WO2022115037A1 - Hair components and methods of isolating them - Google Patents

Abstract

The invention relates generally to methods of isolating hair components, such as cuticle cells, keratin, keratinassociated proteins (KAP) and melanosomes, from hair using a serine protease. The cuticle cells, keratin, KAP and melanosomes isolated using the methods and their applications are also provided. In one embodiment, the serine protease is isolated from a bacterium of Bacillus genus. In another embodiment, the bacterial serine protease is subtilisin 147.

Description

HAIR COMPONENTS AND METHODS OF ISOLATING THEM

CROSS-REFERENCES

[0001] This application claims priority to Singapore patent application 10202011688P, filed on 24 November 2020 and Singapore patent application 10202012926P, filed on 22 December 2020, which are expressly incorporated herein by reference in their entirety, with particular reference to the figures, legends, and claims therein.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of molecular biology. In particular, the present invention relates to the isolation of hair components, such as melanosomes, keratin, keratin- associated proteins (KAP) and cuticle cells.

BACKGROUND

[0003] Hair components have shown great potential in biomedical applications due to their biocompatibility, biodegradability, and more importantly their ability to promote cell adhesion and proliferation. Hair consists of a few main components: cuticle cells, keratin (intermediate filaments proteins), keratin-associated proteins (KAP, matrix proteins) and melanosomes.

[0004] The outer cuticle cells form a scaly layer and possess hydrophobicity, impermeability, chemical resistance, thermal stability, and natural antibacterial properties that function to protect the hair from physical and chemical damage. The unique properties of cuticle cells as a biomaterial promise the potential for various applications.

[0005] Beneath the cuticle layer, the cortex is composed of many spindle-shaped cells that contain two main groups of proteins: keratins and keratin-associated proteins (KAP). Keratins have emerged recently as alternative materials to be used as bioactive coatings to improve the cytocompatibility of surfaces while the abundance of cysteine in keratin-associated proteins (KAP) facilitates disulfide bonding, providing a tough and durable structure to withstand external mechanical forces. Both keratin and keratin-associated proteins (KAP) exhibit prospects for biomedical applications.

[0006] Melanosomes are melanin-rich structures that are tightly held by the keratin filaments within hair shaft. Melanins have unique physicochemical properties in vitro, including broadband light absorption, intrinsic free radical quenching, efficient non-radiative energy transfer and humidity dependent electronic semiconductivity.

[0007] Methods attempting to harvest the hair components have been developed in the past. However, they often result in a limited yield with non-collectable wastage of other useful components. Thus, there is a need for the development of comprehensive methods that are compatible for the isolation of multiple hair components such as cuticle cells, keratin, keratin-associated proteins (KAP) and melanosomes.

SUMMARY OF INVENTION

[0008] In one aspect, the present disclosure refers to a method of isolating melanosomes from hair, wherein the method comprises: exposing hair which has undergone a process of removal of keratin and keratin-associated proteins (KAP) to a solution comprising a bacterial serine protease for at least 0.5 hours to release melanosomes into the solution; and isolating melanosomes from the solution.

[0009] In another aspect, the present disclosure refers to a method of isolating melanosome, keratin, and keratin-associated proteins (KAP) from hair, wherein the method comprises: a) mixing hair in a KAP extraction solution to release the KAP into the KAP extraction solution; b) separating hair and the solution of a); and isolating the KAP from the solution; c) mixing the hair from b) with a keratin extraction solution to release keratin into the keratin extraction solution; d) separating hair and the solution of c); and isolating the keratin from the solution; e) exposing the hair from d) to a solution comprising a bacterial serine protease for at least 0.5 hours to release melanosomes; and f) isolating melanosomes obtained under e).

[00010] In one example, the methods disclosed herein further comprising a process of removal of keratin and keratin-associated proteins (KAP) prior to isolating melanosomes from hair, wherein the process comprises: a) mixing hair in a KAP extraction solution to release the KAP into the KAP extraction solution; b) separating hair and the solution of a); and removing the solution containing KAP; c) mixing the hair from b) with a keratin extraction solution to release keratin into the keratin extraction solution; and d) separating hair and the solution of c); and removing the solution containing keratin.

[00011] In another example of the methods disclosed herein, the bacterial serine protease is isolated from a bacterium of Bacillus genus. In yet another example, the bacterial serine protease is a subtilisin. In yet another example, the bacterial serine protease is subtilisin 147. In yet another example, the bacterial serine protease used in the solution comprising the bacterial serine protease is about at least 125 Anson Unit per gram of hair. In yet another example, the exposure of hair to the solution comprising the bacterial serine protease is at a temperature of 30-70 °C.

[00012] In a further example, the solution comprising the bacterial serine protease can be reused for exposure of a different batch of hair. In another example, the hair is delipidised hair.

[00013] In one aspect, the present disclosure refers to the isolated melanosomes produced by the method disclosed herein, wherein the melanosomes are characterized by non-aggregated appearance.

[00014] In another aspect, the present disclosure refers to a method of isolating cuticle cells from hair, wherein the method comprises: a) exposing hair to a solution comprising a serine protease for 2 to 4 days; b) collecting a foam layer formed on the top of the solution of a); and c) isolating cuticle cells from the collected foam layer of b). [00015] In one example of the method disclosed herein, the serine protease is isolated from a bacterium. In another example, the bacterium is of Bacillus genus. In another example, the serine protease is a subtilisin. In another example, the serine protease is subtilisin 147. In another example, the subtilisin 147 used is 320-1280 Anson Unit per gram of hair. In yet another example, the exposure of hair to the solution comprising the serine protease is at a temperature of 50-60°C.

[00016] In another example, the solution comprises subtilisin 147 as sole enzyme and exposure of hair to the solution is for about 3 days. In yet another example, the solution comprising the serine protease can be reused for exposure of a different batch of hair. In yet another example, the hair is delipidised hair.

[00017] In another aspect, the present disclosure refers to an isolated intact cuticle cell produced by the method disclosed herein, wherein the cuticle cells are characterized by intact flaky appearance.

[00018] In yet another aspect, the present disclosure refers to a film prepared from the cuticle cells isolated in the method disclosed herein, or the isolated intact cuticle cells as disclosed herein.

[00019] In yet another aspect, the present disclosure refers to a method of removing cuticle cells from hair, wherein the method comprises: a) exposing hair to a solution comprising L-cysteine, and at least one or more serine protease for about 4 hours; and b) removing cuticle cells from the solution of a). [00020] In one example of the method disclosed herein, the at least one or more serine protease is isolated from a bacterium. In another example, the bacterium is of Bacillus genus. In another example, the at least one or more serine protease is a subtilisin. In yet another example, the at least one or more serine protease is selected from subtilisin 147 and/or subtilisin 309. In yet another example, the subtilisin 147 used is 320-640 Anson Unit per gram of hair. In yet another example, the concentration of subtilisin 309 used is at least about 320 Anson Unit per gram of hair. In yet another example, the concentration of L-cysteine is about 1-5% (w/v). In yet another example, the exposure of hair to the solution is at a temperature of 50-60°C. In yet another example, the solution comprising L-cysteine and at least one or more serine protease can be reused for exposure of a different batch of hair. In yet another example, the hair is delipidised hair.

[00021] In a further example of the method disclosed herein, the hair is an animal hair with scaly structure. In another example, the hair is keratinous hair. In another example, the hair is a human hair. In yet another example, the hair is wool.

BRIEF DESCRIPTION OF THE DRAWINGS

[00022] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which: [00023] Figure 1 describes the morphology of hair that are untreated (Figure (la)), treated with esperase (Figure (lb)) and savinase (Figure 1(c)) according to methods described herein. Lower panels under Figures 1(a)- Figure 1(c) show a zoomed-in image of respective area marked by rectangle in the upper panels of Figures 1(a)- Figure 1(c), respectively. As shown in panels Figure 1(b) and Figure 1(c), the morphology of hair surfaces changes after treatment with serine proteases, esperase or savinase, compared to Figure 1(a) the untreated hair surface. The differences are clearly observed in the lower panels, where the cuticle scales of both Figure 1(b) and Figure 1(c) are exfoliated, losing the typical overlapping arrangement with high density of cuticle cells of untreated hair as shown under Figure 1 (a). The schematic drawing of Figure 1(d) shows hierarchical structure of a hair.

