation of hydroxytyrosol comprising at least one step of converting tyrosol to hydroxytyrosol catalyzed by an enzyme with hydroxyphenylacetate hydroxylase activity.

4. Process for the in vivo preparation of hydroxytyrosol, characterized in that it uses a microorganism expressing an enzyme with hydroxyphenylacetate hydroxylase activity and which does not express a tyrosol or hydroxytyrosol degrading enzyme.

5. Method according to claim 4, characterized in that it comprises the following steps:

(1) prior step of growth of said microorganism in the presence of HP A;

(2) step of culturing said microorganism in carbon source deficiency medium and in the presence of tyrosol.

6. Method according to claim 5, characterized in that said culture according to step (2) is carried out under stationary growth conditions of said microorganism.

7. The method of claim 5 or 6, characterized in that said microorganism is Escherichia coli.

8. Microorganism genetically modified to express an enzyme with hydroxyphenylacetate hydroxylase activity and not to express an enzyme with aryl dehydrogenase activity.

9. Microorganism according to claim 8, characterized in that it is a strain of Escherichia coli.

10. Hydroxytyrosol obtained by the process according to any one of claims 3 to 7

11. Fraction rich in hydroxytyrosol from all or part of the microbial culture likely to be derived from the process according to any one of claims 4 to 7.