Enzymatically produced glucosylated mycosporine for development of innovative anti-UV products

Abstract

The subject matter of the present invention is the preparation of glucosylated mycosporins by an enzymatic cascade process using an alpha-transglucosylase and sucrose and the use thereof, in particular for their anti-UV properties.

Description

Technical Field

[0001] The present invention relates to the preparation of glucosylated mycosporins by an enzymatic cascade process using α-transglucosylase and sucrose and their use, in particular, for their anti-UV properties (eg, UV absorbing and protective properties, antioxidant properties).

[0002] The present invention is applicable, for example, in the fields of ophthalmology, dermatology, cosmetology, materials and agriculture.

[0003] In the following description, references between brackets [ ] refer to the reference list presented at the end of the text. Background Art

[0004] Prevention and protection are essential to deal with chronic and acute damage to human health, especially to the skin and eyes, caused by UV radiation. Reducing UV exposure and applying sunscreens are therefore strongly recommended to prevent skin diseases. Broad-spectrum inorganic sunscreens composed of titanium dioxide (TiO2) and zinc oxide (ZnO) are commonly used, which work primarily by absorbing radiation and also by diffusing radiation [1]. However, their generally opaque and white appearance is not accepted by all consumers, and the possible cytotoxicity of nanoparticle accumulation in the body has not been fully studied [2]. Organic filters composed of synthetic molecules have been accused of having relatively limited efficacy in protecting the skin, sometimes causing allergies, and being endocrine disruptors [1,2]. Some are even suspected of decomposing into carcinogenic compounds, such as octocrylene [3], which has recently been shown to decompose into benzophenones over time. In addition, the lack of photostability of some compounds can have a counterproductive effect by forming ROS (reactive oxygen species) and increasing oxidative stress. Finally, from an environmental point of view, both types of filters have been accused of accumulating in coastal waters, causing endocrine changes in fish and coral bleaching [4,1]. In addition, in sunscreens, chemical filters such as oxybenzone cause allergies and side effects in humans and are known to damage coral reefs [4]. Therefore, it is necessary to develop new solutions based on natural resources and using environmentally friendly methods.

[0005] Mycosporin and mycosporin-like amino acids (MAAs), hereinafter collectively referred to as mycosporin, are small molecules of approximately 400 Daltons (Da) organized around a cyclohexenone or cyclohexenimine ring. Due to their structure, these zwitterions have unique UV absorption capabilities, with absorption in the range of 309 nm to 362 nm and from 21,800 cm -1 .M -1 Up to 50,000cm -1 .M -1The extinction coefficient of [5]. Therefore, they cover both the UV-A range and the UV-B range. Mycosporins have also been described as potent antioxidants. They are secondary metabolites, and their role as natural sunscreens has been studied for many years. Their potential is promising for several reasons: (i) they can act as UV filters and antioxidants, which is an effective combination for protecting the body from multiple damage caused by photodamage, (ii) they are also being studied for their anticancer, anti-aging and anti-inflammatory properties, and (iii) they have great potential for promoting wound healing [1]. Their mechanism of action is as follows: in the photoexcited state, they undergo rapid internal conversion to the ground electronic state (S0) by releasing heat or by undergoing reversible isomerization [1,6]. Due to this mechanism, more than 90% of the energy received is released to the surrounding environment by heat transfer without generating ROS. Fluorescence emission has not been specifically reported [7].

[0006] Mycosporins are found in a wide variety of organisms. UV radiation affects terrestrial life, but it can also penetrate water at depths of 0.5 to 47 meters, depending on water clarity and weather conditions [1]. Thus, mycosporins have been identified in cyanobacteria, but also in heterotrophic bacteria and marine fungi, microalgae, macroalgae (mainly red and brown algae), phytoplankton (diatoms, dinoflagellates), corals, molluscs and fish [7,8]. They have also been identified in terrestrial organisms such as lichens that live in sometimes very extreme conditions [9]. This is the case of the mycosporin serinol MSer(OH), which is found in particular in the species Lichinapygmaea found in the intertidal zone of the coast of Western Europe.

[0007] In metazoans such as fish, mycosporins accumulate rapidly in tissues of the outer layers (such as the skin on the dorsal surface) and sensitive organs (such as the digestive tract, reproductive organs, and eyes) exposed to sunlight [1, 5, 7, 8]. In the eye, they are particularly concentrated in the cornea and lens, thus protecting the retina from photo-oxidative damage caused by UV rays

[10] .

[0008] The presence of mycosporins in fish ocular tissue is a subject of particular interest for bioinspiration, especially in the development of preventive and / or curative biomedical solutions in the field of ophthalmology. However, the formulation of biomedical products from currently available mycosporins is complicated by their small size, which causes their rapid elimination from mucous membranes, especially the eye.

[0009] Glucosylated mycosporins already exist in nature, which essentially have a degree of polymerization (DP) of 1 to 2 glucosyl units (i.e., xylose, galactose, glucosamine, etc.), with the exception of mycosporin-glycine mycosporins of up to 8 different kinds of glucosyl units (i.e., xylose, galactose, glucosamine, etc.)

[11] . 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, various mycosporins have been identified (shinorine, porphyrin-334, palythine, mycosporin-glycine, mycosporin-glutamicol) associated with different glucosyl units (galactose, glucosamine, xylose, etc.). Although the role of these glucosylated mycosporins has not yet been fully explored, they may play a multifunctional role in protecting against UV radiation as well as against thermal stress or desiccation. The value of the sugars covalently bound to the mycosporins has also not yet been fully elucidated, although it has been strongly suggested that this would improve the interaction with the extracellular polysaccharide matrix of these cyanobacteria

[13] . In addition to the enhanced stability and non-covalent attachment to the extracellular polysaccharide matrix, the high proportion of glucosylated mycosporins undoubtedly demonstrates another useful role: Ishihara et al. thus identified that 86.5% of the total mycosporins of Nostoc sphaericum were β-galactosyl-shinorine-334; 13.2% were hexosyl-shinorine and only 0.2% were free shinorine-334

[14] . Summary of the invention

[0010] The inventors hypothesized that it might be interesting to incorporate long glucosylated chains into mycosporins in order to see whether the resulting molecules would be sufficiently stable and retain their UV resistance properties (photostability) to be useful.

