Enzymatically produced glucosylated mycosporines for developing innovative Anti-uv products

Abstract

The subject of the present invention is that of preparing glucosylated mycosporines by means of an enzymatic cascade method using α-transglucosylases and sucrose, and use thereof in particular for their anti-UV properties.

Description

[0001] ENZYMATICALLY GLUCOSYLATED MYCOSPORINS FOR THE DEVELOPMENT OF INNOVATIVE ANTI-UV PRODUCTS DESCRIPTION

[0002] Technical field

[0003] The present invention relates to the preparation of glucosylated mycosporins by an enzymatic cascade process using α-transglucosylases and sucrose, and their use in particular for their anti-UV properties (e.g. UV absorption and protection properties, antioxidant properties).

[0004] The present invention finds applications, for example, in the fields of ophthalmology, dermatology, cosmetology, materials, agriculture. In the description below, the references in parentheses [ ] refer to the list of references presented at the end of the text.

[0005] State of the art

[0006] Prevention and protection are essential to address chronic and acute damage caused by UV radiation to human health, especially to the skin and eyes. Thus, reducing UV exposure and applying sunscreen is highly recommended for the prevention of skin diseases. Broad-spectrum inorganic sunscreens composed of titanium dioxide (TiC) and zinc oxide (ZnO), acting primarily by absorption but also by scattering radiation, are commonly used [1].However, their generally covering and white appearance is not accepted by all consumers, and the possible cytotoxicity of the accumulation of nanoparticles in the body is not yet sufficiently studied [2]. Organic filters composed of synthetic molecules have been accused of having relatively limited effectiveness for skin protection, of being sometimes allergenic, and of being endocrine disruptors [1, 2]. Some are even suspected of degrading into carcinogenic compounds, such as octocrylene [3], which has recently been shown to degrade over time into benzophenone. In addition, the lack of photostability of certain compounds could create a counterproductive effect by creating ROS (Reactive Oxygen Species) and increasing oxidative stress.Finally, from an environmental point of view, these two types of filters are accused of accumulating in coastal waters, causing endocrine changes in fish and coral bleaching phenomena [4,1]. Furthermore, in sunscreens, chemical filters such as oxybenzone cause allergies and side effects in humans, and are known to damage coral reefs [4]. It is therefore necessary to develop new solutions based on natural resources and implementing environmentally friendly processes.

[0007] Mycosporines and mycosporine-like amino acids (MAAs) (hereinafter both referred to collectively as mycosporines) are small molecules of approximately 400 daltons (Da), organized around a cyclohexenone or cyclohexenimine ring. Due to their structure, these zwitterions exhibit unique UV absorption capabilities, with an absorption range of 309–362 nm and an extinction coefficient of 21,800 cnr 1 .M' 1 at 50,000 cnr 1 .M' 1[5]. They therefore cover both the UV-A and UV-B range. Mycosporins are also described as potent antioxidants. They are secondary metabolites and their role as natural sunscreens has been studied for years. Their potential is promising for several reasons: (i) they can act as UV filters and antioxidants, an effective combination to protect the body against multiple damages of photoinjuries, (ii) they are also studied for their anticancer, anti-aging and anti-inflammatory properties, and (iii) they have a strong potential to promote wound healing [1]. Their mechanism of action is as follows: in a photoexcited state, they undergo a rapid internal conversion to the ground electronic state (S0) by releasing heat or by performing a reversible isomerization [1,6].Through this mechanism, more than 90% of the received energy is released by heat transfer to the surrounding environment without generation of ROS. No fluorescence emission has been particularly reported [7].

[0008] Mycosporins are present in a very wide variety of organisms. UV radiation impacts terrestrial life, but it can also penetrate waters from 0.5 to 47 meters deep, depending on water clarity and weather conditions [1]. Mycosporins have been identified in cyanobacteria, but also in heterotrophic bacteria and marine fungi, microalgae, macroalgae (mostly red and brown algae), phytoplankton (diatoms, dinoflagellates), corals, molluscs and fish [7,8]. They have also been identified in terrestrial organisms, such as lichens living in sometimes very extreme conditions [9]. This is the case of the mycosporine-serinol MSer(OH) present in particular in the species Lichina pygmea, present in the mid-littoral zones of the coasts of Western Europe.

[0009] In metazoans such as fish, mycosporins can accumulate rapidly in the tissues of the outer layers exposed to sunlight, such as the skin of the dorsal surface, as well as sensitive organs such as the digestive tract, reproductive organs, and also the eyes [1,5,7,8]. In the eye, they are particularly concentrated in the cornea and the lens, thus preserving the retina from photooxidative damage caused by UV rays.

[0010] .

[0010] The presence of mycosporins in the ocular tissues of fish is a particularly interesting subject of bioinspiration, especially in the development of preventive and / or curative biomedical solutions in the ophthalmological field. However, the formulation of biomedical products from the currently available mycosporins is complicated due to their small size which leads to their rapid elimination from the mucous membranes (especially the eye).

