2.1 Superoxide dismutase

Some SODs have copper (Cu) and zinc (Zn),^{35, 36} iron (Fe),³⁷ manganese (Mn)^{36, 38} or other metals as cofactors. They are ubiquitous in eukaryotic organisms^38-40 and found in prokaryotic organisms.^{37, 41} Cu-Zn-SOD can be found in peroxisomes, cytosol^{42, 43} and, to a lesser degree, in the intermembrane space of mitochondria⁴³; MnSOD predominantly in the mitochondrial matrix,⁴³ while FeSOD exists in prokaryotic cells and some plants.⁴⁴ Cu-Zn-SOD is present in human epidermis, with higher activity in younger people and tends to decrease with age, especially after 60 years⁴⁵ and has a role in wound healing.⁴⁶ SOD antioxidant\activity appears to be higher in males than in females.⁴⁵

Higher levels of SOD are usually found in skin habitually exposed to UVR such as the face, to inhibit UVR-induced melanogenesis owing to ROS.⁴⁷ However, solar exposure appears to decrease the enzyme levels in the stratum corneum, making it more vulnerable to oxidative stress.⁴⁸ In vivo studies showed no significant variations in SOD levels between photoaged and intrinsically aged skin.⁴⁹ Similar activity levels were found in young and old murine skin. There may be a spectral effect as UVA radiation (320–400 nm) seems to inhibit SOD activity in reconstructed epidermis,⁵⁰ though earlier studies showed no effect in cultured human skin fibroblasts from healthy donors.⁵¹ UVB (290–320 nm) irradiation does not seem to affect SOD.⁵²

MnSOD is naturally evoked by cytokines in psoriatic epidermis⁵³ and modulates the inhibition of tumour growth.⁵⁴ This enzyme may accelerate wound healing⁵⁵ and its deficiency promotes cellular senescence and ageing phenotypes in skin.⁵⁶ SOD activity has also been correlated to a decrease in acne severity.⁵⁷ Grange et al. (2009) found that ROS are rapidly produced by keratinocytes upon stimulation by Cutibacterium acnes (the aetiological agent of acne, previously named Propionibacterium acnes) surface proteins and that SOD has a role in decreasing this effect.⁵⁸ In this sense, some adjuvant therapies that aim to increase SOD serum levels through coenzyme Q10 supplementation for example have been used to treat acne.³³ More specifically, SOD3 was shown to suppress toll-like receptor-2 (TLR-2), nuclear factor κβ (NF-κβ) and p38 expression in C. acnes. It also has an anti-inflammatory role reducing the expression of caspase-1 and inhibiting pro-inflammatory cytokines such as tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and IL-8. SOD is correlated with reduced lipid accumulation and expression of lipogenic regulators in sebocytes affected by C. acnes.⁵⁹ Its anti-inflammatory potential was also shown in vitro to treat atopic dermatitis in mice using mesenchymal stem cells overexpressing SOD3, as well as human keratinocytes.⁴⁷ According to Jankovic et al. (2016), SOD3 can reset the ratio between the superoxide anion and L-arginine in the skin of diabetic rats. Based on this, molecules with SOD mimetic activity have been investigated at least since 1983.⁶⁰

There is no consensus on skin and blood SOD levels in vitiligo patients. A group in India reported that significantly high SOD levels were observed in active vitiligo patients compared with stable vitiligo and healthy controls.⁶¹ Some authors have confirmed these results.⁶² Sravani et al. (2009) showed that vitiligo patients present higher SOD levels than the controls,⁶² while others have not.⁶³