[00024] Figure 2 uses the scanning electron microscopic (SEM) images of hair to show the degree of cuticle isolation in hair treated with different dosages of a serine protease. In this case, the enzymatic concentration of esperase is 8 Anson Unit/pL or 8 Novo protease unit (NPU)/pL. The dosages of esperase used for 2.5 gram of hair sample are: 100 pL (i.e. 320 Anson Units/ gram of hair; Figure 2(a)), 200 pL (i.e. 640 Anson Units/ gram of hair; Figure 2(b)), 300 pL (i.e. 960 Anson Units/ gram of hair; Figure 2(c)), 400 pL (i.e. 1280 Anson Units/ gram of hair; Figure 2(d)) for 72 hours, at conditions as described herein. When the dosage of esperase is, for example, 100 pL (i.e. 320 Anson Units/ gram of hair) , cuticles on the surface of hair shaft are dislodged but some cuticle cells still remain on the hair surface, resulting in a rough surface similar to that in Figure 1(b). Hair without cuticles is shown in the inset image in Figure 2(a). With increasing esperase concentration, the proportion of descaled hair with smooth surfaces greatly increased (Figure 2 (b), Figure 2 (c), Figure 2 (d)). When the dosage of esperase is above 300 pL (i.e. 960 Anson Units/ gram of hair), almost all of the hair shafts present a smooth surface that is void of cuticles.

[00025] Figure 3 provides examples showing the typical morphology of cuticle cells isolated from human hair using esperase as an exemplary serine protease: What is shown in Figure 3(a) is a foamy top layer formed after enzymatic treatment that comprises large number of isolated cuticle cells which can be separated from the mixture after centrifugation. Figure 3(b) shows lyophilized cuticle cell powder. Optical microscopy images of Figure 3(c) and Figure 3(d) show isolated cuticle cells suspended in water and cuticle cells dispersed in ethanol, respectively. Hair shafts as shown in Figure 3(e) and Figure 3(f) before and after treatment with esperase, respectively, shows morphological changes resulting from the loss of overlapping scaly patterns. The scanning electron microscopy (SEM) images in Figure 3(g) of lyophilized isolated cuticle cells shows a homogenous population of intact flaky cuticle cells with consistent shapes and sizes. In a series of zoomed-in panels of Figure 3(h) to Figure 3(j) showing cuticle film surface (70x, 500x, 2000x magnification), the cuticles cells are shown to have polygonal shapes with similar dimensions. In addition, both surfaces of each cuticle cell are distinguishable: the upper side is smoother than the underside, resulting in different contrast under scanning electron microscopy (SEM) as highlighted in Figure 3(j).

[00026] Figure 4 shows transmission electron microscopy (TEM) images of the cross-sections of delipidised human hair and lyophilized hair cuticle cells, in Figure 4(a) and Figure 4 (b), respectively. The cross-sections show three distinctive layers of each hair cuticle cell before enzymatic digestion, which include A-layer, exocuticle layer and endocuticle layer. Figure 4(c) is a schematic showing the mechanism of cuticle isolation using serine protease.

[00027] Figure 5 shows scanning electron microscopy (SEM) images of surface morphology of hair shafts after removal of cuticles by treatment with combined esperase and L-cysteine (Fl-EL) in Figure 5(a); and combined savinase and L-cysteine (Fl-SL) in Figure 5(b). Dispersed cortical cells released from hair shafts are observed after treatment with L-cysteine in combination with exemplary serine proteases. Flowever, the cuticles are degraded and cannot be collected in this method. Thus, the combination of L-cysteine and enzyme is more suitable for removal but not the isolation of hair cuticles. [00028] Figure 6 shows fourier transform infrared spectroscopy (FTIR) spectra of delipidised hair, three descaled hair samples (Fl-E: hair treated with esperase, Fl-EL: hair treated with L-Cysteine in combination with esperase, Fl-SL: hair treated with L-Cysteine in combination with savinase) and isolated cuticles. Since hair mainly consists of keratins, all the IR spectral data show characteristic absorption bands ascribed to polypeptide units (-CONF1-), namely, amide A (3000-3600 cm ¹), the amide B (3056-3075 cm¹), the amide I (1700-1600 cm¹), II (1580-1480 cm ¹) and the amide III (1300- 1220 cm ¹) absorption peaks. Compared to intact hair and three descaled hair samples, the cuticle shows much higher intensity at 1075 cm ¹ and 1041 cm ¹, indicating the cuticle contains a higher content of oxidized cysteine residues resulting from the weathering or exposure to the sunlight (UV), which is consistent with the function of cuticle layer in hair.

[00029] Figure 7 shows solid state ¹³C nuclear magnetic resonance (NMR) spectra and peak fitting of Carbonyl-group (190-160 ppm) in Figure 7(a) and Figure 7(b), respectively, of delipidised hair and isolated cuticle and three descaled hair samples (Fl-E: hair treated with esperase, Fl-EL: hair treated with L-Cysteine in combination with esperase, Fl-SL: hair treated with L-Cysteine in combination with savinase). Flair is mainly composed of proteins (90%) and minor components including lipids (5%) and melanin (1-3%). Therefore, the solid state ¹³C NMR spectrum of hair is essentially dominated by the signals of the most abundant proteins including keratins and keratin-associated proteins (KAPs). The contents of the a-helix structure of intact hair are about 48% and are about 54-61% in the three descaled hair samples, which is about 6%- 13% higher in the descaled hair samples than that of intact hair. The cuticle contains about 73.78% of b-sheet and random coil structure.

[00030] Figure 8 shows a thermal behaviour analysis on intact hair, cuticle, and three descaled hair samples. Thermogravimetric analysis (TGA) of Figure 8(a) and Derivative thermogravimetric (DTG) analysis of Figure 8(b) both show two distinct stages of weight loss, which are attributed to loss of adsorbed water and lateral chain destruction of hair proteins, respectively. Descaled hair showed similar thermal stability while cuticle cells show higher stability due to a higher proportion of b-sheet structure that enhances more intermolecular interactions. Figure 8(c) shows the differential scanning calorimetry (DSC) curves of intact hair and isolated cuticle and three descaled hair samples (Fl-E: hair treated with esperase, Fl-EL: hair treated with L-Cysteine in combination with esperase, Fl-SL: hair treated with L- Cysteine in combination with savinase). The endotherm peak which occurs at around 100 °C is related to the evaporation of water. Due to the impermeability of cuticle to water, there is little bounding water within the cuticle sample, and therefore, the endotherm peak in cuticle sample is above 100 °C.

[00031] Figure 9 shows an estimation of extractability and characteristics of the extracted proteins. In Figure 9(a), the extractability of hair, cuticle, and three descaled hair samples is examined by calculating the percent weight loss during standard KAP and keratin extraction (Fl-E: hair treated with esperase, H- EL: hair treated with L-Cysteine in combination with esperase, H-SL: hair treated with L-Cysteine in combination with savinase). The H-E shows a lower extractability than that of H-EL and H-SL due to the disrupted cortex structures of H-EL and H-SL from the removal of cortical cells. Figure 9 (b) is electrophoresis of extracted proteins (KAPs, and keratin) stained with Coomassie blue. Figure 9(c) shows extracted proteins (KAPs, and keratin) immunoblotted with 8 keratin antibodies (K31, K33, K35, K32, K86, K85, K81, K82) against hair keratins. Keratins extracted from hair, H-E, H-EL, and H-SL show similar characteristic bands at about 45-50 kDa (type I keratin) and 50-60 kDa (type II keratin), respectively, suggesting that keratin structure is preserved in hair samples treated with different serine proteases.

[00032] Figure 10 shows a schematic summary of the procedure to isolate melanosomes from hair using bacterial serine protease after keratin and keratin-associated proteins (KAP) extraction.