[0011] Glycosylated mycosporins are already present in nature with essentially a degree of polymerization (DP) of 1 to 2 glycosylated units (e.g. xylose, galactose, glucosamine, etc.), exceptionally up to 8 glycosylated units of different nature (e.g. xylose, galactose, glucosamine, etc.) for a case of mycosporine of the mycosporine-glycine type

[0011] . However, the most widely described are those found in the exopolysaccharide matrix of terrestrial cyanobacteria of the genus Nostoc (Nostoc commune, Nostoc sphaericum) [12-14]. In these organisms, different mycosporines have been identified (shinorine, porphyra-334, palythine, mycosporine-glycine, mycosporine-glutamicol) linked to different glycosylated units (galactose, glucosamine, xylose, etc.).Although the role of these glycosylated mycosporines has not been fully discovered, they probably provide a multifunctional role of protection against UV rays but also against heat stress or desiccation. The interest of sugars covalently linked to mycosporines is also not fully elucidated, even if it is strongly suggested that this would improve interactions with the exopolysaccharide matrix of these cyanobacteria

[0013] . In addition to the increased stability and non-covalent attachment to the extracellular matrix of glycans, the high proportion of glycosylated mycosporines surely testifies to another useful role: Ishihara et al. thus identified that 86.5% of the total mycosporines of Nostoc sphaericum were p-galactose-porphyra-334; 13.2% were hexose-shinorines and only 0.2% were free porphyra-334

[0014] ,.

[0012] Description of the invention

[0013] The inventors hypothesized that it might be interesting to integrate long glucosylated chains into mycosporins in order to see if the molecules obtained would be sufficiently stable and would retain their anti-UV properties (photostability) to be usable.

[0014] The inventors have therefore developed a new process capable of adding a long glucosyl chain to mycosporins, which has made it possible to obtain new environmentally friendly and highly UV-protective products or biomaterials, replacing current UV filters, capable of extending the retention time of mycosporins in organs, such as the eyes. The nature of the carbohydrate chain (composed solely of glucosyl units) should reduce the risk of allergic reactions.

[0015] The obtained UV-protective biomolecules (glucosylated mycosporin) with extended retention time using exclusively natural precursors can be applied in cosmetic formulations, medical products (e.g. in ophthalmology), biomaterials and outdoor materials as alternatives to common active ingredients.

[0016] For this purpose, although it has never been studied in relation to mycosporins, enzymatic glycosylation has emerged as the most effective process. One family of enzymes has emerged as particularly interesting for this type of enzymatic catalysis. These are the α-transglucosylases of family 70 of Glycoside Hydrolases (GH-70)

[0015] , enzymes that are naturally found in lactic acid bacteria of the genera Leuconostoc, Lactobacillus, Streptococcus, Oenococcus or Weissela sp.

[0016] . These enzymes are of significant industrial interest due to their catalytic mechanism, illustrated in Figure 16. From sucrose, a very cheap substrate derived from renewable resources (beet and sugar cane), these enzymes are capable of forming various products depending on the nature of the acceptor molecule.After forming a covalent intermediate (step 1 in Figure 16), GH-70s can transfer the glucosyl unit to .

[0017] :

[0017] - water (step 3 in Figure 16) to carry out a hydrolysis reaction, in this case there is formation of free glucose (taking place mainly at the start of the reaction for the very good polymerases of the GH-70 family).

[0018] - a glucose and progressively glucose oligosaccharides of increasing size (step 4 in Figure 16), to achieve the synthesis of homopolymers of aD-glucosyl units, called a-glucans. These represent a wide diversity of polymers, ranging from 10 3 at 10 9Da and composed of osidic bonds of different natures (a-1,2; a-1,3; a-1,4 and / or a-1,6). Among these a-glucans, one of the best known is dextran. It is a more or less linear polymer containing a majority of glucosyl units linked in a-1,6 and which has numerous applications in industry (food, cosmetic and pharmaceutical fields for example).

[0019] - fructose (step 5 in Figure 16), there is then formation of sucrose isomers, such as leucrose for example. These products are nevertheless in the minority.

[0020] - an exogenous acceptor molecule added to the medium at the start of the reaction (glycone or aglycone), possessing at least one hydroxyl function (step 6 of Figure 16), this catalytic action is called the acceptor reaction.

[0021] The ability to transfer glucosyl units to these exogenous molecules depends on several factors, including the structure of the acceptor and that of the enzyme's active site. An acceptor deemed efficient will be able to redirect almost all of the transfer of glucosyl units not towards the formation of polymers but towards its own glucosylation. To date, a wide variety of acceptors have already been tested, including, for example, flavonoids such as quercetin and luteolin

[0018] .

[0022] The present invention therefore aims to produce glucosylated derivatives of mycosporines, in particular myscosporin-serinol (hereinafter referred to as MSer(OH)) derived in particular from the marine lichen Lichina pygmea, using an enzymatic cascade process using α-transglucosylases of the Glycoside Hydrolase family 70 (GH-70) and sucrose. Naturally, these enzymes use sucrose from beet or sugar cane to synthesize (polymerases) or decorate (branching sucrases) polymers of high molar mass glucosyl units (α-glucans). For comparison, some sucrose phosphorylases that also use sucrose as a glucosyl unit donor, as well as commercial CGTases belonging to the GH-13 family that use starch (another natural and abundant substrate) as a glucosyl unit donor, have also been tested, but without success.

[0023] The enzymatic cascade method described here (Figure 1) has the advantage of not involving a chemical reaction; this avoids several steps of protection / deprotection of the hydroxyl functions of sugars which generally require the use of metal catalysts or strong acids. The latter are known to generate large quantities of waste to be treated and can lead to uncontrolled side reactions.

[0024] The production of glucosylated mycosporins is carried out in a completely aqueous medium without the addition of organic solvent.

[0025] Glucosylation can be performed in a single step (co-synthesis or one-pot synthesis) or in two steps as follows:

[0026] 1 / glucosylation of a mycosporine (eg mycosporine-serinol, MSer(OH)) by the enzyme GS-D (a-transglucosylase of the GH-70 family) from sucrose, allowing the production of intermediate glucosylation products called mycosporine-glu (eg MSer(OH)-glu);

[0027] 2 / elongation of the intermediate glucosylation products mycosporine-glu (eg MSer(OH)-glu) by other α-transglucosylases of the GH-70 family from sucrose, allowing the production of final glucosylation products called mycosporine-polym (eg MSer(OH)-polym).