[0011] Therefore, the present inventors have developed a new method that enables the addition of long glucosyl chains to mycosporins, thereby creating new environmentally friendly highly UV protective products or biomaterials to replace current UV filters, capable of prolonging the retention time of mycosporins in organs such as the eye. The nature of the sugar chains (consisting only of glucosyl units) should reduce the risk of allergic reactions.

[0012] UV protective biomolecules (glucosylated mycosporins) obtained exclusively using natural precursors with prolonged retention time can be applied as an alternative to current active ingredients in cosmetic preparations, medical products (eg in ophthalmology), biomaterials and outdoor materials.

[0013] For this purpose, enzymatic glucosylation has emerged as the most effective method, although it has never been studied in connection with mycosporins. For this type of enzymatic catalysis, a family of enzymes of particular interest has emerged. These are the α-transglucosylases of the glycoside hydrolase family 70 (GH-70)

[15] , enzymes naturally present in lactic acid bacteria of the genera Leuconostoc, Lactobacillus, Streptococcus, Oenococcus or Weissela sp.

[16] . These enzymes are of considerable industrial interest due to their catalytic mechanism, such as Fig.16 These enzymes are able to form a variety of products from sucrose, a very cheap substrate derived from renewable resources (sugar beets and sugar cane), based on the nature of the acceptor molecule. Fig.16 After step 1) in , GH-70 can transfer the glucosyl unit to

[17] :

[0014] -water( Fig.16 Step 3 in ), to carry out a hydrolysis reaction in which free glucose is formed (for very good polymerases of the GH-70 family, mostly at the beginning of the reaction).

[0015] -glucose and gradually transfer to oligosaccharides of increasing size ( Fig.16 Step 4) above to achieve the synthesis of homopolymers of α-D-glucosyl units (called α-glucans). These are shown in 10 3 Up to 10 9 There are a variety of polymers in the range of 1,2 to 2,000 Da and composed of glycosidic bonds of different nature (α-1,2; α-1,3; α-1,4 and / or α-1,6). Among these α-glucans, one of the best known is dextran. It is a more or less linear polymer containing a large proportion of α-1,6-linked glucosyl units and has many applications in industry (e.g., in the food, cosmetic and pharmaceutical fields).

[0016] -fructose( Fig.16 Step 5) in the reaction, for example, sucrose isomers such as leucrose are formed. However, these products are in the minority.

[0017] - an exogenous acceptor molecule (glycosyl or aglycone) added to the medium at the beginning of the reaction, which has at least one hydroxyl functional group ( Fig.16 Step 6 in ); this catalytic reaction is called the acceptor reaction.

[0018] The ability to transfer glucosyl units to these foreign molecules depends on several factors, including the structure of the receptor and the structure of the active site of the enzyme. An effective receptor will be able to redirect almost all of the glucosyl units transferred to its own glucosylation instead of polymer formation. To date, a variety of receptors have been tested, for example, flavonoids such as quercetin and luteolin

[18] .

[0019] The object of the present invention is therefore to produce glucosylated derivatives of mycosporin, in particular mycosporin-serinol (hereinafter referred to as MSer(OH)), derived in particular from the marine lichen Lichina pygmaea, by an enzymatic cascade process using α-transglucosylases of the glycoside hydrolase family 70 (GH-70) and sucrose. Naturally, these enzymes use sucrose derived from sugar beet or sugar cane to synthesize (polymerases) or modify (branching sucrases) high molar mass polymers of glucosyl units (α-glucans). By comparison, some sucrose phosphorylases, which also use sucrose as donor of glucosyl units, and commercial CGTases belonging to the GH-13 family, which use starch (another natural and abundant substrate) as donor of glucosyl units, have also been tested, but without success.

[0020] The enzymatic cascade method described herein ( Figure 1 ) has the advantage of not involving any chemical reactions; this avoids several steps of protection / deprotection of the hydroxyl functional groups of the sugars, which usually require the use of metal catalysts or strong acids. These metal catalysts or strong acids are known to generate a large amount of waste to be handled and can cause uncontrolled parasitic reactions.

[0021] Glucosylated mycosporins are produced in a completely aqueous medium without the addition of organic solvents.

[0022] Glucosylation can be performed in a single step (co-synthesis or one-pot synthesis) or in two steps as follows:

[0023] 1 / Glucosylation of mycosporin (e.g., mycosporin-serinol, MSer(OH)) from sucrose by GS-D enzyme (α-transglucosylase of the GH-70 family), thereby producing a glucosylated intermediate called mycosporin-glu (e.g., MSer(OH)-glu);

[0024] 2 / Elongation of the mycosporin-glu glucosylation intermediate (eg, MSer(OH)-glu) from sucrose by other α-transglucosylases of the GH-70 family to produce the final glucosylation product known as mycosporin polymer (eg, MSer(OH)-polymer).

[0025] This second step in the enzymatic cascade allows the synthesis of mycosporins covalently linked to different lengths of glucosylation chains and glycosidic bonds (α-1,2; α-1,3; α-1,4 and / or α-1,6) to be "tailored" depending on the enzyme and the reaction conditions achieved. Depending on the enzyme used, mycosporin-glu intermediates (e.g., MSer(OH)-glu) can carry up to 4 glucosyl units with an average degree of polymerization (DP) mainly concentrated between 1 and 2 glucosyl units, while mycosporin-polymer end products (e.g., MSer(OH)-polymers) can have 2 to 100,000 glucosyl units with an average degree of polymerization (DP) concentrated between 3 and 1,000,000, 3 to 800,000, 3 to 500,000, 3 to 24,000, 3 to 60 or 3 to 41 glucosyl units.