An ointment formulation of SOD and interleukin-1β was found to be effective on reparative processes of burn wounds, decreasing inflammation and accelerating wound-healing.⁶⁴ This was supported in a study of experimentally scalded rats in which recombinant human CuZnSOD in three different formulations—native enzyme, enzyme-loaded gel and enzyme-loaded liposomes—reduced oedema, wound size and tissue necrosis. The liposome formulation was also efficient in ROS scavenging, accelerating wound-healing processes.⁶⁵ SOD seems to ameliorate the healing process in cold burns.⁶⁶ Similarly, hydrogels containing CuZnSOD enhanced repair of chronic wounds.⁶⁷ Other drug-delivery systems were investigated as alternatives for SOD. For example, SOD and catalase (which will be further discussed) immobilized in silver and gold nanoparticles were found to reduce UV radiation-induced oxidative stress in rat skin. Nonetheless, the immobilized enzymes had the same effect of free enzymes on the tested parameters (DNA and lipids oxidation products, total antioxidant capacity and expression of antioxidant enzymes).⁶⁸ More recently, the same enzymes immobilized on gold and silver nanoparticles were shown to enhance DNA repairing systems of rat skin after exposure to UV radiation.⁶⁹ Three-day application of free or immobilized SOD and catalase decreased the levels of the most common DNA damage products generated by UV irradiation, namely cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6–4) photoproducts (6–4PP). The enzymes were more effective when immobilized on gold nanoparticles than on silver nanoparticles.

The use of SODs in dermocosmetic products is limited by its loss of activity in the external environment. Therefore, researchers have investigated different organisms as sources of enzymes with improved stability.⁷⁰ MnSOD from Deinococcus radiodurans expressed in E. coli, for example, presents higher resistance to UVR.⁷¹ SOD expressed by different organisms and proteoforms show variation in molecular weight, kinetics and stability. Saccharomyces cerevisiae CuZnSOD, for example, has a molecular weight of 32 kDa, similar to Bos taurus CuZnSOD (32.5 kDa).^{72, 73} B. taurus CuZnSOD has a kM value of 3.55 ± 0.08 x 10⁻⁴ M,⁷⁴ with optimal temperature and pH at 25°C and 7.8–8.0, respectively.^{75, 76} The isoelectric point (pI) also varies with origin and has a wide range of values from 1.0 to 10.5. Similarly, pH stability varies from 1 to 12.⁷⁷

2.2 Catalase

This enzyme is common in aerobic organisms with a cytochrome system (with some exceptions) and has a major role in the prevention and repairing of cell damage by hydrogen peroxide excess.^78-80 Studies have shown that catalase can prevent skin ageing by reverting alterations related to MAP kinases.⁸¹ Usually, it is isolated from bacteria and mammalian tissue and/or organs where it occurs at higher concentrations, such as the liver.^{79, 80, 82} Catalase from Aspergillus niger, suggested for dermatological use, has a molecular weight of 385 kDa⁸³ and pI of 6.5.⁸⁴ Optimal temperature and pH are 60°C and 7.0, respectively, but the enzyme is stable at pHs from 4.0 to 8.3 and a temperature range from 20°C to 60°C.⁸⁵ Catalase has an important role in aged skin. Jankovic et al. (2019)⁸⁶ detected increased catalase in the skin of elderly rats, accompanied by degenerative structural changes in epidermal and dermal compartments along with low skin renewal capacity. Its activity abruptly declined through adulthood and in middle-aged rat skin but increased in the oldest groups (18-month and 21-month-old groups). Also, exposure to UVA decreases catalase activity in the stratum corneum, leading to vulnerability to oxidative stress.⁴⁸ In a more recent publication, UVB exposure was also associated with lower levels of catalase activity in the skin that may contribute to carcinogenesis.⁸⁷

Catalase has been associated with several skin conditions. In vitiligo patients, increased oxidative stress is observed in skin because of lower levels of the enzyme.⁶² Significantly down expression of catalase was also observed in fibroblasts of Hutchinson-Gilford Progeria Syndrome patients⁸⁸ and low levels were found in piebaldism patients.⁸⁹ One congenital disorder, acatalasemia or Takahara's disease, is specifically related to the lack or major reduction in catalase expression.⁹⁰

Despite this potential, only products containing pseudocatalase are currently available in the market to treat vitiligo. This is a transition metal bicarbonate complex activated by UVB radiation.^{91, 92} However, pseudocatalase formulations have not been proved sufficiently effective owing to the limited activity in comparison with the natural enzyme. According to the Braunschweig Enzyme Database (BRENDA), one catalase molecule catalyses the decomposition of 2.8 million of H₂O₂ molecules per second; corresponding to one of the highest turnover numbers (K_cat) of all enzymes.⁹³