[0028] This second step of the enzymatic cascade allows the “custom” synthesis of mycosporine covalently linked to glucosylated chains of varying lengths and osidic bonds (a-1,2; a-1,3; a-1,4 and / or a-1,6) depending on the enzymes and reaction conditions used. Mycosporine-glu intermediates (eg MSer(OH)-glu) can carry up to 4 glucosyl units with an average degree of polymerization (DP) mostly centered between 1 and 2 glucosyl units, while mycosporine-polym final products (eg MSer(OH)-polym) can have between 2 and 1,000,000 glucosyl units, with an average degree of polymerization (DP) centered between 3 and 1,000,000, between 3 and 800,000, between 3 and 500,000, between 3 and 24,000, between 3 and 60, or between 3 and 41 glucosyl units, depending on the enzymes used.Since mycosporins are described as natural sunscreens, the mycosporin derivatives developed by the process of the present invention composed of mycosporins having a long glucosylated chain could easily be used for the prevention and protection of cells (dermal and ocular) and have cosmetic, medical and / or pharmaceutical applications. In particular, applications in ophthalmological products for the protection of the eye (e.g., prevention of cataracts) or for the protection of people with genetic and hereditary abnormalities such as albinism and xeroderma pigmentosum, can be envisaged. Another application would be to integrate these glucosylated mycosporins into bio-sourced, biodegradable sunscreens without environmental consequences such as coral bleaching.Finally, these glucosylated myscosporins can also be used for the protection of materials used outdoors

[0019] .

[0029] The present invention therefore relates to a process for producing a glucosylated mycosporine, said process comprising a step a) of glucosylation of a mycosporine by the enzyme α-transglucosylase GS-D from sucrose to obtain an intermediate glucosylation product being a glucosylated mycosporine having a degree of polymerization ranging from 1 to 4 glucosyl units, preferably an average degree of polymerization of 1 to 2 glucosyl units.

[0030] According to a particular embodiment of the present invention, the method may further comprise a step b) of elongation of the carbohydrate part of the intermediate glucosylation product obtained in step a) from sucrose by at least one α-transglucosylase other than α-transglucosylase GS-D to obtain a glucosylated mycosporine having an average degree of polymerization ranging from 3 to 1,000,000 glucosyl units, from 3 to 800,000 glucosyl units, from 3 to 500,000 glucosyl units, from 3 to 24,000 glucosyl units (for a very good polymerase such as, for example, the dextransucrase enzyme DSR-OK A1), from 3 to 60 glucosyl units, from 3 to 41 glucosyl units; preferably from 9 to 1,000,000 glucosyl units, from 9 to 800,000 glucosyl units, from 9 to 500,000 glucosyl units, from 9 to 24,000 glucosyl units, from 9 to 60 glucosyl units, or from 9 to 41 glucosyl units.

[0031] According to a particular embodiment of the process of the present invention, steps a) and b) can be carried out concomitantly, namely that said process comprises the glucosylation of a mycosporin and the elongation of its carbohydrate part by the enzyme α-transglucosylase GS-D and at least one other α-transglucosylase different from α-transglucosylase GS-D from sucrose, to obtain a glucosylated mycosporin having an average degree of polymerization ranging from 3 to 1,000,000 glucosyl units, from 3 to 800,000 glucosyl units, from 3 to 500,000 glucosyl units, from 3 to 24,000 glucosyl units (for a very good polymerase such as for example the enzyme dextran-sucrase DSR-OK A1), from 3 to 60 glucosyl units, from 3 to 41 glucosyl units; preferably from 9 to 1,000,000 glucosyl units, from 9 to 800,000 glucosyl units, from 9 to 500,000 glucosyl units, from 9 to 24,000 glucosyl units, from 9 to 60 glucosyl units, or from 9 to 41 glucosyl units.

[0032] For the purposes of the present invention, the term "average degree of polymerization (DP)" means the degree of polymerization determined at the apex of the peaks obtained by high-performance size exclusion chromatography analysis coupled with a UV detector and calibration / calibration of the column using commercial dextrans with a molar mass of between 342 g / mol and 70,000 g / mol.

[0033] According to a particular embodiment of the method of the present invention, said at least one α-transglucosylase of step b) is chosen from the group consisting of: glucansucrases GS-A, GS-B, GS-C, GS-F A1, and GS-FS; dextransucrases DSR-M A1, DSR-M A2 W624A, DSR-G A1, DSR-S vardel A4N, and DSR-OK A1; alternansucrase ASR A1.

[0034] According to a particular embodiment of the process of the present invention, the mycosporine of step a) is chosen from the group consisting of: pure or purified mycosporine, Lichina pygmea lichen extract enriched with mycosporine from 0 to 50% by weight of extract, preferably from 5 to 35% by weight of extract, preferentially from 15 to 25% by weight of extract, most preferably from 10% by weight of extract. Preferably, the mycosporine used in step a) is mycosporine-serinol (MSer(OH)). The latter can be isolated from extracts of cyanolichens such as Lichina pygmae and Lichina confiais, different species of Peltigera, Stereum hirsutum, Alternaria chrysanthemi, Ascochyta pisi, Ophiobolus graminis, Pleospora herbarum, or Pyronema omphalodes. It can also be produced from its precursor (gadusol) by synthetic biology.

[0035] The present invention also relates to a glucosylation intermediate product obtained by the process of the present invention, said glucosylation intermediate product being a glucosylated mycosporine having an average degree of polymerization ranging from 1 to 4 glucosyl units (alpha-type bonds), preferably from 1 to 2 glucosyl units (alpha-type bonds).