[0026] Since mycosporins are considered natural sunscreens, mycosporin derivatives consisting of mycosporins with long glucosylation chains developed by the process of the present invention can be easily used for the prevention and protection of cells (epidermal cells and ocular cells) and have cosmetic, medical and / or pharmaceutical applications. In particular, applications can be envisaged for eye protection in ophthalmic products (e.g., cataract prevention) or for the protection of people with genetic and hereditary abnormalities (such as albinism and xeroderma pigmentosum). Another application would be to incorporate these glucosylated mycosporins into biodegradable sunscreens of biological origin without environmental consequences such as coral bleaching. Finally, these glucosylated mycosporins can also be used to protect materials used outdoors

[19] .

[0027] Therefore, the object of the present invention is a process for producing a glucosylated mycosporin, said process comprising the step a): glucosylating the mycosporin from sucrose by the enzyme α-transglucosylase GS-D to obtain a glucosylated intermediate, the glucosylated mycosporin having a degree of polymerization ranging from 1 to 4 glucosyl units, preferably an average degree of polymerization of 1 to 2 glucosyl units.

[0028] According to a specific embodiment of the present invention, the method may further comprise the step b): elongating the sugar portion of the glucosylated intermediate obtained in step a) from sucrose by at least one α-transglucosylase other than α-transglucosylase GS-D to obtain a glycosylated intermediate having 3 to 1,000,000 glucosyl units, 3 to 800,000 glucosyl units, 3 to 500,000 glucosyl units, 3 to 24,000 glucosyl units (for very good polymers) synthase, such as, for example, DSR-OKΔ1 dextransucrase), a glucosylated mycosporin with an average degree of polymerization in the range of 3 to 60 glucosyl units, 3 to 41 glucosyl units; preferably 9 to 1,000,000 glucosyl units, 9 to 800,000 glucosyl units, 9 to 500,000 glucosyl units, 9 to 24,000 glucosyl units, 9 to 60 glucosyl units or 9 to 41 glucosyl units.

[0029] According to a specific embodiment of the method of the present invention, steps a) and b) can be performed simultaneously, that is, the method comprises: removing sucrose from the sucrose by the enzyme α-transglucosylase GS-D and at least one other enzyme different from α-transglucosylase GS-D. α - The transglucosylase glucosylates the mycosporin and elongates its sugar moieties to obtain a glucosylated mycosporin with an average degree of polymerization in the range of 3 to 1,000,000 glucosyl units, 3 to 800,000 glucosyl units, 3 to 500,000 glucosyl units, 3 to 24,000 glucosyl units (for very good polymerases such as, for example, DSR-OKΔ1 dextransucrase), 3 to 60 glucosyl units, 3 to 41 glucosyl units; preferably 9 to 1,000,000 glucosyl units, 9 to 800,000 glucosyl units, 9 to 500,000 glucosyl units, 9 to 24,000 glucosyl units, 9 to 60 glucosyl units or 9 to 41 glucosyl units.

[0030] For the purposes of the present invention, "average degree of polymerization (DP)" means the degree of polymerization determined at the apex of the peak obtained by high performance size exclusion chromatography coupled to a UV detector and column calibration using commercial dextran having molar masses ranging from 342 g / mol to 70,000 g / mol.

[0031] According to a specific embodiment of the method of the present invention, the at least one α-transglucosylase of step b) is selected from the group consisting of: dextran sucrases GS-A, GS-B, GS-C, GS-FΔ1 and GS-FS; dextran sucrases DSR-MΔ1, DSR-MΔ2W624A, DSR-GΔ1, DSR-S vardelΔ4N and DSR-OKΔ1; alternan sucrase ASRΔ1.

[0032] According to a particular embodiment of the process of the invention, the mycosporin of step a) is selected from the group consisting of: a pure or purified mycosporin, an extract of Lichina pygmaea enriched with 0% to 50% mycosporin by weight of the extract, preferably 5% to 35% by weight of the extract, preferentially 15% to 25% by weight of the extract, most preferentially 10% by weight of the extract. Preferably, the mycosporin used in step a) is mycosporin-serinol (MSer(OH)). The mycosporin-serinol can be isolated from an extract of cyanolichen (such as various species of Lichina pygmaea and Lichina confinis, Peltigera, Stereum hirsutum, Alternaria chrysanthemi, Ascochytapisi, Ophiobolus graminis, Pleospora herbarum or Pyronema omphalodes). It can also be produced from its precursor (gadusol) by synthetic biology.

[0033] Another object of the present invention is a glucosylated intermediate obtained by the process of the present invention, said glucosylated intermediate being a glucosylated mycosporin having an average degree of polymerization ranging from 1 to 4 glucosyl units (α-type bonds), preferably from 1 to 2 glucosyl units (α-type bonds).

[0034] Another object of the present invention is a glucosylated mycosporin obtained by the process of the present invention, said glucosylated mycosporin having between 3 and 100,000 glucosyl units (α-type bonds), between 3 and 800,000 glucosyl units (α-type bonds), between 3 and 500,000 glucosyl units (α-type bonds), between 3 and 24,000 glucosyl units (α-type bonds), between 3 and 60 glucosyl units (α-type bonds) or between 3 and Preferably, the average degree of polymerization is within the range of 9 to 1,000,000 glucosyl units (α-type bonds), 9 to 800,000 glucosyl units (α-type bonds), 9 to 500,000 glucosyl units (α-type bonds), 9 to 24,000 glucosyl units (α-type bonds), 9 to 60 glucosyl units (α-type bonds) or 9 to 41 glucosyl units (α-type bonds).

[0035] Another object of the present invention is the cosmetic use of the glucosylated mycosporin according to the invention as an anti-UV agent for (i) protecting the skin from UV rays, (ii) combating photoaging.