Recently, our group showed that catalase-loaded polymersomes of Pluronic L-121 provided antioxidant skin protection in vitro (pig ear skin model), especially in the deepest layers of the skin compared to free catalase, with the added benefit of low cytotoxicity for topical application.⁹⁴ Further, enzymatic radical scavenger activity with catalase encapsulation in sugar ester vesicles was observed in in vivo experiments performed in post-burn skin, with enhanced kinetic parameters, thermal stability, reusability and protection against trypsin digestion.⁹⁵ Our group also investigated the bioconjugation of catalase with polyethylene glycol (PEG) as a potential treatment for vitiligo and UVR-induced skin damag.e⁹⁶ We showed that PEGylation increased enzyme stability and provided extra safety since the PEGylated enzyme concentrates in the external layers of the skin, preventing permeation.

2.3 Peroxiredoxin

Prx is found in plants,¹⁰⁰ bacteria,¹⁰¹ mammals¹⁰² and other organisms. It presents several isoforms such as Prx I, II, III,¹⁰³ IV,⁹⁹ V¹⁰⁴ and VI.⁹⁸ According to the literature, purified Prx from several species,—for example, yeast and bacteria—does not contain common redox centres such as metal ions.^{97, 105, 106} The mechanism of action is correlated to the disulphide linkage in Cys47, which can be oxidized by hydrogen peroxide.^{105, 107} Despite the higher expression in mammal tissues, Prx presence in skin was only detected in 1999, more precisely different isoforms were found in rat skin and hair follicles.^{108, 109} Since then, it has been associated with protection against UVB-induced apoptosis of epidermal keratinocytes.¹¹⁰ Increased levels of Prx II were detected in psoriasis and associated to biological regulation, defence and inflammatory responses, and ROS regulation.¹¹¹

Experiments in mice indicate Prx II has an important role in promoting the growth of primary mesenchymal stem cells, which are important to dermal thickness and wound healing.^{101, 112} Also, in vivo and in vitro studies suggest that Prx I deficiency in mice is correlated with skin cancer.¹¹³ Another study with Prx VI indicates a dual role in skin cancer development: a tumour-preventive effect, but acceleration of tumour progression at higher levels of expression.¹¹⁴ Transgenic mice overexpressing the human Prx IV gene have shown fibroblast proliferation and migration, especially in adult and aged mice, also more resistant to H₂O₂ cytotoxicity.¹¹⁵ In addition, Prx IV can protect the skin during skin cancer treatment.¹¹⁶ It improves the recovery after membrane lipid peroxidation.⁹⁸

Human PrX is about 34 kDa and optimal temperature is 37°C.^{117, 118} This enzyme is stable to 60°C¹¹⁷ while stability pH can vary significantly from pH = 4.5 for Mycobacterium tuberculosis¹¹⁹ to 7.8–10.2 for Taiwanofungus camphoratus.¹²⁰

2.4 Methionine sulfoxide reductase

Methionine sulfoxide reductases (Msr) (EC 1.8.4.11) are part of mammalian antioxidant defence¹²¹ and can be divided in two monomeric classes: MsrA and MsrB.^{122, 123} They catalyse the reduction of free methionine and methionine residues in peptides and proteins, repairing methionine oxidation by H₂O_2.¹²⁴ Therefore, Msr are important not only for antioxidant defence but also for cellular regulation.¹²⁵ MsrA is found in epidermis, hair follicles and eccrine glands as sebaceous glands; it is related to melanogenesis, DNA repair systems and photoprotection.¹²⁶ MSRB2 expression is found in melanocytes, while both MSRB1 and MSRB3 are found to be expressed by vascular endothelial cells.¹²⁷

According to the literature, MsrA knockout mice presented enhanced sensitivity to oxidative stress, shorter lifespan, atypical walking pattern and higher tissue levels of oxidized protein, indicating that MsrA plays an important role in ageing.^{121, 128} On the contrary, MsrA gene overexpression in Drosophila sp. increased lifespan.¹²⁵ Vitiligo patients were observed to have low levels of MsrA and MsrB,¹²⁹ and research indicates that both enzymes are deactivated at high concentrations of hydrogen peroxide.¹³⁰