[0036] The present invention also relates to a glucosylated mycosporine obtained by the process of the present invention, said glucosylated mycosporine having an average degree of polymerization ranging from 3 to 100,000 glucosyl units (alpha-type bonds), from 3 to 800,000 glucosyl units (alpha-type bonds), from 3 to 500,000 glucosyl units (alpha-type bonds), from 3 to 24,000 glucosyl units (alpha-type bonds), from 3 to 60 glucosyl units (alpha-type bonds), or from 3 to 41 glucosyl units (alpha-type bonds); preferably from 9 to 1,000,000 glucosyl units (alpha-type bonds), from 9 to 800,000 glucosyl units (alpha-type bonds), from 9 to 500,000 glucosyl units (alpha-type bonds), from 9 to 24,000 glucosyl units (alpha-type bonds), from 9 to 60 glucosyl units (alpha-type bonds), or from 9 to 41 glucosyl units (alpha-type bonds).

[0037] The present invention also relates to the cosmetic use of a glucosylated mycosporine according to the present invention, as an anti-UV agent, to (i) protect the skin from UV rays, (ii) combat photo-aging.

[0038] The present invention also relates to a cosmetic composition comprising as active ingredient a glucosylated mycosporin according to the present invention.

[0039] The present invention also relates to the cosmetic use of a cosmetic composition according to the present invention, for (i) protecting the skin from UV rays, (ii) combating photo-aging.

[0040] The present invention also relates to a glucosylated mycosporin according to the present invention for use as a medicament, for use in the prevention or treatment of, for example, cataracts, skin diseases associated with UV sensitivity such as xeroderma pigmentosum, hereditary genetic diseases such as albinism, or skin cancer.

[0041] In conclusion, the invention implements green processes and mobilizes natural resources, namely mycosporins, notably from a lichen growing on the seashore, and sucrose. The latter resource, serving as a substrate for the GH-70 family enzymes, is available in large quantities and at low cost. The enzymatic reactions are processes in mild conditions and do not produce (toxic) waste. Some glucosylated chains have dextran signatures, an a-glucan widely used in the cosmetic and biomedical fields, because it is very well tolerated by the body. The dextran chain of the MSer(OH)-polym products being modular "to order" (e.g. in terms of osidic bonds, carbohydrate chain size), it can easily be adapted to the type of application targeted.In nature, in the mucus and lens of fish for example, mycosporins are bound to proteins [7,20,21]. In the present invention, this phenomenon has been mimicked by producing MSer(OH)-polym mycosporine derivatives having an elongated carbohydrate chain. Compared to free mycosporine molecules, the glucosylated mycosporins obtained by the method of the present invention have better properties, especially photostability.

[0042] Compared to commercial UV protection molecules, such as oxybenzone (a UVA and UVB blocker) which causes bleaching of coral reefs [4], glucosylated mycosporins would not cause such damage.

[0043] BRIEF DESCRIPTION OF THE FIGURES

[0044] Figure 1 represents the principle of the enzymatic cascade allowing the glucosylation of mycosporines from sucrose and involving α-transglucosylases of the GH-70 family: a first glucosylation step carried out by the enzyme GS-D then an elongation of the glucosidic chain by different enzymes of the family, allowing both control of the size and the structure of this chain (different types of α-osidic bonds depending on the enzyme chosen).

[0045] Figure 2 shows the UV chromatograms (λ=310 nm) of the GH-70 family α-transglucosylase enzymes screened for MSer(OH) glucosylation. Only the GS-D enzyme was able to efficiently glucosylate MSer(OH).

[0046] Figure 3 shows the UV chromatograms (λ=310 nm) of commercial CGTase and sucrose phosphorylase enzymes screened for MSer(OH) glucosylation.

[0047] Figure 4 shows the UV chromatograms (λ = 310 nm) of the different glucosylation products of 5 mM MSer(OH) by GS-D, named MSer(OH)-glu. The numbering 1 -8 identifies the different MSer(OH)-glu glucosylation products that were obtained at the end of the first step of the enzymatic cascade. Figure 5 shows (A) the chemical structure of MSer(OH) with carbon numbering and illustration of the possible glucosylation positions. The hexagons represent glucosyl units linked to MSer(OH) (B) examples of structures of DP2 produced by the glucosylation of MSer(OH) by the enzyme GS-D (C) examples of structures of DP3 produced by the glucosylation of MSer(OH) by the enzyme GS-D (D) examples of structures of products resulting from the enzymatic cascade (glucosylation+elongation).

[0048] Figure 6 shows the NMR spectra 1H (spectrum A) and HMBC (spectrum B) confirming the glucosylation of MSer(OH) by GS-D into a glucosylation intermediate (MSer(OH)-glu) with an average degree of polymerization ranging from 1 to 2 glucosyl units.

[0049] Figure 7 shows the comparison of UV chromatograms (λ=310 nm) of MSer(OH) glucosylation reactions by GS-D, at 5 and 50 mM MSer(OH).

[0050] Figure 8 shows the UV chromatograms (λ=310 nm) of the GS-D enzymatic cascade polymerizations followed by a polymerase (i.e. another α-transglucosylase of the GH-70 family). The enzymes used in the second step are indicated on the chromatograms.

[0051] Figure 9 shows the UV chromatograms (λ=310 nm) of 6 of the 11 different reactions, separated by high-performance size exclusion chromatography (HPSEC). The chromatogram annotated DEX 10,000 Da is that of a commercial dextran with an average molar mass of 10,000 g / mol. The asterisks identify the majority population(s) for each reaction. Peaks DP1 and DP2 correspond to Peak 1 and Peak 2, respectively, in Figure 4.