[0036] Another object of the present invention is a cosmetic composition comprising as active ingredient a glucosylated mycosporin according to the invention.

[0037] Another subject of the present invention is the cosmetic use of the cosmetic composition according to the invention for (i) protecting the skin from UV rays, (ii) combating photoaging.

[0038] Another object of the invention is the glucosylated mycosporin according to the invention for use as a medicament for 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.

[0039] In summary, the present invention implements a green process and mobilizes natural resources, namely mycosporins (especially derived from lichens growing on the coast) and sucrose. This latter resource, which serves as a substrate for enzymes of the GH-70 family, is available in large quantities and at low cost. The enzymatic reaction is a process under mild conditions and does not produce (toxic) waste. Some of the glucosylated chains have the characteristics of dextran (which is an α-glucan widely used in the cosmetic and biomedical fields because it is very well tolerated by the body). Since the dextran chains of the MSer(OH)-polymer product can be "custom-adjusted" (i.e., in terms of glycosidic bonds, sugar chain size), it can be easily adapted to the type of target application. In nature, mycosporins are bound to proteins, for example in the mucus and lens of fish [7, 20, 21]. Within the scope of the present invention, this phenomenon has been mimicked by producing MSer(OH)-polymer mycosporin derivatives with elongated sugar chains. The glucosylated mycosporins obtained by the process of the present invention have better properties, in particular photostability, compared to the free mycosporin molecules.

[0040] In contrast to commercial UV protection molecules such as, for example, oxybenzone (a UVA and UVB blocker) [4], which cause bleaching of coral reefs, glucosylated mycosporins do not cause such damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The principle of the enzymatic cascade for the glucosylation of mycosporin from sucrose and involving α-transglucosylases of the GH-70 family is shown: the initial glucosylation step is performed by the GS-D enzyme, after which the glucosidic chain is elongated by different enzymes of this family, allowing control over both the size and structure of the chain (different types of α-glycosidic bonds depend on the enzyme chosen).

[0042] Figure 2 Depicted is the UV chromatogram (λ=310 nm) of α-transglucosylases of the GH-70 family screened for MSer(OH) glucosylation. Only the GS-D enzyme can efficiently glucosylate MSer(OH).

[0043] Figure 3 Depicted are UV chromatograms (λ=310 nm) of commercial CGTase and sucrose phosphorylase screened for MSer(OH) glucosylation.

[0044] Figure 4 Depicted is the UV chromatogram (λ=310 nm) of different glucosylation products of 5 mM MSer(OH) by GS-D, referred to as MSer(OH)-glu. Numbers 1 to 8 identify the different MSer(OH)-glu glucosylation products obtained at the end of the first step of the enzymatic cascade.

[0045] Figure 5 Shown are (A) a diagram of the chemical structure of MSer(OH) with carbon numbering and possible glucosylation positions. The hexagons represent glucosyl units bound to MSer(OH) (B), which are example structures of DP2 produced by glucosylation of MSer(OH) by GS-D enzyme (C), which are example structures of DP3 produced by glucosylation of MSer(OH) by GS-D enzyme (D), which are example structures of products from the enzymatic cascade (glucosylation + elongation).

[0046] Figure 6 Shows 1 H NMR (spectrum A) and HMBC (spectrum B) spectra, which confirmed the glucosylation of MSer(OH) by GS-D to a glucosylated intermediate (MSer(OH)-glu) having an average degree of polymerization in the range of 1 to 2 glucosyl units.

[0047] Figure 7 Shown is a comparison of UV chromatograms (λ=310 nm) of the glucosylation reaction of MSer(OH) by GS-D at 5 mM and 50 mM of MSer(OH).

[0048] Figure 8 Shown is a UV chromatogram (λ=310 nm) of a GS-D enzymatic cascade polymerization followed by a polymerase (ie, another α-transglucosylase of the GH-70 family). The enzyme used in the second step is indicated on the chromatogram.

[0049] Fig. 9 Depicted are UV chromatograms (λ=310 nm) of 6 of 11 different reactions separated by high performance size exclusion chromatography (HPSEC). The chromatogram labeled DEX 10,000Da is the chromatogram of a commercial dextran with an average molar mass of 10,000 g / mol. The asterisks identify the major population for each reaction. Peaks DP1 and DP2 correspond to Figure 4 Peak 1 and Peak 2 in .

[0050] Fig.10 Depicted are UV chromatograms (λ=310 nm) of 3 different reactions out of 11 different reactions separated by high performance size exclusion chromatography (HPSEC).The chromatogram labeled DEX 70,000 Da is that of a commercial dextran with an average molar mass of 70,000 g / mol.

[0051] Fig.11 The Synergi coupled with a UV detector (λ=310 nm) is shown. TMComparison of HPLC analysis of polymerization of MSer(OH)-glu products by DSR-MΔ2 enzyme and its DSR-MΔ2W624A mutant on a Fusion-RP C18 column.

[0052] Fig.12 Shown are absorbance scans of MSer(OH) and MSer(OH)-glu and MSer(OH)-polymer products for wavelengths ranging from 200 nm to 800 nm (region of interest shown: 200 nm to 400 nm).

[0053] Fig.13 Shown is a comparison of UV chromatograms (λ=310 nm) of GS-D glucosylation reactions of up to 10% of purified MSer(OH) present in a crude extract of the lichen Lichina pygmaea in reactions with 6.37 mM MSer(OH) and 292 mM sucrose.

[0054] Fig.14 Shown is a comparison of UV chromatograms (λ=310 nm) of the glucosylation reaction of GS-D and DSR-MΔ1 on MSer(OH) purified and present in a crude extract derived from the lichen Lichina pygmaea.

[0055] Fig.15 Chromatograms of co-synthesized polymers are shown. The MSer(OH)-glu product is consumed less efficiently by the polymerase and less MSer(OH)-polymer is produced.

[0056] Fig.16 Schematic diagram showing different catalytic mechanisms of GH-70.