[00033] Figure 11 describes the morphology of melanosomes isolated from hair using bacterial serine protease as referred to herein. Figure 11 (a) and Figure 11 (b) show scanning electron microscopy (SEM) images of hair melanosomes obtained using the method disclosed herein at different magnifications. Morphological observations of the melanosomes showed that the isolated melanosomes are separate from one another with well-defined rod shapes and consistent lengths and diameters. Figure 11(c) and Figure 11(d) show transmission electron microscopy (TEM) images of hair melanosomes at different magnifications. The morphology and size recorded are consistent with native melanosomes reported previously.

[00034] Figure 12 shows transmission electron microscopy (TEM) images of longitudinal and transverse sections of melanosomes isolated from human hair using an exemplary bacterial serine protease (esperase) in Figure 12(a) and Figure 12(b), respectively (MS: membrane-like structure; V: spherical vesicles with a diameter of 10-20 nm; MT: inner matrix with sheet-like structures; SA: sheet like array structure in the cross-section). Ultra-structures observed are consistent with those in native melanosomes reported in the literature. The transmission electron microscopy (TEM) images show the presence of spherical nanoparticles within melanosomes, as reported before. This observation demonstrates that the integrity of the melanosomes isolated from hair using the method described herein is well preserved.

[00035] Figure 13 shows scanning electron microscopy (SEM) images of isolated melanosomes using the method disclosed herein compared to melanosomes isolated using conventional methods. Figure 13(a) to Figure 13(c) are representative images of melanosomes isolated using the method disclosed herein. The isolated melanosomes are characterised by non-aggregated appearance. Figure 13(a) to Figure 13(c) are isolated melanosomes washed and collected with different centrifugation speeds: Figure 13(a): 2000 rpm, Figure 13(b): 3000 rpm, and Figure 13(c): 4000 rpm. In contrast, other methods known in the art yield isolated melanosomes displaying aggregated appearance. For example, images of isolated melanosomes published by Liu et al. in Figure 13 (d) or an acid/based method as shown in Figure 13 (e)-Figure 13 (f) (Pigment cell Res. 16:355-365.2003) demonstrate aggregation characteristic under microscope, indicating the presence of remaining associated hair matrix material that was not removed by the conventional methods.

[00036] Figure 14 shows the Fourier transform infrared spectroscopy (FTIR) spectra of the melanosomes isolated from human hair, compared to intact human hair. Since hair mainly consists of keratin, the spectrum of hair shows higher intensities in the regions of 2850-2950 cm ¹, 1455 cm ¹, 1360- 1380 cm ¹ and 1050-1100 cm ¹. Melanosome mainly contains indole structure. The spectrum of isolated melanosome shows the absorption peak of indolic and pyrrolic groups can be observed at 3000-3600 cm ¹, 2950-2980, 1610-1690 cm ¹, 1520-1540 cm ¹, 1400-1500 cm ¹, 1360-1380 cm ¹, 1200-1240 cm ¹ and 1050-1100 cm ¹. Thus, the presence of large number of melanosomes in the isolated sample is confirmed.

[00037] Figure 15 shows solid state ¹³C nuclear magnetic resonance (NMR) spectra of intact hair and the melanosomes isolated from human hair. Hair fibres are mainly composed of proteins (90%) and therefore the solid state ¹³C NMR spectrum of hair is essentially dominated by the signals of the most abundant proteins including keratins and keratin-associated proteins (KAPs). As proteins mainly contribute to the carboxyl and aliphatic signals while melanosome mainly contributes to the aromatic signal, the comparison of the aromatic-to-carbonyl signal area ratio (93-160 ppm and 160-190 ppm, respectively) based on NMR data presented herein could be used to qualitatively evaluate the relative content of melanin and proteins in the melanosomes extracted from hair. The aromatic-to-carbonyl signal area ratio of the isolated melanosome sample is 2.54 for the method disclosed herein, showing the enrichment of melanosome content in the sample.

[00038] Figure 16 shows the thermogravimetric analysis of the melanosomes isolated from human hair. Figure 16(a) shows thermogravimetric analysis (TGA) curve and Figure 16(b) shows derivative thermogravimetric (DTG) curve of the melanosomes obtained using the method disclosed herein compared to those of intact hair. Up till 500°C, the melanosomes only lost about 48.84% of their total mass. This demonstrates that the isolated melanosomes are characterised by strong thermal stability. [00039] Figure 17 describes the UV-vis absorption in Figure 17(a) and UV-vis-transmittance in Figure 17(b), respectively, of the human hair melanosomes suspension at various concentrations (UVA region: 400 nm-320 nm; UVB region: 320 nm-280 nm; UVC region, 280 nm-220 nm). The melanosomes isolated using the method disclosed herein demonstrates high absorption intensities at UV region, as compared to visible light region, which is dependent on increasing concentrations of melanosomes. [00040] Figure 18 shows digital pictures of remaining hair debris and solubilised melanosomes after repeated enzymatic treatment of KAP and keratin-extracted hair. The solution containing esperase of 20 pL dosage has been collected and reused repeatedly for four times on 4 different batches of hair (1 g, KAP and keratin removed) for the isolation of melanosomes using the method disclosed herein. Samples (batches 1 to 4) are subsequently centrifuged and inverted to reveal the amount of undigested debris after each round of treatment. The solubilised melanosomes released from hair cortex are shown in the dark solution in the bottom of the tubes. It is observed that the amount of debris increased with the repeated use of the same solution containing esperase. However, the solutions remain dark in colour, indicating large amount of melanosomes dissolved in the solution.

DEFINITIONS

[00041] As disclosed herein, the term “keratin” refers to a fibrous structural protein of hair, nails, horn, hoofs, wool, feathers, and of the epithelial cells in the outermost layers of the skin. Keratin proteins can be categorised into alpha-keratins and beta-keratins based on their secondary structure. Alpha-keratins are often found in, for example, the hair, the skin, and the wool of mammals, and are primarily fibrous and helical in structure. Keratin forms a packed arrangement of filament structure, termed keratin intermediate filament (KIF), in hair.

[00042] As disclosed herein, the term “keratin-associated proteins (KAP)” refers to a major component of the hair and plays a crucial role in forming a strong hair shaft through a cross-linked network with keratin. Keratin-associated proteins (KAP) are located in the matrix around the keratin intermediate filament (KIF) and are therefore also called matrix proteins.

[00043] As disclosed herein, the term “melanosome” refers to the melanin-containing organelle that is responsible for pigmentation of, for example, the hair and skin of mammals.

[00044] As disclosed herein, the term “melanin” refers to “eumelanin” and “pheomelanin”, which are the pigments occurring in, for example, the hair, skin, and iris of the eye in people and animals. While there are three types of melanin known in the art, including eumelanin, pheomelanin and neuromelanin, only eumelanin and pheomelanin are considered in the present disclosure. Eumelanin pigment is characterised by the dark brown to black colour and pheomelanin pigment shows yellow to red colour variations. The relative amount of eumelanin and pheomelanin contained in the hair determines the exhibited colour of hair.

[00045] As disclosed herein, the term “cuticle cells” refers to the type of flattened and hardened dead cells that are arranged in a scale-like pattern in the same direction along the hair shaft and are overlapping with the exposed edge of cuticle cells facing away from the follicle. Each cuticle cell contains three layers, the A-layer, exocuticle and endocuticle. As disclosed herein, the term “cuticle” refers to the outermost part of the hair made from single or multiple layers of cuticle cells that strengthen and protect the hair.

[00046] As disclosed herein, the term “Anson Unit” refers to the enzyme units quantified by the amount of enzyme that liberates 1 pmol of TCA-soluble, Folin -positive amino acids within 1 minute at pH 7.5 and 37°C, using haemoglobin as a substrate. Anson units (that use haemoglobin as a substrate) are considered equivalent to commonly used protease units (that use casein as a substrate) when defining the activity of a proteolytic enzyme. As disclosed herein the term “Novo protease unit (NPU)” is defined to be equivalent to Anson Unit (AU). That is, one Anson Unit (AU) is identical to one Novo protease unit (NPU). Thus, the terms “Anson Unit (AU)” and “Novo protease unit (NPU)” are used interchangeably in this disclosure. The activity of exemplary enzymes used herein is measured by either Anson Unit (AU) or Novo protease unit (NPU).