[0052] Figure 10 shows the UV chromatograms (λ=310 nm) of 3 of the 11 different reactions, separated by high-performance size exclusion chromatography (HPSEC). The chromatogram annotated DEX 70,000 Da is that of a commercial dextran with an average molar mass of 70,000 g / mol.

[0053] Figure 11 shows the comparison of HPLC analyses on a Synergi™ Fusion-RP C18 column coupled with a UV detector (λ=310 nm) of the polymerization reactions of MSer(OH)-glu products by the DSR-M A2 enzyme and its DSR-M A2 W624A mutant.

[0054] Figure 12 shows the absorbance scans of MSer(OH) and the products MSer(OH)-glu and MSer(OH)-polym for wavelengths ranging from 200 nm to 800 nm (area of ​​interest shown: 200 to 400 nm). Figure 13 shows the comparison of the UV chromatograms (λ=310 nm) of the glucosylation reactions of GS-D on the purified MSer(OH) present at 10% in a crude extract of Lichina pygmea lichen, during reactions carried out with 6.37 mM MSer(OH) and 292 mM sucrose.

[0055] Figure 14 represents the comparison of the UV chromatograms (λ=310 nm) of the glucosylation reactions of GS-D then DSR-M A1 on the purified MSer(OH) and that present in the crude extract from the lichen Lichina pygmea.

[0056] Figure 15 shows the chromatograms of cosynthesis polymerizations. MSer(OH)-glu products are less efficiently consumed by polymerases, less MSer(OH)-polym are produced.

[0057] Figure 16 shows the schematic of the different catalytic mechanisms of GH-70.

[0058] Figure 17 represents the quantum yield of MSer(OH) and the different glucosylation products MSer(OH)-glu and Mser(OH)-polym obtained by the process of the invention.

[0059] Figure 18 represents the TEAC score of MSer(OH) and the products of the enzymatic cascade of the process of the invention, with and without MSer(OH).

[0060] EXAMPLES

[0061] EXAMPLE 1: EXAMPLE OF IMPLEMENTATION OF THE METHOD OF THE INVENTION

[0062] First screening:

[0063] A collection of α-transglucosylases from the GH70 and GH13 families was screened for their ability to glucosylate the mycosporine-serinol molecule (MSer(OH)) from sucrose or possibly starch. Initially, the work was carried out using pure MSer(OH) obtained from an extract of Lichina pygmea. The enzymes listed in Table 1 below were all produced in recombinant form in Escherichia coli, except for CGTases and sucrose-phosphorylase (enzymes no. 22, 23 and 24) which are commercial formulations.

[0064] Table 1

[0065] L.=Leuconostoc Lb.=Lactobacillus Bb.=Bifidobacterium P=Polymerase; B=Branching sucrase; Bif=bifunctional; GT=a-glucano transferases; S- Php=sucrose phosphorolysis; ND=not determined.

[0066] The enzymes were produced from E. coli BL21 Star (DE3) cells transformed with the plasmid containing the target enzyme gene (Table 2). 300 μL of the transformation mixture was used to seed a 30 mL volume of LB (Lysogeny Both), supplemented with 100 μg / mL ampicillin. The medium was incubated overnight at 37 °C to prepare a preculture. 1 L cultures in modified ZYM5052 medium

[0022] , whose characteristics are presented in Table 2, were seeded at an initial OD (λ=600 nm) of 0.05 from the previous day's preculture, then incubated for 26 hours at 21 °C and 150 rpm.

[0067] At the end of fermentation, the culture media were centrifuged (15 minutes, 800 g, 8 °C) and the pellets were concentrated to an OD (λ = 600 nm) of 80 in 50 mM sodium acetate buffer at pH = 5.75. The cells were then disrupted with ultrasound according to the following protocol: 5 cycles of 20 seconds at 30% of the maximum power of the probe, cold, spaced by 4 minutes of rest in ice. The sonication supernatants containing the soluble enzymes of interest were then recovered after 30 minutes of centrifugation (15,000 g, 6 °C) and stored at 4 °C. [Table 2]

[0068] Gly=Glycerol; Glu=Glucose; a-Lac=a-Lactose; L-Ara=L-arabinose

[0069] Enzyme activity was determined by measuring the initial rate of production of reducing sugars using the dinitrosalicilic acid (DNS) method

[0023] . One enzyme unit represented the amount of enzyme that liberates one pmole of fructose per minute, at 30 °C, from 100 g / L of sucrose in 50 mM sodium acetate buffer, pH=5.75. During a kinetic run, 100 pL of reaction medium was withdrawn and the reaction was stopped by adding an equivalent volume of DNS. The samples were then heated for 5 minutes at 95 °C, cooled in ice, diluted half-way in water, and the absorbance was read at λ=540 nm. A standard range of 0 to 2 g / L of fructose made it possible to establish a link between absorbance value and concentration of reducing sugars.

[0070] The first screening, which involved all the enzymes in Table 1, was carried out in a volume of 25 pL containing 5 mM MSer(OH), 292 mM sucrose and 50 mM sodium acetate buffer at pH=5.75. The reaction was incubated at 30 °C with shaking at 500 rpm. After 24 hours, the reaction mix was incubated at 70 °C for 10 minutes to inactivate the enzymes. For analysis, the reaction media were diluted 1 / 10 in water and centrifuged for 5 minutes at 2000 g.