Msr activity may influence cutaneous ageing and carcinogenesis,¹²⁷ and the topical application of MsrA has been shown to protect against UVB-induced damage in skin models.¹³¹ Despite the dermatological potential, studies in the literature about Msr are scarce.^{122, 132} The optimal pH is 7.4 for the enzyme from B. taurus and H. sapiens.¹³³ Its isoeletric point is 5.7 in Nicotiana tabacum¹³⁴; temperature stability is 53.6°C for MsrA from Treponema denticola¹³⁵ and 85°C from Thermococcus kodakarensis.¹³⁶ The H. sapiens MsrA has a molecular weight of approximately 23–25 kDa,^{124, 137} while the enzyme from M. musculus is 32 kDa138 and from B. taurus is 25 kDa.¹³⁷

3.1 Keratinase

Keratin is a sulphur-containing fibrous and structural polypeptide and an intrinsic part of the epidermis and appendages including hair, nails, feathers, wool, as well as animal parts like horns, hooves and squama.¹⁴⁵ It presents a rigid structure owing to the disulphide crosslinks along the polypeptide chain.¹⁴⁶ Keratin is an abundant biomass in nature, only surpassed by chitin and cellulose.¹⁴⁷ It is considered a major environmental problem due industrial waste.¹⁴⁸ Microbial proteolytic enzymes responsible for degradation of keratin degradation and other insoluble substrates are called keratinases (EC 3.4.4.25).¹⁴⁹ Common sources of these extracellular enzymes are actinomycetes, bacteria and fungi.¹⁵⁰

Microbial keratinases have invaluable potential in different processes including the production of food, organic fertilizers, detergents, cosmetics, as well as medicine. Keratinases can be used to treat skin conditions involving hyperkeratinization such as acne, nail disorders, and removal of corns and calluses.¹⁵¹ Hyperkeratinization is common in acne patients, in which keratinized cells accumulate in the sebaceous canal and develop into comedones or acne lesions.¹⁵² Corns and calluses are common on toes and feet, due to repetitive friction and pressure, they are characterized as hyperkeratotic lesions.¹⁵³ This process is a natural and protective response from the body to stressors; however, it usually causes discomfort.¹⁵⁴ Keratinases could replace the salicylic acid, commonly used in the treatment of calluses, in a sustainable manner.¹⁵⁵ Hyperkeratosis is also present in epidermolysis bullosa simplex with mottled pigmentation—a disease characterized by punctate hyperkeratosis of the palms, soles and dystrophic nails¹⁵⁶—and other conditions such as bullous congenital icthyosiform erythroderma, ichthyosis bullosa of siemens, epidermolytic palmoplantar keratoderma, monilethrix, etc.¹⁵⁷ Keratinases have also been used in topical preparations for hair removal.¹⁵⁸ Sanghvi et al. (2016) investigated the dehairing efficiency of keratinase from B. subtilis DP1and the keratinase-based cream was more effective in hair removal than the chemical-based counterpart.¹⁵⁹ Keratinase from Bacillus licheniformis is present in commercial products for the treatment of nail disorders and removal of corns and calluses.¹⁵¹ Also, keratinase from Paecilomyces lilacinus was investigated in cryogel patches for antimicrobial treatment.¹⁶⁰

Most of the documented keratinases are monomeric enzymes with molecular weight up to 58 kDa.¹⁴⁷ The enzyme from Bacillus licheniformis presents molecular weight and pH stability of 31.4 kDa and 8.5, respectively. It is stable at pH 5–12, and the optimal temperature is 50°C–55°C.¹⁶¹ Keratinase from Doratomyces microsporus presents isoelectric point at 9, while the optimal pH and temperature are 8–9 and 50°C, respectively. The enzyme presents 100% relative activity when human stratum corneum is used as a substrate in comparison with 22% in human nails and 14% in porcine nails.¹⁶² The k_M value for the enzymes from Paecilomyces marquandii and Doratomyces microsporus are 0.17 ± 0.02 and 1.03 ± 0.17 mM, respectively.¹⁶³