[00047] As disclosed herein, the terms “aggregative”, “aggregating”, or grammatical equivalents thereof, refer to a condition where a subject is formed in a mass by several fragments or particles clustering and adhering together, with the fragments or particles being otherwise distinct and separate units. Examples of “aggregative” melanosomes are shown in Figure 13(d)-Figure 13(f). “Non- aggregative”, “non-aggregating”, or their grammatical equivalents, refers to a condition where the subjects are individually isolated from one another without or with minimal adhereance. A typical example of non-aggregating appearance of isolated melanosomes is shown in Figure 13(a)-Figure 13(c). [00048] As used herein, the term “enzyme” refers to one or more proteins that have the ability to catalyse biological reactions. In some examples, enzymes require a co-factor, co-enzyme, or a (co-) substrate in order to initiate the biological reaction in question. In other examples, the enzyme is self-catalysing. The enzyme’s specificity to initiate a specific reaction is based on its amino acid sequence. From a chemical aspect, enzymes are like any chemical catalyst and are not consumed in chemical reactions. Enzymes, just like catalysts, also do not alter the equilibrium of a reaction. Enzymes are much more specific with regard to the reaction they catalyse, compared to catalysts. Among other factors such as time, temperature and pH, enzyme activity can be affected by other molecules, such as, but not limited to, inhibitors (which are molecules that decrease enzyme activity), and activators (which refer to molecules that increase enzyme activity). Such factors may reversibly or irreversibly alter the enzyme’s catalytic ability. Enzymes are commonly classified by the reaction that they catalyse. Also, in order to catalyse any reaction, enzymes must bind (to) their substrates. Thus, enzymes have two structural sites that are important for their function, which are referred to as the enzyme’s active sites when referenced in combination. One is the substrate (binding) site, the other being the catalytic site, whereby the former binds the substrate, and the latter reduces the chemical activation energy required to perform the reaction being catalysed. Due to the substrate binding site, enzymes are highly specific as to what substrates they bind and then the reaction catalysed. The high specificity is achieved by the substrate binding sites (also referred to as “pockets”) having complementary shape, charge and hydrophilic/hydrophobic characteristics to the substrates. Enzymes can therefore distinguish between very similar substrate molecules to be chemoselective, regioselective and stereospecific.

[00049] Most enzyme active sites comprise a set of three coordinated amino acids, usually referred to as the catalytic triad, and are usually found in hydrolase and transferase enzymes (such as, but not limited to, proteases, amidases, esterases, acylases, lipases and b-lactamases). These residues of the catalytic triad form a charge -relay network to polarise and activate the nucleophile, which attacks the substrate, forming a covalent intermediate which is then hydrolysed to release the product and regenerate free enzyme. The nucleophile is most commonly a serine or cysteine amino acid, but can also be a threonine or a selenocysteine. The three-dimensional (tertiary) structure of the enzyme brings together the triad residues in a precise orientation, despite potentially being far apart in the linear sequence (primary structure).

[00050] As used herein, the term “serine protease” refers to enzymes that cleave peptide bonds in proteins. Such serine proteases are found ubiquitously in both eukaryotes and prokaryotes and fall within two broad categories based on their structure, namely, chymotrypsin-like (trypsin-like) or subtilisin-like. Serine proteases are characterised by a distinctive structure, consisting of two beta-barrel domains that converge at the catalytic active site. As with all enzymes, serine proteases can be further categorised based on their substrate specificity.

[00051] As used herein, the term “bacterial serine protease” refers to the serine protease isolated from a bacterium. One example of a bacterial serine protease is a subtilisin (E.C. 3.4.21.62), which refers to an alkaline non-specific serine protease from Bacillus spp. (for example, but not limited to subtilis, amyloliquefaciens, licheniformis, or lentus ) that initiates the nucleophilic attack on a peptide bond through a serine residue at the active site. In other words, subtilisin catalyses the hydrolysis of proteins and peptide amides. Subtilisin is a type of serine endopeptidase of MEROPS family S8, and, while being structurally unrelated to the chymotrypsin-clan of serine proteases, uses the same type of catalytic triad in the active site. The active site of subtilisin features a charge -relay network involving Asp-32, His-64, and active site Ser-221 arranged in its’ catalytic triad. In summary, Ser-221 acts as a nucleophile and cleaves peptide bonds with its partially negative oxygen atom, which is possible due to the nature of the charge -relay site of subtilisin. Analogue catalytic triads involving, for example, Asp-His-Cys function similarly, with Cysteine taking over the role of serine. Both esperase and savinase, as disclosed herein, are enzymes that are functionally equivalent to subtilisin, whereby the term “functionally equivalent” refers to the fact that the use of esperase and/or savinase brings about comparable results compared to the use of subtilisin.

[00052] As used herein, the term “esperase” refers to an endopeptidase with a broad specificity which performs well in alkaline (elevated pH) conditions and at elevated temperatures, compared to other microbial serine proteases. Esperase is used to catalyse the hydrolysis of internal peptide bonds. The terms “esperase” and “subtilisin 147” are used interchangeably and refer to an extracellular serine endopeptidase originally isolated from the bacteria Bacillus lentus.

[00053] As used herein, the term “savinase” refers to a serine peptidase which catalyses stereoselective hydrolysis of some esters, as well as strained amides, under alkaline conditions. Specifically, savinase can be used for the stereoselective hydrolysis of esters, such as amino esters. In addition, savinase is also suitable for hydrolysis of proteins and the hydrolysis of (steric) strained amides. The terms “savinase” and “subtilisin 309” are used interchangeably and refer to an extracellular serine endopeptidase isolated from the bacteria Bacillus lentus, which is a N85S variant of another extracellular serine endopeptidase, maxacal, that was originally isolated in Bacillus alcalophilus PB92. [00054] As disclosed herein, the term “foam layer” refers to the specific mixture formed by cuticle cells, air and a small amount of solution, found floating on the surface of the solution after exposing hair which has undergone the removal of keratin and keratin-associated proteins (KAP), with serine protease. The foam layer is characterised by the white colour appearance, water insolubility, relatively lower density than water and the clear physical separation from the solution below.

[00055] As disclosed herein, the term “flaky” refers to the condition of isolated cuticle cells, which are of intact individual flattened scale-like shapes that do not adhere to each other easily or can be separated with minor physical disturbance into individual cell pieces.

[00056] As sued herein, the term “extractability” refers to the percentage of weight loss of hair before and after treatment. The extractability of hair is used to estimate the efficiency of a method of treatment used. For example, the weight of intact hair is W1 , after keratin-associated proteins (KAP) extraction, the weight of KAP free hair is W2. Subsequently the hair undergoes Keratin extraction, the weight of the hair residues after Keratin extraction is measured to be W3. The keratin-associated proteins (KAP) extractability is calculated as follows:

Extractability (KAP)=(W 1 - W2)/W 1*100%; while the Keratin extractability is calculated as follows:

Extractability (Keratin)=(W2-W3)/W 1 * 100% .

[00057] As used herein, the term “delipidation” or “delipidisation” refers to a pre-treatment method used for hair samples collected. Methods of delipidising hairs are known in the art. A typical method for delipidisation of hair comprises washing hair samples with 70 % of an alcohol, such as ethanol (2- 3 times); immersing washed hair into chloroform/methanol (2:1, v/v) solution (for about 24h); rinsing hair sample with water and drying the rinsed hair; and cutting the hair samples into small fragments (for example, 2 mm in length) for subsequent treatments. The term “intact hair” as used in this disclosure refers to hair that was not treated to remove any of the hair components. As used in this disclosure, the terms “intact hair” and “delipidised hair” refers to the same hair sample and are used interchangeably.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[00058] Major hair components including cuticle cells, keratin (intermediate filament proteins), keratin- associated proteins (KAP, matrix proteins) and melanosomes have shown potential in biomedical applications. However, applications of hair components have been inadequately developed due to difficulties in obtaining quality components. Therefore, there is a need for methods of isolating hair components which reduces wastage of hair material in the process. [00059] The application as disclosed herein provides comprehensive methods that are suitable for the isolation of multiple hair components such as cuticle cell, keratin, keratin-associated proteins (KAP) and melanosome, with minimal damage to the structure and physicochemical properties of the isolated component.