[0071] The separation of the different products (MSer(OH)-glu) was carried out in reverse phase with a C18 Synergi™ Fusion-RP 250 mm x 2 mm column (porosity: 80 Å, particle size: 4 μm, Phenomenex, USA). This column was maintained at 30 °C on a Thermo Ultimate 3000 HPLC system equipped with a UV detector (Thermo Ultimate 3000) at Å=310 nm and a Corona Véo CAD (Charged Aerosol Detector) detector (nebulization temperature: 50 °C). The mobile phase consisted of a mixture of ultrapure water (solvent A) / LC-MS grade acetonitrile (solvent B) at a flow rate of 0.2 mL / min. The separation was achieved in 75 minutes by a linear gradient, as defined in Table 3.

[0072] [Table 3]

[0073] During this step, the GS-D enzyme proved to be by far the most efficient (Figures 2 and 3), converting 95% of MSer(OH) into 8 different glucosylated products, while for all other enzymes tested, the conversion rate was between 0% and 6.5%. Representing 72.3% of the area of ​​all MSer(OH)-glu products, the majority peaks are the peaks numbered 1 and 2 in Figure 4. Their analyses by liquid chromatography coupled with a mass detector (LC-MS) showed that they had a degree of polymerization (DP) of 1 and 2 glucosyl units respectively. The minority peaks (products 3 to 8 in Figure 4) would have a degree of polymerization (DP) of 3 to 4 glucosyl units.

[0074] In order to identify the position of glucosyl units on MSer(OH)-glu, Nuclear Magnetic Resonance (NMR) analyses were performed. The spectra 1 H, 13C, JMod, HSQC and HMBC were recorded on a Bruker Avance 500 MHz equipment at 298K with a BBI 5 mm z-gradient H-BB-D probe. The data were acquired and processed using TopSpin 3.5 software. All these analyses highlighted that glucosylation took place on the hydroxyls carried by the C8 carbon and the equivalent C10 / C11 carbons of MSer(OH) (Figure 5A). Figures 5B-D specify the structure of the products obtained by the method of the invention, and that the number of substitutions by glucosyl units can vary from 1 to 2, possibly 3, and the variety of glucosylated products that the culture medium can include.

[0075] To assess the possibility of industrial application, the MSer(OH) glucosylation reaction by GS-D was tested at a 50 mM MSer(OH) concentration, a concentration 10 times higher than during screening. The protocol followed was otherwise identical to that described previously. In particular, since the sucrose concentration was kept at 292 mM, the sucrose:MSer(OH) molar ratio decreased from 60:1 to 6:1. The comparison of the chromatograms for these two reactions is shown in Figure 7. The conversion rate of MSer(OH) to MSer(OH)-glu increased from only 95% with 5 mM MSer(OH) to 90% with 50 mM MSer(OH). This new satisfactory conversion rate showed that it was possible to increase the quantities of glucosylated product during a batch reaction.It was observed that the relative proportion between peaks 1 and 2 changed slightly, with the product corresponding to diglucosylated MSer(OH) (peak 2) being favored when the initial concentration was 5 mM (i.e. when the sucrose:MSer(OH) ratio was highest).

[0076] Second screening:

[0077] Following these initial results, the extension of glucosylated MSer(OH)-glu products from sucrose by certain polymerases of the GH-70 family was the subject of a second screening (see Table 1). An MSer(OH)-polym product was obtained whose glucosidic part was very clearly extended compared to that obtained by GS-D.

[0078] MSer(OH)-glu production was carried out under conditions similar to those described in the first screening but in larger quantities. The reaction was thus carried out in a volume of 50 pL containing 25 mM of pure MSer(OH), 292 mM of sucrose, 1 U.mL' 1of GS-D, and 50 mM sodium acetate buffer at pH=5.75. This was incubated at 30 °C with stirring at 500 rpm. After 24 hours, the reaction medium was incubated at 70 °C for 10 minutes to inactivate the enzyme. The extension of the MSer(OH)-glu intermediate products by other enzymes of the same family was carried out without a prior purification step of MSer(OH)-glu: 5 pL of reaction medium from step 1 was incorporated into a new 25 pL reaction so that the final molar concentration of MSer(OH)-glu was equivalent to 5 mM of free MSer(OH). To this were added the equivalent of 292 mM of sucrose and 1 U.mL' 1of enzyme selected for the second step of the enzymatic cascade (see Table 1). This new medium also contained 50 mM sodium acetate buffer at pH=5.75. The reactions were incubated at 30 °C with shaking at 500 rpm for 24 hours, then stopped by heating at 70 °C for 10 minutes.

[0079] The separation of the different products (MSer(OH)-polym) was carried out in the same way as for the MSer(OH)-glu during the first screening.

[0080] As shown in Figure 8, the 11 polymerases tested were able to elongate the MSer(OH)-glu intermediates with chains of glucosyl units of varying size and nature depending on the binding specificity of the enzyme tested. Across all reactions, at least 67.6% and up to 97.7% of the MSer(OH)-glu intermediates were elongated (Table 4). Peaks 1 and 2 of the MSer(OH)-glu intermediates (the very majority species in the medium) were mainly consumed by the polymerases, the percentage of conversion of each of these two peaks being able to vary depending on the specificity of the enzyme used (Table 4). For most enzymatic cascades, peak 2 (diglucosylated MSer(OH)) is consumed preferentially and sometimes almost entirely.

[0081] The average mass of carbohydrate chains of elongated MSer(OH)-glu ( / .e. MSer(OH)-polym) was analyzed by high-performance spectroscopy (HPSEC) on a Shodex 802.5 column. Elution was performed isocratically at a water flow rate of 0.3 mL / min and a temperature of 70°C. In the case of elongation of MSer(OH)-glu intermediates by DSR-OK A1 and DSR-S vardel A4N enzymes that form much larger natural polymers, HPSEC analysis was performed by combining a Shodex 805 column and a Shodex 802.5 column in series. Elution was performed with 450 mM NaNOs, 1% v / v ethylene glycol.