[0057] Fig.17 The quantum yields of MSer(OH) and various MSer(OH)-glu and Mser(OH)-polymer glucosylation products obtained by the method of the present invention are shown.

[0058] Fig.18 Shown are the TEAC scores of MSer(OH) and the enzymatic cascade products (with and without MSer(OH)) of the process of the invention. DETAILED DESCRIPTION

[0059] Example

[0060] Example 1: Example implementation of the method of the present invention

[0061] First Screening

[0062] A collection of α-transglucosylases of the GH70 and GH13 families were screened for their ability to glucosylate mycosporin-serinol (MSer(OH)) molecules from sucrose or, optionally, starch. Initially, work was performed using pure MSer(OH) obtained from a Lichina pygmaea extract. The enzymes listed in Table 1 below have all been produced in recombinant form in Escherichia coli, with the exception of CGTase and sucrose phosphorylase (enzymes 22, 23, and 24), which are commercial preparations.

[0063] Table 1

[0064]

[0065]

[0066] = L. = Leuconostoc; Lb. = Lactobacillus; Bb. = Bifidobacterium; P = polymerase; B = branching sucrase; Bif = bifunctional; GT = α-glucanotransferase; S-Php = sucrose phosphorolysis; ND = not determined.

[0067] The enzyme was produced from E. coli BL21 Star (DE3) cells transformed with a plasmid containing the target enzyme gene (Table 2). 300 μL of the transformation mixture was used to inoculate a 30 mL volume of LB (Lysogeny Both) supplemented with 100 μg / mL ampicillin. The culture medium was incubated overnight at 37°C to prepare a preculture. A 1 L culture in modified ZYM5052 medium

[22] (characteristics of which are presented in Table 2) was inoculated from the preculture of the previous day at an initial OD (λ=600 nm) of 0.05 and then incubated at 21°C and 150 rpm for 26 hours.

[0068] At the end of the fermentation, the culture medium was centrifuged (15 minutes, 800 g, 8°C) and the precipitate was concentrated to an OD of 80 (λ=600 nm) in 50 mM sodium acetate buffer pH=5.75. The cells were then disrupted by ultrasound according to the following protocol: 5 cycles of 20 seconds at 30% of the maximum power of the probe, cooling, and separation by standing in ice for 4 minutes. The ultrasound-treated supernatant containing the soluble enzyme of interest was then recovered after 30 minutes of centrifugation (15,000 g, 6°C) and stored at 4°C.

[0069] Table 2

[0070]

[0071]

[0072] Gly = glycerol; Glu = glucose; α-Lac = α-lactose; L-Ara = L-arabinose

[0073] Enzyme activity was determined by measuring the initial rate of production of reducing sugars using the dinitrosalicylic acid (DNS) method

[23] . One enzyme unit represents the amount of enzyme that releases one μmol of fructose per minute from a solution of 100 g / L sucrose in 50 mM sodium acetate buffer (pH = 5.75) at 30°C. During the kinetics, 100 μL of the reaction medium was removed and the reaction was stopped by adding an equivalent volume of DNS. The sample was then heated at 95°C for 5 minutes, cooled in ice, diluted by half in water, and the absorbance was read at λ = 540 nm. A standard range of 0 g / L to 2 g / L fructose was used to establish the relationship between the absorbance value and the reducing sugar concentration.

[0074] The first screening involving all enzymes in Table 1 was performed in a volume of 25 μL 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 stirring at 500 rpm. After 24 hours, the reaction mixture was incubated at 70°C for 10 minutes to inactivate the enzymes. For analysis, the reaction medium was diluted 1:10 in water and centrifuged at 2000 g for 5 minutes.

[0075] Use 250mm×2mm C18 Synergi TM Fusion-RP column (porosity: Particle size: 4 μ m, Phenomenex, USA) is carried out in reverse phase to separate various products (MSer (OH) -glu). The column is maintained at 30 ° C on a Thermo Ultimate 3000 HPLC system equipped with a UV detector (Thermo Ultimate 3000) and a Corona VéoCAD (Charged Aerosol Detector) detector (atomization temperature: 50 ° C) at λ = 310 nm. The mobile phase is composed of a mixture of ultrapure water (solvent A) / LC-MS grade acetonitrile (solvent B) at a flow rate of 0.2 mL / min. Separation is achieved in 75 minutes using a linear gradient as defined in Table 3.

[0076] Table 3

[0077] Time (min) ％A ％B 0 100 0 12 100 0 70 95 5 75 95 5

[0078] During this step, the GS-D enzyme proved to be the most efficient by far ( Figure 2 and Figure 3 ), which converted 95% of MSer(OH) into 8 different glucosylation products, whereas for all other enzymes tested, the conversion ranged from 0% to 6.5%. Only 72.3% of the total area of ​​MSer(OH)-glu products was represented, with the main peak being Figure 4 The peaks are numbered 1 and 2 in the HPLC. Their analysis by liquid chromatography coupled to a mass spectrometer (LC-MS) showed that they had a degree of polymerization (DP) of 1 and 2 glucosyl units, respectively. Figure 4 Products 3 to 8 in FIG. 5A are believed to have a degree of polymerization (DP) of 3 to 4 glucosyl units.

[0079] To identify the position of the glucosyl units on MSer(OH)-glu, nuclear magnetic resonance (NMR) analysis was performed. The NMR spectra were recorded on a Bruker Avance 500 MHz instrument at 298 K using a 5 mm BBI H-BB-D probe with a Z gradient. 1 H. 13 C, JMod, HSQC and HMBC spectra. TopSpin 3.5 software was used to acquire and process the data. All of these analyses showed that glucosylation occurred on the hydroxyl groups carried by the C8 carbon and C10 / C11 equivalent carbons of MSer(OH) ( Figure 5 A). Figure 5 Figures B to D show the structures of the products obtained by the method of the invention, and the number of substitutions by glucosyl units can vary from 1 to 2, possibly 3, and the diversity of glucosylated products that the culture medium may contain.