[00060] Hair has a typical hierarchical structure: a central cortex consisting of closely packed cortical cells arranged parallel to the hair long axis and an outer sheath of scaly cuticle cells. Thus, the outmost cuticle layer of cells is the first to be isolated.

[00061] Cuticle cells can be obtained using mechanical methods, such as scraping with razor blades or vigorous sonication. However, such methods are laborious with low yield and quality. Chemical methods, such as ionic liquid mixtures, can remove cuticle cells from the hair, but with damaged structure of cuticle cells that lose the intact flaky structure. Disclosed in this application is an enzymatic method of isolating cuticle cells from hair. In some examples, the present disclosure describes a method of isolating cuticle cells from hair, wherein the method comprises exposing hair to a serine protease. In one example, the serine protease is comprised in a solution. As used herein, “serine protease” refers to a group of enzymes which cleave peptide bonds in proteins. Thus, in one example, the solution comprises an enzyme that cleaves peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the enzyme's active site. In another example, the solution comprises a serine endopeptidase. As shown in Figure 1 in panels (b) and (c), the morphology of hair surfaces changes after treatment with exemplary serine proteases, as compared to (a) intact hair. The cuticle cells are dislodged from the hair under magnification. As defined herein, serine proteases can be isolated from a bacterium or eukaryotes. In some examples, the serine protease is isolated from a bacterium of Bacillus genus. In one example, the serine protease as disclosed herein is isolated from, but is not limited to, Bacillus subtilis, Bacillus amyloliquefaciens , Bacillus licheniformis, or Bacillus lentus. In another example, the serine protease is subtilisin or a functional equivalent thereof. In another example, the serine protease is subtilisin isolated from Bacillus subtilis. In one example, the serine protease is an esperase (also known as subtilisin 147). In another example, the serine protease is a savinase (also known as subtilisin 309). In another example, the serine protease is a combination of proteases as disclosed herein, for example, but not limited to, esperase and savinase. Therefore, in a further example, the serine protease is a subtilisin, which refers to an alkaline non-specific serine protease from Bacillus spp. In one example, the serine protease is an esperase (also known as subtilisin 147). In another example, the serine protease is a savinase (also known as subtilisin 309).

[00062] In some examples, the method disclosed herein comprises exposing hair to a solution comprising a serine protease for about 2 to 4 days or 2 days, about 3 days, or about 4 days. As exemplified in Figure 2, exposing hair to a solution comprising a serine protease for 3 days at appropriate concentrations, for example, is sufficient to isolate cuticle cells from hair.

[00063] In one example, it is referred to a method of isolating cuticle cells from hair, wherein the method comprises: exposing hair to a solution comprising a serine protease for 2 to 4 days; collecting a foam layer formed on the top of the solution of a); and isolating cuticle cells from the collected foam layer of b).

[00064] In a method disclosed herein, the temperature of exposing hair to the solution comprising a serine protease is at about 30 to 70°C or 50 to 60°C. For example, as disclosed herein, the temperature used for isolation of cuticle cells using an exemplary serine protease is 50°C. A person skilled in the art would be able to understand that the temperature allowing enzymes to function is based on the intrinsic properties of the enzymes and would be able to understand that the working temperature of a particular enzyme could be a range of temperatures as referred to herein. For example, the temperature can be about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, or about 60°C, about 65°C, or about 70°C.

[00065] Serine proteases, as a person skilled in the art would understand, are alkaline proteases, which means they are active in neutral to alkaline pFi range. As shown in the Experimental Section, the buffer pH for the solution used in the method disclosed herein can be from 8 to 12 or about 9. In other examples, the buffer pH for the solution used can be about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, or about 11.5 or about 12.

[00066] As demonstrated in Figure 2, for example, the efficiency of cuticle cell isolation positively correlates with the concentration of the serine protease used. In some examples, the concentration of the serine protease is about 960 Anson Units per gram of hair. A person skilled in the art would be able to understand that the concentration of a particular enzyme to function is based on the intrinsic properties of the enzymes and would be able to understand that the working concentration of a particular enzyme could be a range of concentrations as referred to herein. For example, the concentration can be between about 320 to 1280 Anson Units, 320 to 960 Anson Units, about 320 Anson Units, about 640 Anson Units, about 960 Anson Units, or about 1280 Anson Units.

[00067] After enzymatic digestion of hair through exposure in solution comprising a serine protease, the dislodged cuticle cells from hair are collectable by forming a foam layer on the top of the solution, as visualised in Figure 3 (a). The foam layer can be collected and dried by methods known in the art, for example, into lyophilised powder of cuticle cells as shown in Figure 3(b). In one example, the isolation of cuticle cells further comprises: (a) ultrasonicating the suspension; (b) filtering the suspension after a) and collecting a filtrate; (c) centrifuging the filtrate of b) at a first speed for a first time period and removing a supernatant generated from centrifugation from a precipitate; (d) resuspending the precipitate generated in c) in a suspension; (e) centrifuging the suspension of d) at a second speed for a second time period, and collecting a supernatant generated from centrifugation from a precipitate; and (f) collecting the supernatant of e), wherein a), b) and d) to f) are repeated at least three or more times. In other examples, the cuticle cells within the foam can be isolated through other clean-up processes. A person skilled in the art would easily come up with routine experimental procedures and parameters for isolation of the insoluble cuticle cells from the foam layer. [00068] The method disclosed herein for the isolation of cuticles is applicable for combinations of serine proteases, or a single serine protease. One example provides a method of isolating cuticle cells from hair, wherein the method comprises exposing hair to a solution comprising an esperase (subtilisin 147) as the sole enzyme for 2 to 4 days; and collecting a foam layer formed on the top of the solution. [00069] The method of isolating cuticle cells from hair as disclosed herein has a higher efficiency in terms of the amount of enzymes used, wherein the solution comprising one or more serine proteases can be reused for exposure of a different batch of hair, thus making the method disclosed herein economically competitive over other methods.

[00070] The disclosure also provides an isolated intact cuticle cell produced by the method as disclosed herein, wherein the cuticle cells are characterized by intact flaky appearance. As demonstrated in Figure 3 (g) to (j), the isolated intact cuticle cells produced by the method disclosed herein are characterised by a homogenous population of flaky appearance with structural integrity and little or no impurities. The cuticle cells have irregular polygonal shapes with dimensions of about 48.6 ± 7.5 pm by 25.6 ± 2.8 pm, with upper side and under side clearly distinguishable.

[00071] Using the cuticle cells isolated in the method as disclosed herein, there could be various applications. For example, a coated polymer film can be prepared from the isolated cuticle cells by depositing the cuticle cells onto the polymer film. In one example, a standard vacuum suction method is used to deposit cuticles cells onto the polymer film.

[00072] The paragraphs above provide methods of isolating intact cuticle cells from hair. In some cases, cuticle cells are not essential to the intended application and are desired to be removed quickly from the remaining hair components, regardless of the collectability of the dislodged cuticle cells from hair. [00073] Thus, disclosed herein is a method of removing cuticle cells from hair. In some examples, the present disclosure describes a method of removing cuticle cells from hair wherein the method comprises exposing hair in a solution comprising L-cysteine. In some examples, the concentration of L-cysteine is about 1%, about 2%, about 3%, about 4%, or about 5% (weight to volume or w/v%).