3.2 Papain

Papain (EC 3.4.22.2) is a cysteine hydrolase globular protein extracted from the papaya fruit latex (Carica papaya) with molecular weight of approximately 23.4 kDa and a pI value of 4.4.¹⁶⁴ Papain is heat stable at temperatures from 60 to 80°C, the highest being best. Optimal pH is 6–9 but the enzyme is stable from pH 6 to 11.¹⁶⁵ The presence of four disulphide bridges contributes to the preserved stability and activity under harsh conditions of temperature and pH.¹⁶⁶ The catalytic site is located between two protein domains, an ɑ-helix and an antiparallel β-sheet, and is composed of cysteine-25 and histidine-159.¹⁶⁶

Papain is commonly used in some cultures and countries for wound healing since the 1950s owing to its effectiveness and low cost.^{167, 168} The easiest and cheaper topical use of papain consists of papaya pulp directly in the wound,^{169, 170} although the most common formulation corresponds to a papain dried extract.¹⁶⁷ The enzyme powder is dissolved into distilled water, applied to the wound and protected with a dry gauze. Some care must be taken to avoid metallic material near the enzyme that could result in oxidation. Nonetheless, papain home-based preparations are considerably unstable.¹⁶⁹ The fermented phytopreparation from papaya is also used topically to treat burns in most regions of Africa.^{170, 171} Research indicates that papain modified with polyethylene glycol presents increased stability in comparison with free papain.¹⁷² Papain holds several beneficial properties such as degradation of proteins including collagen,¹⁷³ anti-inflammatory activity¹⁷⁴ and is an antioxidant.^{174, 175} In 1993, Velasco investigated a gel formulation of 0.4% w/v of papain in patients and found good therapeutic effect, easy application, reduced cost, high enzymatic stability and good wound adherence.¹⁷⁶ In fact, papain is extensively used for wound healing not only on viable tissue but also on necrotic tissue, in patients with diabetic ulcers, pressure ulcers and venous ulcers. In all cases, treatment is associated with an improvement in wound healing and reduction of the wound area.^177-181 Liposome-formulated papain has been shown to present advantages over the free protein. It not only protects the enzyme but also promotes higher skin drug deposition compared to free papain. This formulation inhibited hypertrophic scar in rabbit ears, by inhibiting angiogenesis and fibroblast invasion, and reducing collagen synthesis.¹⁷³ A poly (ɣ-glutamic acid) (ɣ-PGA) chitooligosaccharide hydrogel with papain was also investigated to treat hypertrophic scars in rabbit ears. The hydrogel inhibited excessive collagen deposition during wound healing, while preserving enzyme activity.¹⁸² Papain formulations can also be used as depilatory agents in a safe way.^{183, 184}

According to FDA, approximately 35 unapproved papain-based topical products were on the USA market in 2015 and the Agency demonstrated concern regarding the safety of these products since several reports of adverse effects related to hypersensitivity were registered.¹⁸⁵ Based on that, research on the safety and delivery of papain increased. For example, biocompatible wound dressings incorporating papain were investigated and found to preserve enzyme stability over 28 days¹⁸⁶ and a papain-based wound cleanser exhibited accelerated healing process in rats (97% in comparison with untreated skin), promoting complete re-epithelization and collagen deposition under the wound.¹⁸⁷ Besides, papain can also be used as absorption enhancer in human skin.¹⁸⁴

3.3 Collagenase

Collagenase (EC 3.4.21.32) is a serine endopeptidase, a matrix metalloproteinase produced by fibroblasts.^{188, 189} As others, matrix metalloproteinases are responsible for degrading collagen to help tissue renewal.¹⁹⁰ This enzyme is essential to natural wound healing and is present at high concentrations in early proliferative phases of inflammatory wound, decreasing during remodelling of scar tissue and after complete epithelialization.^{164, 191} Collagenase activity can be both desirable and not. Matrix metalloproteinase-1 (MMP-1) is the main collagenase present in the skin, and its upregulation is triggered by repeated sun UVB exposure^{192, 193} and UVA radiation.^{194, 195} This increase is responsible to accumulate degraded fibrillar collagens (types I and III in skin), characterizing the photoageing process—wrinkling of photodamaged skin,¹⁹⁶ since collagen is related with skin elasticity, hydration and its absence can be observed with the presence of fine lines and wrinkles.¹⁹⁷ MMP-1 is the major collagenase implicated in wound healing, enabling keratinocytes migration to re-epidermisation process.¹⁹⁸ Further, clinical studies showed that exogenous collagenase in ointment formulations may prevent burn wound conversion, diminish hospital stay, overall need for surgery and blood transfusions.¹⁹⁹