[0080] To estimate the possibility of industrial application, the glucosylation reaction of MSer(OH) by GS-D was tested at a MSer(OH) concentration of 50 mM (i.e., 10 times the concentration during screening). The protocol followed was the same as previously described. In particular, the sucrose:MSer(OH) molar ratio was decreased from 60:1 to 6:1, while the sucrose concentration was maintained at 292 mM. Figure 7A comparison of the chromatograms for these two reactions is depicted in . The conversion of MSer(OH) to MSer(OH)-glu just changed from 95% in the case of 5 mM MSer(OH) to 90% in the case of 50 mM MSer(OH). This new satisfactory conversion indicates that it is possible to increase the amount of glucosylated product in batch mode reactions. It was observed that the relative ratio between Peak 1 and Peak 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).

[0081] Second screening

[0082] Based on these initial results, the extension of the products of MSer(OH)-glu glucosylation from sucrose by certain polymerases of the GH-70 family was the subject of a second screen (see Table 1). MSer(OH)-polymer products were obtained whose glucoside moieties were significantly elongated relative to those obtained by GS-D.

[0083] MSer(OH)-glu was produced under similar conditions to those described in the first screen but in greater amounts. The reaction contained 25 mM pure MSer(OH), 292 mM sucrose, 1 U.mL- 1 The reaction was carried out in a volume of 50 μL of 50 mM GS-D and 50 mM sodium acetate buffer at pH = 5.75. It 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.

[0084] Without prior purification of MSer(OH)-glu, elongation of the MSer(OH)-glu intermediate was performed by other enzymes of the same family: 5 μL of the reaction medium from step 1 was added to a new 25 μL reaction, so that the final molar concentration of MSer(OH)-glu was equal to 5 mM of free MSer(OH). To this was added an equivalent of 292 mM sucrose and 1 U.mL- 1 The enzyme selected for the second step of the enzymatic cascade (see Table 1). The new medium also contained 50 mM sodium acetate buffer at pH = 5.75. The reaction was incubated at 30°C with stirring at 500 rpm for 24 hours and then stopped by heating at 70°C for 10 minutes.

[0085] Separation of the different products (MSer(OH)-polymers) was performed in the same way as for MSer(OH)-glu in the first screen.

[0086] like Figure 8As shown, the 11 polymerases tested were able to extend MSer (OH) -glu intermediates with glucosyl unit chains of different sizes and properties according to the binding specificity of the enzymes tested. Across all reactions, at least 67.6% and up to 97.7% of MSer (OH) -glu intermediates were elongated (Table 4). Peaks 1 and 2 of MSer (OH) -glu intermediates (the vast majority of substances in the culture medium) were mainly consumed by polymerases, wherein the conversion percentage of each of the two peaks varied according to the specificity of the enzyme used (Table 4). For most enzyme cascades, Peak 2 (di-glucosylated MSer (OH)) was preferentially consumed and sometimes almost completely consumed.

[0087] The average mass of the elongated MSer(OH)-glu sugar chains (i.e., MSer(OH)-polymers) was analyzed by size exclusion chromatography (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 the MSer(OH)-glu intermediate by the DSR-OKΔ1 and DSR-S vardelΔ4N enzymes, which form much larger native polymers, HPSEC analysis was performed by combining a Shodex 805 column and a Shodex 802.5 column in series. Elution was performed with a mixture of 450 mM NaNO3, 1% v / v ethylene glycol.

[0088] The separation calibration is based on a standard range of commercial dextran (polymers of α-1,6-linked glucosyl units) of increasing size from 342 Da to 70,000 Da. The average mass MM of the product is determined at the apex of the elution peak. av The average degree of polymerization (DP) visible under UV light av ) is estimated by the following relationship: DP av =MM av / 162.

[0089] Depending on the polymerase used in this second step, chains of glucosyl units ranging from 3 to 41 units on average were identified (Table 4, Fig. 9 and Fig.10 ).

[0090] Table 4

[0091]

[0092] Peak 1 = MSer(OH)-monoglucosylated; Peak 2 = MSer(OH)-diglycosylated; ND: not determined

[0093] Notably, the DSR-MΔ1, DSR-MΔ2, and DSR-MΔ2W624A enzymes were very efficient in extending MSer(OH)-glu, achieving 80.2% and 90.4% conversion of MSer(OH)-glu, respectively. They produced dextran-type motifs consisting only of glucosyl units linked by α-1,6 bonds (the natural motif of these enzymes), thus conferring them a lower risk of anaphylactic shock. Fig.11 As shown, it is possible to control polymer size by enzyme engineering, DSR-MΔ2W624A is a variant of the DSR-MΔ1 enzyme

[24] . The W624A mutation produces MSer(OH)-polymers with a reduced average molar mass (10 units), whereas reaction with the DSR-MΔ1 enzyme produces MSer(OH)-polymers with a higher average molar mass, containing an average of 37 glucosyl units.

[0094] Low molar mass dextran are already marketed for pharmaceutical applications, especially in certain ophthalmic products. In free form, they are used as ocular lubricants or artificial tears (see https: / / www.dextran.com / application- areas / eye ). Their coupling with MSer(OH) with high UV absorption capacity will allow the development of new products, formulations, (bio)materials and pharmaceutical products combining the properties of sugars and mycosporins.

[0095] The absorption capacity of mycosporin is not affected by the grafting of polysaccharide chains. In fact, the absorbance scans between λ=200 nm and λ=800 nm of the MSer(OH)-polymer products obtained with all the tested enzymes, performed using a CARY 3500 spectrophotometer, showed that the glucosylation and elongation of the glucosyl chains on MSer(OH) do not cause the maximum wavelength λ of the absorbance. 最大 Any changes in Fig.12 ).