[00074] For example, as described herein, L-cysteine can also be used in combination with one or more proteases as disclosed herein for the removal of cuticle layer, that is, for descaling of hair samples. In one example, the solution further comprises at least one or more serine proteases. In some examples, the serine protease is isolated from a bacterium. In further examples, the serine protease is isolated from a bacterium of Bacillus genus. In one example, the serine protease as disclosed herein is isolated from, but is not limited to, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis , or Bacillus lentus. In another example, the serine protease is subtilisin or a functional equivalent thereof. In another example, the serine protease is subtilisin isolated from Bacillus subtilis. In one example, the serine protease is an esperase (also known as subtilisin 147). In another example, the serine protease is a savinase (also known as subtilisin 309). In another example, the serine protease is a combination of proteases as disclosed herein, for example, but not limited to, esperase and savinase. Exemplary combinations of L-cysteine with serine protease include, but are not limited to, combinations of L- cysteine and esperase, L-cysteine and savinase. As disclosed herein for some examples, samples treated using L-cysteine and esperase are labelled as “H-EL”. Samples treated using L-cysteine and savinase are labelled herein as “H-SL”.

[00075] As shown in Figure 5(a) and 5(b), the treated hair samples under scanning electron microscopy (SEM) show smooth surface morphology with cuticle cells removed with the method disclosed herein for about 4 hours. Thus, in one example, the present disclosure describes a method of removing cuticle cells from hair wherein the method comprises exposing hair in a solution comprising L-cysteine and esperase for about 4 hours; and removing cuticle cells from the solution. In some examples, the cuticle cells are removed from the solution. In another example, the present disclosure describes a method of removing cuticle cells from hair wherein the method comprises exposing hair in a solution comprising L-cysteine and savinase for about 4 hours; and removing cuticle cells from the solution. In some examples, the cuticle cells are removed from the solution.

[00076] The exposure time of hair to the solution can be about 3 to 5 hours or about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, or about 5 hours. In some examples, the temperature of the exposure of hair to the solution can be about 50 to 60°C or at about 50 °C, about 55 °C, or about 60 °C. In some examples, the concentration of the esperase can be 320 to 1280 Anson Unit per gram of hair. In some examples, the concentration of the savinase can be at least about 320 Anson Unit per gram of hair. In yet another example, the concentration of savinase is about 320-1280 Anson Unit per gram of hair. A person skilled in the art would easily understand that minor alterations in the parameters would not affect the efficiency of cuticle cell removal.

[00077] The method of removing cuticle cells from hair as disclosed herein is suitable for upscaling given that the solution can be reused for exposure to a different batch of hair.

[00078] After isolation or removal of the layer of cuticle cells, the hair cortical cells are exposed. The inset in Figure 5(a), for example, are images of the dispersed cortical cells from hair, after the removal of cuticle cells. Major components of hair cortical cells are keratin filaments embedded in the matrix of keratin-associated proteins (KAP), as shown in Figure 1(d). Melanosomes are small rod-like structures that are dispersed within cortical cells. It is provided in this disclosure, a method of isolating melanosomes, keratin, and keratin-associated proteins (KAP) from hair.

[00079] As described herein, conceptually simplified for the ease of understanding, is a method of isolating keratin-associated proteins (KAP), keratin, and melanosomes sequentially from hair. The method comprises a first step of isolating keratin-associated proteins (KAP) from hair, a second step of isolating keratin from hair residue with keratin-associated proteins (KAP) removed from the first step, and a third step of isolating melanosomes from the hair residue after the removal of keratin-associated proteins (KAP) and keratin.

[00080] As the matrix of cortical cells, keratin-associated proteins (KAP) are removed for easy access to other hair components. In one example, the present disclosure provides a method of isolating melanosomes, keratin, and keratin-associated proteins (KAP) from hair, wherein the method comprises: mixing hair in a KAP extraction solution to release the KAP into the solution. The KAP extraction solution, as disclosed herein, contains urea, a reducing agent, and alcohol, such as ethanol, prepared in an appropriate buffer. For a person skilled in the art, it is understandable that a reducing agent can be routinely selected from, for example, DTT (dithiothreitol), glutathione, DTE (dithioerythritol) or tris(2- carboxyethyl)phosphine (TCEP); or a suitable buffer can be routinely selected based on the type of reactions, for example, a Tris-HCL buffer. In one particular example as described herein, the KAP extraction solution contains urea, DTT and ethanol in Tris-HCl buffer.

[00081] For the KAP extraction solution as disclosed herein, the exposure time of hair to the solution can be between about 48 hours to 96 hours or about 48 hours. In further examples, the exposure time can be about 48 hours, about 54 hours, about 60 hours, about 66 hours, about 72 hours, about 78 hours, about 84 hours, about 90 hours, or about 96 hours.

[00082] In some examples, the temperature of exposing hair to the KAP extraction solution is about 30 to 70 °C. In some examples, the temperature of exposing hair to the KAP extraction solution can be about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, or about 70°C. In other examples, the buffer pH for the KAP solution used in the method disclosed herein can be between about 8 to 12 or about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5 or about 12. It is understood for a person skilled in the art that for chemical reactions to work, the allowable temperature and pH are usually not limited to a particular value, but a range of workable variations.

[00083] As disclosed herein, the method further comprises: separating hair and the KAP extraction solution exposed with hair; and isolating the KAP from the solution. In one example, the KAP extraction solution exposed with hair is separated from hair by filtration, as shown in the schematic of Figure 10. As demonstrated in this disclosure, the mixture can be filtered with nylon cloth, as an example, or any other material of appropriate functions to separate solution and insoluble content.

[00084] The collected hair, after removal of keratin-associated proteins (KAP) using the method disclosed herein, is subject to the removal of keratin and melanosomes. As disclosed herein, the method further comprises mixing the collected hair after removal of keratin-associated proteins (KAP) with a keratin extraction solution to release keratin into the solution. In some examples, the keratin extraction solution contains thiourea, urea and a reducing agent. In this disclosure, for example, the keratin extraction solution used contains thiourea, urea, and DTT in Tris-HCl buffer. In some examples, the exposure time with keratin extraction solution can be between about 6 to 36 hours or about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, or about 36 hours. For any of the exposure times disclosed herein, the exemplary temperature of exposing hair to the keratin extraction solution can be between about 40 to 60°C or about 40 °C, about 45 °C, about 50 °C, about 55 °C, about 60 °C. In further examples, the buffer pH for the solution used in the method disclosed herein can be between about 8 to 12 or about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5 or about 12. It is understood for a person skilled in the art that for chemical reactions to work, the allowable temperature and pH are usually not limited to a particular value, but a range of workable variations.

[00085] Keratin extraction solution as described herein releases keratin into the solution, at the conditions disclosed herein. Subsequently, the method further comprises separating hair and the keratin extraction solution exposed with hair; and isolating the keratin from the solution. In some examples, the keratin extraction solution exposed with hair is separated from hair by filtration. In further examples, the filtered hair is air-dried and collected for the isolation of melanosome.

[00086] The isolation of melanosomes from hair requires the isolation or removal of surrounding keratin-associated proteins (KAP) and keratin form the hair cortical cells. In one example, the present disclosure provides a method of isolating melanosomes from hair, wherein the hair has undergone a process of isolating keratin and keratin-associated proteins (KAP) using the methods as disclosed herein. In another example, the present disclosure provides a method of isolating melanosomes from hair, wherein the hair has undergone a process of isolating keratin and keratin-associated proteins (KAP) using other methods of removal of those components.

[00087] As disclosed herein, the method of isolating melanosomes, keratin, and keratin-associated proteins (KAP) from hair, further comprises exposing the collected hair after removal of keratin- associated proteins (KAP) and keratin, to a bacterial serine protease to release melanosomes. It is understood by a person skill in the art that a bacterial serine protease can be, for example, an enzyme that cleaves peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the enzyme's active site, or a serine endopeptidase. In one example, the bacterial serine protease is a subtilisin. In a further example, the bacterial serine protease is isolated from a bacterium of Bacillus genus. In some examples, the bacterial serine protease can be comprised in a solution. As exemplified in this disclosure, the bacterial serine protease can be an esperase (also known as subtilisin 147). As shown in Figure 13(a)-Figure 13(c), the specific use of a bacterial serine protease results in the isolation of intact, non-aggregated melanosomes.