[0082] The separation calibration was based on a standard range of commercial dextrans (polymers of glucosyl units linked in a-1,6) of increasing sizes between 342 and 70,000 Da. The average masses MM moyof the products were determined at the apex of the elution peaks. The average degree of polymerization (DPmoy) visible in UV was estimated using the following relationship: DPmoy = MM moy / 162.

[0083] Depending on the polymerase used during this second step, chains of glucosyl units ranging on average from 3 to 41 units could be identified (Table 4, Figures 9 and 10).

[0084] [Table 4]

[0085] Peak 1 = MSer(OH)-monoglucosylated; Peak 2 = MSer(OH)-diglucosylated; nd: not determined

[0086] Notably, the DSR-M A1, DSR-M A2 and DSR-M A2 W624A enzymes elongated MSer(OH)-glu very efficiently, achieving a conversion of MSer(OH)-glu of 80.2% and 90.4%, respectively. They produced a dextran-like motif, composed exclusively of glucosyl units linked by α-1,6 bonds (a natural motif of these enzymes); which gives them a lower risk of anaphylactic shock. As illustrated in Figure 11, it was possible to control the size of the polymers by enzyme engineering, DSR-M A2 W624A being a variant of the enzyme DSR-M A1

[0024] . The W624A mutation in fact made it possible to obtain MSer(OH)-polyms with a reduced average molar mass (10 units) while the reaction with the enzyme DSR-M A1 produced MSer(OH)-polyms with a higher average molar mass, comprising on average 37 glucosyl units.

[0087] Low-molecular-weight dextrans are already marketed for pharmaceutical applications, particularly in certain ophthalmic products. In free form, they are used as ocular lubricants or artificial tears (see https: / / www.dextran.com / application-areas / eve). Their coupling to an MSer(OH) with a high UV absorption capacity should allow the development of new products, formulations, (bio)materials, and drugs combining the properties of sugars and mycosporins.

[0088] The absorption capacity of mycosporines was not affected by the grafting of a polysaccharide chain. Indeed, absorbance scans between λ=200 nm and λ=800 nm of the MSer(OH)-polym products obtained with all the enzymes tested, carried out using a CARY 3500 spectrophotometer, showed that glucosylation and elongation of the glucosyl chains on the MSer(OH) did not cause a change in the maximum absorbance wavelengths λMAX (Figure 12).

[0089] Finally, the glucosylation reaction was also tested on a crude extract of Lichina pygmea, more easily sourced, which can be obtained from an aqueous or hydroalcoholic extraction, possibly followed by lyophilization. The extract is derived directly from the lichen Lichina pygmea, not purified but enriched in MSer(OH) up to 10% of the total mass of the extract. The first glucosylation step by GS-D gave results identical to the reactions carried out on pure MSer(OH), i.e. 95% glucosylated MSer(OH) (Figure 13). The second step involving the polymerases was also as efficient as when carried out with purified MSer(OH) (Figure 14 with the enzyme DSR-M A1). Thus, the enzymes are not inhibited by other components of the crude medium. This is particularly advantageous for an industrial approach to the process which avoids the costly MSer(OH) purification step.

[0090] Advantage of the 2-step enzymatic cascade vs. “one-pot” synthesis:

[0091] The use of GS-D and another polymerase in synergy (one-pot synthesis or co-synthesis for simultaneous glucosylation and elongation of MSer(OH)) was evaluated and compared with the glucosylation yields obtained in two-step enzymatic cascades.

[0092] However, as illustrated by the comparison of Figure 8 (enzyme cascade) and 15 (co-synthesis), the overall conversions of MSer(OH) obtained are less interesting in co-synthesis than in the enzymatic cascade. In co-synthesis, the MSer(OH)-glu products are less consumed by the polymerases, so there is a larger population of MSer(OH)-glu and a smaller population of MSer(OH)-polym.

[0093] EXAMPLE 2: PROPERTIES OF MSer(OH)-glu and MSer(OH)-polym

[0094] The antioxidant properties and photostability of the MSer(OH); MSer(OH)-glu and MSer(OH)-polym products generated by the process of the invention were tested on reaction media purified from the DNA present in the cell extracts used for the glucosylation of MSer(OH), in order to avoid interference from DNA in the phenomena studied. This purification was carried out by anion exchange chromatography, with a Diaion resin.

[0095] Photostability of synthesized products:

[0096] Photostability was assessed on the different MSer(OH) glucosylation products. For this, the samples were irradiated in a Sun Test XLS solar simulator (Atlas Material Testing Solutions, USA). The solar filter was chosen to reproduce conventional solar exposure. The concentration of remaining molecules was monitored by measuring the absorbance at λ=310 nm. The photodegradation kinetics obtained allow the calculation of the quantum yield of photodegradation of the products. This quantum yield represents the ratio between the photo-degraded molecules and the number of photons received by these molecules, and is calculated according to the following formula: where ro is the photodecomposition rate (mol.L' 1 .s' 1 ), V is the volume of the solution (L), NA is Avogadro's number (mol -1), lo the number of photons emitted in the absorbance band domain of MSer(OH) and A the absorbance of the solution of the different samples. The experiments were carried out in triplicate.

[0097] All MSer(OH) glucosylation and elongation products exhibit high photostability, with quantum yields in the order of 10 -5 (Figure 17), comparable to those of certain sunscreens as well as other mycosporines [25, 26],

[0098] Mycosporine glucosylation therefore does not impact photostability. This is due to the fact that glucosylation positions do not prevent the delocalization of the electron cloud, allowing the dissipation of the received energy in the form of heat. Also, the length of the carbohydrate chain does not affect the photostability of the products.