The enzyme is commercially available in several commercial formulations, all derived from Clostridium histolyticus. It is used to promote debridement of necrotic tissue in burns, skin ulcers^{200, 201} and to treat Dupuytren's contracture,^{202, 203} Peyronie's disease^{204, 205} and cellulite.²⁰⁶ Collagenase can be used not only in adults for wound debridement but also in infants and neonates in a safe and effective way.²⁰⁷ Even in eschar tissue, contaminated or not, it has a great potential to debride most chronic wounds.^{200, 208, 209} It can also be used to treat fibrosis²¹⁰ and to reduce keloids.²¹¹ Collagenase formulated in polymeric nanoparticles of acrylamide, aminoethyl methacrylate, ethylene glycol dimethacrylate and methylenebis(acrylamide) has been investigated to treat fibrosis.²¹⁰ A single dose of this formulation substantially decreased the collagen area in comparison with the same dose of free collagenase in mice.

Collagenase from Clostridium histolyticus presents optimal activity at pH 7.4 in phosphate buffer, and it is stable at pH 5.6.²¹² The molecular weight is approximately 76–96 kDa.²¹³ The k_M value for rat type I, bovine type II and human type III collagen, is 4.0, 4.0 and 5.0 μM, respectively.^213-264

4.1 T4 endonuclease V

T4 endonuclease V (T4N5) (EC 3.1.21.7) was originally isolated from Escherichia coli infected with T4 bacteriophage.²¹⁷ This enzyme can cleave the nucleotides in the internal regions of the DNA, specifically the UVR-induced CPDs, initiating their removal, which when unrepaired contribute to mutations that result in actinic keratoses and keratinocyte cancers. T4N5 repairs CPD by catalysing two reactions, the first uses glycosylase, which releases one thymine (from the CPD). In addition, another reaction involving AP lyase incises the phosphodiester backbone at the site of the missing base, causing a single stranded break.²¹⁸ Its k_M value for 49-mer oligonucleotide is 0.004–0.024 mM,²¹⁹ the molecular weight is 20 kDa,²²⁰ stability pH ranging from 6 to 9 and optimal temperature ranging from 70 to 90°C.²²¹ T4N5 from Pyrococcus furiosus has a pI value of 9.8.²²²

Early work with T4 endonuclease V showed that topically applied T4N5-loaded liposomes efficiently removed DNA photoproducts in human and mouse skin and reduced the incidence of skin photocarcinogenesis in mice.^{223, 224} In clinical trials, topical application of T4 endonuclease V-loaded liposomes reduced the incidence of basal cell carcinomas by 30% and that of actinic keratoses by at least 68% in xeroderma pigmentosum patients. Adverse effects were rare and allergic or irritant contact dermatitis not observed.²²⁵

4.2 Photolyase

Photolyases (EC 4.1.99.13) repair DNA photodamage by employing the energy of blue light (350–500 nm).^{226, 227} They are found in bacteria, plants, reptiles, amphibians and marsupials but not in placental mammalian cells. Two types of photolyases have been identified with different substrate specificity: CPD and (6–4)-photolyase.^{226, 227} The observation that bacterial DNA repair enzymes could be used to repair damage in human cells first happened nearly 45 years ago.²¹⁸ In 2015, Aziz Sancar was recognized with the Nobel Prize in Chemistry for his work elucidating the DNA repair mechanisms by photolyase.²²⁸ The enzyme binds to the dimer with high affinity and specificity, which can occur in the dark. Following, photoreduction or dimer reversal takes place but only in the presence of light, specifically blue light. Photolyase presents a non-covalently bound chromophore/cofactor that can be a folate or a deazaflavin, and a two electron-reduced FAD as a catalytic cofactor.²²⁹