[00088] The exposure time of hair to the bacterial serine protease can be between about 0.5 to 4 hours, or about 0.5 hours, about 1 hour, about 2 hours, about 3 hours, or about 4 hours. In some examples, the temperature of exposing hair to the solution comprising a bacterial serine protease can be between about 30 to 70 °C or about 30 °C, about 35 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C, about 60 °C, about 65 °C, or about 70 °C. For the exemplary temperatures disclosed herein, the buffer pH for the solution used in the method disclosed herein can be between about 8 to 12 or about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, or about 11.5 or about 12. In some examples, the concentration of the bacterial serine protease is about 125 Anson Unit per gram of hair. In some examples, the concentration of the bacterial serine protease can be about 250 Anson Unit, about 375 Anson Unit, about 500 Anson Unit, about 625 Anson Unit per gram of hair or more.

[00089] As disclosed herein, the method of isolating melanosomes, keratin, and keratin-associated proteins (KAP) from hair, further comprises isolating melanosomes from the solution. In some examples, the released melanosomes in the solution are removed by filtering followed by centrifugation and washing. In some examples, centrifugation takes place at a speed of 6000 to 8500 rpm or 8000 rpm for about 5 to 15 minutes or 10 minutes. In further examples, the centrifugation and wash steps are repeated twice, three times, four times, five times, or six times. In some examples, the melanosomes are isolated from the solution in the washed sediments from centrifugation. In one specific example, the isolation of melanosomes further comprises: a) filtering the solution and collecting a filtrate; b) centrifuging the filtrate of a) at a speed for a time period and removing a supernatant generated from centrifugation from a precipitate; c) resuspending the precipitate generated in b) in a suspension; d) centrifugating the suspension of c) at the same speed and for the same time period of b) and removing the supernatant generated from centrifugation from the resulting precipitate; and e) collecting the precipitate of d), wherein c) and d) are repeated at least six or more times. It is understood that various other methods for isolating insoluble melanosomes from a solution can be routinely carried out by a person skilled in the art without burden.

[00090] The disclosure also provides the isolated melanosomes produced by the method as disclosed herein, wherein the melanosomes are characterized by non-aggregated appearance. The melanosomes isolated using the method disclosed herein are in rod-shape as shown in Figure 11 (a)- Figure 11 (d). The characteristic non-aggregated appearance, as compared to previous methods, are shown in, for example, Figure 13. As disclosed herein, the term “non-aggregated” refers to a condition where the melanosomes are individually isolated from one another without adhereance.

[00091] In the methods disclosed herein, the hair is delipidised hair. As disclosed herein, the term “delipidisation” refers to a pre-treatment cleaning step on hair before the isolation of hair components. An exemplary method of delipidisation comprises washing hair with ethanol and removing external lipids using a mixture of chloroform/ methanol (2:1, v/v) for about 24 hours. It is understood by a person skilled in the art that, as disclosed herein, the term “hair” refers to any of the fine threadlike strands growing from the skin of humans, mammals, and some other animals. Therefore, the hair can be any animal hair that has a scaly structure. In one example, the hair is a keratinous hair. In another example, the hair is a human hair. In another example, the hair can be wool.

[00092] As disclosed herein, the application provides methods that are suitable for the isolation of multiple hair components such as cuticle cell, keratin, keratin-associated proteins (KAP) and melanosome using bacterial serine proteases. The enzymatic methods provide improved yield and quality of the isolated components with minimal damage to their structure and physicochemical properties.

[00093] The disclosure illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[00094] The disclosure has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

EXPERIMENTAL SECTION

[00095] Disclosed herein are exemplary process of isolating cuticle cell, keratin, keratin-associated proteins (KAP) and melanosome from hair using exemplary serine proteases.

Morphology of Hair Treated with Serine Proteases

[00096] Disclosed herein are examples of removing cuticles of human hair using esperase (Sigma, Product No. P5860; enzyme activity: 8 Anson Unit/pL) and savinase (Sigma, Product No. P3111; enzyme activity: 16 Anson Unit/pL). Delipidised hair were treated with serine protease, for example, esperase or savinase of appropriate dosages at about 50-60°C with a pH of about 9 to 9.5 for about 72 hours. The morphology of hair surfaces after the protease treatment (at the same dosage) are shown in Figure 1. Results show that the cuticle scales of the hair treated with both proteases were exfoliated to some extent. Untreated hair shows a typical overlapping cuticle layer with high density (Figure la). After treating with esperase, the scaly layer is mostly dislodged and exfoliated (Figure lb). Although some cuticles remained on the hair shafts, these cuticles are mostly loosely attached. On hair treated with savinase, more intact cuticles are observed, where it seems the exposed edges of cuticles are dislodged more (Figure lc). Compared to untreated hair, the density of intact cuticles on hair shafts are lower for hair treated with savinase, and the lowest for hair shafts treated with esperase. Corroborating with this observation, during the cuticle collection step, a distinct white foamy layer can be found suspended on top of the esperase treated samples, which is the isolated population of cuticle cells. This was not observed in the savinase treated group. These results suggest that esperase is more suitable and efficient in removing and preserving the cuticle cells of human hair, while savinase is able to remove cuticle cells from hair after treatment at the same dosage.

Cuticle Isolation Using Esperase

[00097] Exemplary hair samples treated with esperase are observed with scanning electron microscopy (SEM) to judge the degree of cuticle removal over a range of esperase dosages (Figure 2). Hair samples are treated with esperase (8 Anson Unit (AU) per pL) of dosages ranging from 100 pL to 300 pL. The treatments are carried out at about 50-60 °C for about 72 hours, at a pH of about 9-9.5. When the dosage of esperase is 100 pL, cuticles on the surface of hair shaft are dislodged but remain on the hair surface, resulting in a rough surface as shown in Figure 1(b). Hair without cuticles are shown in the inset image in Figure 2(a). With increasing esperase dosage, the proportion of descaled hair with smooth surfaces greatly increased (Figure 2b, c, d). When the dosage of esperase is above 300 pF, almost all of the hair shafts present a smooth surface that is void of cuticles.

Morphology of Isolated Cuticle and Descaled Hair Samples

[00098] In one example, hair cuticles are released using esperase treatment and purified by centrifugation. This is due to the tendency of cuticle cells floating to the mixture surface, forming a clear, separated white foamy layer (Figure 3a). The foam is easily collected and lyophilized into a fine powder (Figure 3b). For example, the cuticle cells can be collected from the foam through the following steps: (a) ultrasonicating the mixutre; (b) filtering the mixture after a) and collect a filtrate; (c) centrifuging the filtrate of b) at a first speed for a first time period and removing a supernatant generated from centrifugation from a precipitation; (d) resuspending the precipitation generated in c) in a suspension; (e) centrifuging the suspension of d) at a second speed for a second time period, and collecting a supernatant generated from centrifugation from a precipitation; and (f) collecting the supernatant of e), wherein a), b) and d)-f) are repeated at least three or more times. In other examples, the cuticle cells within the foam can be isolated through other clean-up processes. Interestingly, the flaky cuticle cells can easily form a foam whenever it is resuspended in water, but not in ethanol. Pockets of air bubbles are evidently trapped between the cuticle cells when suspended in water, hence the ease of separation as a foamy layer as described. (Figure 3c). When ethanol is dropped into the foamy suspension, the air bubbles disappeared due to low surface tension (Figure 3d).

[00099] After enzymatic digestion of hair through exposure in solution comprising a serine protease, the dislodged cuticle cells from hair are collectable by forming a foam layer on the top of the solution, as visualised in Figure 3 (a). The foam layer can be collected and dried by methods known in the art, for example, into lyophilised powder of cuticle cells as shown in Figure 3(b).