[0096] Finally, the glucosylation reaction was also tested on a crude extract of Lichina pygmaea, a more readily available source, obtainable from an aqueous or hydroalcoholic extract (optionally followed by lyophilization). This extract was derived directly from the lichen Lichinapygmaea, without purification but enriched in MSer(OH) up to 10% of the total extract mass. The first step of glucosylation by GS-D gave the same results as the reaction carried out on pure MSer(OH) (i.e., 95% glucosylated MSer(OH)) ( Fig.13 The second step involving the polymerase was also as efficient as when performed with purified MSer(OH) ( Fig.14, with DSR-MΔ1 enzyme). Therefore, the enzyme is not inhibited by other components of the crude culture medium. This is particularly advantageous for an industrial approach to the process, avoiding expensive purification steps of MSer(OH).

[0097] Advantages of a 2-step enzymatic cascade over a one-pot synthesis

[0098] The coordinated use of GS-D and another polymerase (one-pot synthesis or co-synthesis for simultaneous glucosylation and elongation of MSer(OH)) was evaluated and compared with the glucosylation yields obtained in a two-step enzymatic cascade.

[0099] However, if through Figure 8 (enzymatic cascade) and Fig.15 The overall MSer(OH) conversion obtained in the co-synthesis is less interesting than in the enzymatic cascade, as shown by a comparison of the (co-synthesis) and (co-synthesis). In the co-synthesis, the MSer(OH)-glu product is less consumed by the polymerase, so there is a larger MSer(OH)-glu population and a smaller MSer(OH)-polymer population.

[0100] Example 2: Properties of MSer(OH)-glu and MSer(OH)-polymers

[0101] Antioxidant properties and photostability of MSer(OH); MSer(OH)-glu and MSer(OH)-polymer products produced by the process of the invention were tested on reaction media purified from DNA present in cell extracts used for MSer(OH) glucosylation to avoid DNA interference in the phenomenon studied. The purification was performed by anion exchange chromatography using Diaion resin.

[0102] Photostability of the synthesized products

[0103] The photostability of various MSer(OH) glucosylation products was estimated. To this end, samples were irradiated in a Sun Test XLS solar simulator (Atlas Material Testing Solutions, USA). A solar filter was selected to reproduce conventional solar exposure. The concentration of the remaining molecules was monitored by measuring the absorbance at λ = 310 nm. The photodegradation kinetics obtained can be used to calculate the photodegradation quantum yield of the product. The quantum yield represents the ratio between the photodegraded molecules and the number of photons received by these molecules, and is calculated according to the following formula:

[0104]

[0105] Where r0 is the rate of photolysis (mol.L- 1 .s- 1 ), V is the volume of the solution (L), NA Avogadro constant (mol -1 ), I0 is the number of photons emitted in the MSer(OH) absorbance band, and A is the absorbance of the solutions of different samples. The experiment was performed in triplicate.

[0106] All MSer(OH) glucosylation and elongation products showed high photostability with a range of approximately 10- 5 The quantum yield ( Fig.17 ), comparable to those of some sunscreens and other mycosporins [25,26].

[0107] Therefore, the glucosylation of mycosporin has no effect on photostability. This is because the glucosylation position does not prevent the delocalization of the electron cloud, allowing the received energy to be dissipated as heat. In addition, the length of the sugar chain does not affect the photostability of the product.

[0108] However, preliminary tests have highlighted the role of DNA in the photostability of the products via the generation of reactive species. This translated into poor photostability of the products from the MSer(OH)+GS-D+DSR-MΔ2W624A cascade, for which DNA purification was the least efficient.

[0109] Finally, it appears that the product of the MSer(OH)+GS-D+DSR-OKΔ1 cascade has greater photostability than free MSer(OH). This could be explained by the physical protection provided by the long dextran chains that shield MSer(OH) from radiation.

[0110] Antioxidant Capacity

[0111] The antioxidant capacity of MSer(OH)-glu and MSer(OH)-polymer glucosylation products was determined according to the TEAC (Trolox Equivalent Antioxidant Capacity) method. The TEAC test is well established for estimating free radical scavenging properties and allows comparison of TEAC scores with a wide range of references. This spectrophotometric method is easy to implement, based on the reduction of previously oxidized ABTS+ forms

[27] . The reduction of ABTS+ causes a decrease in absorbance at λ = 734 nm and is proportional to the concentration of the antioxidant molecule. The TEAC score is established by comparing the directing agent coefficients of inhibition % = f (antioxidant concentration) with those obtained with the reference antioxidant molecule Trolox. The experiments were performed in triplicate.

[0112] The scores obtained for MSer(OH) and its glucosylated derivatives were all greater than 1 ( Fig.18). Thus, the products tested showed a strong antioxidant capacity and this capacity was maintained for all the glycosylated products obtained, in contrast to what was observed when flavonoids such as luteolin were glucosylated

[28] . The antioxidant capacity of the products derived from the enzymatic cascade without MSer(OH) was estimated to be virtually zero, demonstrating that this biological property is actually due to the mycosporin part of the molecule.

[0113] To check whether the test was performed correctly, we also checked the TEAC scores of well-known antioxidants, ascorbic acid or vitamin C. The value obtained (0.8 TEAC) is consistent with those reported in the literature

[29] . Therefore, according to our test, all products in the enzymatic cascade have a greater antioxidant capacity than ascorbic acid.