As mentioned above, photolyase is not innately present in human cells; the homologous human gene has been changed by evolution and we use the blue light for the circadian rhythm.²¹⁸ Humans have other DNA repair mechanisms, such as the nucleotide excision repair (NER) pathway capable of repairing UVR-induced DNA damage. Nonetheless, this mechanism is more effective in recognizing and repairing pyrimidine–primidone type lesions and relatively inefficient in repairing CPDs.²²³ According to studies in photolyase-transgenic mice, the enzyme can remove CPDs from the genome of basal keratinocytes and substantially diminish the incidence of skin tumours; however, it does not affect the UVB-mediated immunosuppression.²³⁰ On the other hand, studies in human skin show that photolyase topical application induces dimer reversion and leads to immunoprotection.^{231, 232} The possibility of incorporating photolyase into a sunscreen and therefore repair DNA photodamage is exciting; in the words of Stege et al. ‘Exogenous application of photolyase differs from conventional photoprotection through its ability to remove damage that has already occurred. This enzyme therapy approach could thus be ideally combined as an after-sun strategy with conventional sunscreens to provide photoprotection and repair at the same time’.²²⁴

Photolyases are monomeric proteins of 50–70kDa.¹⁷³ Regarding the activity and stability parameters, the enzyme from Agrobacterium fabrum presents an optimal temperature of 20–25°C and stability at pH 7–7.5.^{233, 234} A pI value of 8.86 is reported for the Chlamydomonas sp. enzyme.²³⁵ Measuring the kinetic parameters of photolyase is not an easy task and literature data are scarce. Nonetheless, a k_M value of 32 μM is reported for E. coli photolyase.²³⁶

Different studies show the advantages of photolyase compared to traditional sunscreens. A study of a sunscreen formula containing photolyase applied prior to UVR exposure over a week resulted in 93% reduction of CPD formation while the traditional sunscreens resulted in a 62% reduction.²³⁷ The treatment of human skin with the enzyme before UVR exposure reduced telomere shortening associated with premature senescence of cells.²³⁸ Photolyase-based products are already available on the market in sunscreen formulations and after-sun lotions. Nonetheless, none of them contains the purified enzyme; they combine plankton extract (source of photolyase) encapsulated into liposomes with UV filters. As a result, the real concentration of enzyme is not known and may even vary from batch to batch. Recent studies debate the beneficial effects of the plankton extract in the treatment of actinic keratosis, a benign skin neoplasm induced mainly by UV radiation and with the potential to transform into carcinoma.^{223, 239} Puig et al. (2019) cite 11 clinical studies with a total of 228 volunteers and demonstrate the beneficial effect of a photolyase commercial formulation in the treatment of mild to moderate actinic keratosis.²³⁹ Nonetheless, it is important to keep in mind that all clinical trials were opened, and the number of patients reduced. Moscarella et al. (2017) conducted a parallel randomized double-blind clinical trial employing a photolyase commercial formulation and a commercially available sunscreen SPF (sun protection factor) 50 as a comparator. Thirty-six patients were investigated and after 6 months, both groups showed significant improvement in actinic keratosis lesions, but the effect was more pronounced in the group treated with photolyase.²⁴⁰

Yarosh et al. (2019) evaluated 125 scientific publications over the last 20 years with the aim of answering the most prominent questions about the effectiveness of DNA repair enzymes for active photoprotection. The authors concluded that the topical use of DNA repair enzymes effectively promotes the removal of DNA damage, reduces the appearance of new manifestations of actinic keratosis and promotes regression of existing lesions. Additionally, according to the authors, literature data support photolyase potential of preventing photoageing and skin cancer, but further studies could either strengthen the theory or refute it.²¹⁸ One of the factors that can make it difficult to evaluate the real potential of photolyase refers to the use of the plankton extract, without effective control of the enzyme concentration. In this sense, our group has been working on the biotechnological production of photolyase to be incorporated in topical formulations.