[0099] However, preliminary tests have highlighted the interaction of DNA in the photostability of the products, through the generation of reactive species. This results in a lower photostability of the product from the MSer(OH)+GS-D+DSR-M A2 W624A cascade, whose DNA purification was the least efficient.

[0100] Finally, it appears that the product of the MSer(OH)+GS-D+DSR-OK A1 cascade has greater photostability than free MSer(OH). This can be explained by the physical protection role provided by the long dextran chain, which protects MSer(OH) from irradiation.

[0101] Antioxidant power:

[0102] The antioxidant power of the glucosylation products MSer(OH)-glu and Mser(OH)-polym was determined using the TEAC (Trolox Equivalent Antioxidant Capacity) method. The TEAC test is well established for the evaluation of radical scavenging properties and allows comparison of TEAC scores with a large literature. Based on the reduction of the previously oxidized ABTS+ form, this spectrophotometric method is easy to implement

[0027] . The reduction of ABTS+ results in a decrease in absorbance at λ=734 nm and is proportional to the concentration of antioxidant molecule. The TEAC score is established by comparing the leading coefficients of %inhibition = ^antioxidant concentration) with those obtained with Trolox, a reference antioxidant molecule. The experiments were carried out in triplicate.

[0103] The scores obtained for MSer(OH) and its glucosylated derivatives are all greater than 1 (Figure 18). The tested products therefore have a strong antioxidant power and this power is preserved for all the glucosylation products obtained, contrary to what had been observed during the glucosylation of flavonoids such as luteolin

[0028] . The antioxidant power of products resulting from enzymatic cascades without MSer(OH) was evaluated as almost zero, which attests that this biological property is indeed due to the mycosporine part of the molecule.

[0104] In order to verify the correct execution of the test we also checked the TEAC score of a known antioxidant, ascorbic acid or vitamin C. The value obtained (0.8 TEAC) is in agreement with those of the bibliography

[0029] . Thus, according to our test, all the products of the enzymatic cascade have an antioxidant power greater than that of ascorbic acid.

[0105] Liste de références

[0106] 1. Lawrence, K. P., Long, P. F. & Young, A. R. Mycosporine-Like Amino Acids for Skin Photoprotection. Curr. Med. Chem. 25, 5512-5527 (2018)

[0107] 2. de la Coba, F. et al. UVA and UVB Photoprotective Capabilities of Topical Formulations Containing Mycosporine-like Amino Acids (MAAs) through Different Biological Effective Protection Factors (BEPFs). Mar. Drugs 17 (2019)

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[0114] 9. La Barre, S., Roullier, C. & Boustie, J. Mycosporine-Like Amino Acids (MAAs) in Biological Photosystems, in Outstanding Marine Molecules: Chemistry, Biology, Analysis (ed. Stéphane La Barre, J.-M. K. (eds. )) 333-360 (Wiley-CH Verlag GmbH, 2014). doi:10.1002 / 9783527681501 ,ch15

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Claims

CLAIMS 1) Process for producing a glucosylated mycosporine, said process comprising a step a) of glucosylation of a mycosporine by the enzyme a-transglucosylase GS-D from sucrose, to obtain an intermediate glucosylation product which is a glucosylated mycosporine having an average degree of polymerization ranging from 1 to 2 glucosyl units. 2) Method according to claim 1 further comprising a step b) of elongation of the carbohydrate part of the intermediate glucosylation product obtained in step a) by at least one α-transglucosylase GH-70 different from α-transglucosylase GS-D from sucrose, to obtain a glucosylated mycosporine having an average degree of polymerization ranging from 3 to 1,000,000 glucosyl units. 3) Method according to claim 2, in which steps a) and b) are carried out concomitantly and comprise the glucosylation of a mycosporine by the enzyme α-transglucosylase GS-D and at least one α-transglucosylase GH-70 different from α-transglucosylase GS-D from sucrose, to obtain a glucosylated mycosporine having an average degree of polymerization ranging from 3 to 1,000,000 glucosyl units. 4) Method according to claim 2 or 3, wherein said at least one α-transglucosylase of step b) is selected from the group consisting of: glucan-sucrases GS-A, GS-B, GS-C, GS-F A1 and GS-FS, dextran-sucrases DSR-M A1, DSR-M A2 W624A, DSR-G A1, DSR-S vardel A4N, and DSR-OK A1, alternan-sucrase ASR A1. 5) Method according to any one of claims 1 to 4, where the mycosporine of step a) is chosen from the group consisting of: pure mycosporine, Lichina pygmea lichen extract enriched with mycosporine from 0 to 10% by weight of extract. 6) A method according to claim 5, wherein the mycosporine is mycosporine-serinol (MSer(OH)). 7) Glucosylated mycosporin obtained by a process according to claim 1, said glucosylated mycosporin having an average degree of polymerization ranging from 1 to 2 glucosyl units. 8) Glucosylated mycosporin obtained according to any one of claims 2 to 6, said glucosylated mycosporin having an average degree of polymerization ranging from 3 to 1,000,000 glucosyl units, preferably from 3 to 800,000 glucosyl units. 9) Cosmetic composition comprising as active ingredient a glucosylated mycosporin as defined in claim 7 or 8. 10) Glucosylated mycosporin according to claim 7 or 8, for use as a medicament. 11) Glucosylated mycosporin according to claim 7 or 8, for use in the prevention or treatment of cataracts, albinism, xeroderma pigmentosum, skin cancer.

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