[0114] References

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[0116] 2. de la Coba, F. et al. UVA and UVB Photoprotective Capabilities ofTopical Formulations Containing Mycosporine-like Amino Acids (MAAs) throughDifferent Biological Effective Protection Factors (BEPFs). Mar.Drugs 17 (2019)

[0117] 3. Downs, CA, DiNardo, JC, Stien, D., Rodrigues, AMS & Lebaron, P. Benzophenone Accumulates over Time from the Degradation of Octocrylene inCommercial Sunscreen Products. Chem. Res. Toxicol. 34, 1046-1054 (2021)

[0118] 4. Vuckovic, D. et al. Conversion of oxybenzone sunscreen to phototoxic glucoside conjugates by sea anemones and corals. Science 376, 644 - 648 (2022)

[0119] 5. Chrapusta, E. et al. Mycosporine - Like Amino Acids: Potential Health and Beauty Ingredients. Mar. Drugs 15 (2017)

[0120] 6. Woolley, J.M. et al. Unravelling the Photoprotection Properties of Mycosporine Amino Acid Motifs. J. Phys. Chem. Lett. 9, 3043 - 3048 (2018)

[0121] 7. Carreto, J.I. & Carignan, M.O. Mycosporine - Like Amino Acids: Relevant Secondary Metabolites. Chemical and Ecological Aspects. Mar. Drugs 9, 387 - 446 (2011)

[0122] 8. Claverie, M., McReynolds, C., Petitpas, A., Thomas, M. & Fernandes, S.C.M. Marine - Derived Polymeric Materials and Biomimetics: An Overview. Polymers 12, 1002 (2020)

[0123] 9. La Barre, S., Roullier, C. & Boustie, J. Mycosporine-Like Amino Acids (MAAs) in Biological Photosystems in Outstanding Marine Molecules: Chemistry, Biology, Analysis (edited by Stéphane La Barre, J.-M.K. (editor)) 333 - 360 (Wiley-CH Verlag GmbH, 2014). doi:10.1002 / 9783527681501.ch15

[0124] 10. Shick, J.M. & Dunlap, W.C. Mycosporine-like amino acids and related Gadusols: biosynthesis, accumulation, and UV-protective functions in aquatic organisms. Annu. Rev. Physiol. 64, 223 - 262 (2002)

[0125] 11. G.A., Pfleiderer, W., P. & Scherer, S. Structure of a novel oligosaccharide - mycosporine - amino acid ultraviolet A / B sunscreen pigment from the terrestrial cyanobacterium Nostoc commune. J. Biol. Chem. 270, 853 - 8539 (1995)

[0126] 12. Matsui, K. et al. Novel glycosylated mycosporine-like amino acids with radical scavenging activity from the cyanobacterium Nostoc commune. J. Photochem. Photobiol. B 105, 81 - 89 (2011)

[0127] 13. Nazifi, E. et al. Characterization of the chemical diversity of glycosylated mycosporine-like amino acids in the terrestrial cyanobacterium Nostoc commune. J. Photochem. Photobiol. B 142, 154 - 168 (2015)

[0128] 14. Ishihara, K. et al. Novel glycosylated mycosporine-like amino acid, 13-O-(β-galactosyl)-porphyra-334, from the edible cyanobacterium Nostoc sphaericum - protective activity on human keratinocytes from UV light. J. Photochem. Photobiol. B 172, 102 - 108 (2017)

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[0131] 17. Vuillemin, M. et al. Characterization of the First α-(1 - 3) Branching Sucrases of the GH70 Family. J. Biol. Chem. 291, 7687 - 7702 (2016)

[0132] 18. Malbert, Y. et al. Engineering a branching sucrase for flavonoid glucoside diversification. Sci. Rep. 8, 15153 (2018)

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[0134] 20. Dunlap, W.C., Williams, D.M., Chalker, B.E. & Banaszak, A.T. Biochemical photoadaptation in vision: U.V.-absorbing pigments in fish eye tissues. Comp. Biochem. Physiol. Part B Comp. Biochem. 93, 601 (1989)

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Claims

1. A method for producing a glucosylated mycosporin, the method comprising the step a) of glucosylating the mycosporin from sucrose by the enzyme α-transglucosylase GS-D to obtain a glucosylated intermediate, the glucosylated mycosporin having an average degree of polymerization in the range of 1 to 2 glucosyl units.

2. The method according to claim 1, further comprising the step b) of elongating the sugar moiety of the glucosylated intermediate obtained in step a) from sucrose by at least one α-transglucosylase GH-70 different from α-transglucosylase GS-D to obtain a glucosylated mycosporin having an average degree of polymerization in the range of 3 to 1,000,000 glucosyl units.

3. The method according to claim 2, wherein step a) and step b) are performed simultaneously and comprise glucosylating mycosporin from sucrose by the enzyme α-transglucosylase GS-D and at least one α-transglucosylase GH-70 different from α-transglucosylase GS-D to obtain a glucosylated mycosporin having an average degree of polymerization in the range of 3 to 1,000,000 glucosyl units.

4. The method according to claim 2 or 3, wherein the at least one α-transglucosylase of step b) is selected from the group consisting of: dextransucrases GS-A, GS-B, GS-C, GS-FΔ1 and GS-FS, dextransucrases DSR-MΔ1, DSR-MΔ2W624A, DSR-GΔ1, DSR-SvardelΔ4N and DSR-OKΔ1, alternansucrase ASRΔ1.

5. The process according to any one of claims 1 to 4, wherein the mycosporin of step a) is selected from the group consisting of pure mycosporin, Lichina pygmaea lichen extract enriched with 0% to 10% mycosporin by weight of the extract.

6. The method of claim 5, wherein the mycosporin is mycosporin-serinol (MSer(OH)).

7. A glucosylated mycosporin obtained by the process according to claim 1, said glucosylated mycosporin having an average degree of polymerization ranging from 1 to 2 glucosyl units.

8. A glucosylated mycosporin obtained according to any one of claims 2 to 6, said glucosylated mycosporin having an average degree of polymerization in the range of 3 to 1,000,000 glucosyl units, preferably 3 to 800,000 glucosyl units.

9. A cosmetic composition comprising the glucosylated mycosporin according to claim 7 or 8 as an active ingredient.

10. The glucosylated mycosporin according to claim 7 or 8 for use as a pharmaceutical product.

11. The glucosylated mycosporin according to claim 7 or 8 is used for preventing or treating cataract, albinism, xeroderma pigmentosum, and skin cancer.

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