[000100] Untreated hair shafts show overlapping scales that adhere tightly to the hair surface (Figure 3e). After removal of hair cuticles by esperase, the overlapping scales disappeared (Figure 3f). Assessment of the lyophilized cuticle powder shows a homogenous population of flaky cuticle cells at high purity and structural integrity (Figure 3g). To examine the shape and size of the cuticle cells, they were laid down by vacuum suction to form a cuticle film that is observed using scanning electron microscopy (SEM). Results show that the cuticle cells have irregular polygonal shapes with dimensions of about 48.6+ 7.5 pm by 25.6 ± 2.8 pm (Figure 3g), which is consistent with the average values of 40- 60 pm in length and breadth reported. In addition, both surfaces of each cuticle cell are distinguishable: the upper side is smoother than the underside, resulting in different contrast under scanning electron microscopy (SEM), as shown in Figure 3(h-j). Each cuticle cell contains three layers: the outmost epicuticle, exocuticle and the inner endocuticle. The epicuticle is a thin hydrophobic layer on the surface, which makes the hair water resistant. The exocuticle has the highest content of cysteines and crosslinking degree of peptide chains. The endocuticle contains a lower number of cysteines but a higher content of acidic, basic and polar amino acids, allowing this layer to swell in water. The upper side of the cuticle cell is the same as the cuticle surface of untreated hair (Figure 3e), indicating resilience of the cuticle surface to enzymatic attack despite delipidisation.

[000101] Ultrastructural studies of hair by transmission electron microscopy (TEM) have shown that the multiple overlapping cuticles have laminated structures. Two-thirds of the cuticle thickness, from the top surface, is the exocuticle comprising of the exocuticle A-layer and exocuticle (exocuticle B-layer). The remaining one-third is the endocuticle layer. The exocuticle A-layer is about 100 nm thick, while the exocuticle and endocuticle are of varied thicknesses, with a poorly defined boundary between them (Figure 4(a)). According to the transmission electron microscopy (TEM) images of the isolated cuticle cells, the endocuticle layer has largely been removed, leaving essentially the A-layer and exocuticle. The underside of the exocuticle of each isolated cuticle cell is uneven, resembling the native interface between endocuticle and exocuticle (Figure 4(b)). These results indicate that the method described herein releases the cuticle cells without the endocuticle. The thickness of intact cuticle cells is about 430 ± 29 nm, while that of the isolated cuticle cells is about 295 ± 53 nm due to removal of the endocuticle (Figure 4(b)). The exocuticle (including both exocuticle A-layer and exocuticle) is about two-thirds of the cuticle thickness, which makes up about 10% of whole hair mass. Therefore, the theoretical mass proportion of total exocuticles is about 6.7% of total hair mass. The method disclosed herein yields about 3.5%±0.2%. of total hair mass, which works out to be about 52.5% efficiency. [000102] Examination of hair shafts and cuticle sections using transmission electron microscopy

(TEM) revealed the mechanism of cuticle release - via endocuticle degradation by proteases, for example, esperase (Figure 4(c)). Since the exocuticle is highly crosslinked by disulfide and isopeptide structures, these components are unlikely to swell and degrade. The endocuticle contains residual amounts of cysteines but has a higher-than-average content of acidic and basic amino acid residues, which are more prone to swelling in water and can be degraded by proteases such as esperase. In addition, the shaking action in oven provides sufficient energy to strip off the cuticles from the hair shaft, aided by further sonification. The imaging observations suggest intact flaky cuticle cells are obtained using the method disclosed herein. Surface Morphology Comparison of Untreated and Descaled Hair Samples

[000103] Although protease such as esperase can remove cuticles from hair shafts, the process is lengthy (72 h) and requires a high dosage of esperase (300 pL, i.e. 2400 Anson Units for 2.5 gram of hair). L-cysteine is an efficient reducing regent that can cleave disulfide bonds in hair keratins. Therefore, it is used in combination with proteases to accelerate the removal of cuticles and facilitate enzymatic degradation. For example, hair sample can be treated with a combined L-cysteine and a serine protease at appropriate dosages at the temperature of 50-60 °C for about 4 hours, at a pH of about 9-9.5.

[000104] The morphologies of human hair samples treated with combined L-cysteine and proteases, for example, esperase (H-EL) or savinase (H-SL) are shown in Figures 5(a) and (b), respectively, after about four hours of treatment. The surfaces of H-EL and H-SL are similar, indicating the removal of cuticle cells and release of some spindle-shaped cortical cells. However, the cuticles are degraded and cannot be collected in this method. Thus, the combination of L-cysteine and enzyme is more suitable for removal but not the isolation of hair cuticles.

Secondary Structure Characterisation of Hair Cuticle and Descaled Hair Samples

[000105] The FTIR spectra of intact hair and isolated cuticle, and three descaled hair samples are shown in Figure 6. Since hair mainly consists of keratins, all the IR spectral data show characteristic absorption bands ascribed to polypeptide units (-CONH-), namely, amide A (3000-3600 cm ¹), the amide B (3056-3075 cm¹), the amide I (1700-1600 cm¹), II (1580-1480 cm ¹) and the amide III (1300- 1220 cm ¹) absorption peaks.

[000106] The absorption peak at 3278 cm ¹ and 3073 cm ¹ is assigned to the stretching vibration of N-H and O-H (amide A), and aromatic C-H (amide B), respectively. A strong peak at 1635 cm ¹ should be ascribed to the C=0 stretching vibration (amide I), a medium peak at 1523 cm ¹ is assigned to N-H in-plane bending and C-N stretching vibrations (amide II), while the weak band in the range of 1238-1240 cm ¹ is related to the C-N and C-0 stretching vibrations (amide III). Those characteristic bands show little difference. The peaks around 2922, 2851 cm ¹ originate from C-H symmetric and asymmetric stretching vibrations of C¾ groups of proteins and lipids, respectively. The vibrations at 2872 and 2959 cm ¹ are assigned to C-H symmetric and asymmetric stretching modes of C¾ groups, respectively. The peak near 1450 cm ¹ is mainly ascribed to C-H bending vibrations of C¾ groups of proteins and lipids, while the peak at 1400 cm ¹ is considered to originate from C-H bending vibrations of terminal C¾ groups of proteins and lipids. Hair cuticle shows higher intensity at 1451cm ¹ than other hair samples, which is due to the lipid of the isolated cuticle The corresponding bands of S-0 are in the region of 1000-1200 cm⁴. The peaks near 1044 cm ¹ and 1076 cm ¹ are assigned to the cysteic acid and cystine monoxide, respectively. While the peaks at 1121 and 1022 cm ¹ are assigned to the cystine dioxide and S-sulfonate (or cysteine-S-thiosulphate), respectively. Compared to virgin hair and three descaled hair samples, the cuticle shows much higher intensity at 1075 cm ¹ and 1041 cm ¹, indicating the cuticle contains a higher content of oxidized cysteine residues resulting from the weathering or exposure to the sunlight (UV).

[000107] The solid-state ¹³C NMR spectra of intact hair, cuticle and three descaled hair samples are given in Figure 7(a). Hair is mainly composed of proteins (90%) and minor components including lipids (5%) and melanin (1-3%). Therefore, the solid state ¹³C NMR spectrum of hair is essentially dominated by the signal of the most abundant proteins including keratins and keratin-associated proteins (KAPs). Intense signals at region of 165-180 ppm are mainly due to backbone carbonyls belonging to peptide bonds, and to side chain carboxyl and amino groups. The carbonyl region is related to the secondary structure of the keratin, and the deconvolution of the carbonyl peaks is carried out using Gaussian fitting function. There are two major peaks in the deconvoluted form, one is attributed to the a-helix at around 175 ppm, and another is related to the b-sheet and random coil structure at 172 ppm. As shown in Figure 7 (b), the a-helix structure in intact hair is about 48%, and the published results show there is about 50% a-helix structure in human hair. The cuticle contains about 73.78% of b-sheet and random coil structure. The contents of the a-helix structure of the three descaled hair samples are about 54-61%, which is about 6%-13% higher than that of intact hair. The signals at 115-160 ppm with maxima at 123 ppm belong to aromatic residues of phenylalanine tyrosine, tryptophan, and histidine. Spectra of intact hair and three descaled hair samples in this region show higher intensity at 116 ppm, 129 ppm, and 157 ppm than cuticle samples.

[000108] The intense signal in the 45-80 ppm range due to backbone a-carbons and Thr/Ser b- carbons. The signal at 56 ppm and 53 ppm with shoulder frequencies at 61 ppm and 48 ppm represent the a- carbon. Compared to other hair samples, the signal for cuticle samples is broader and more intense, which is related to the higher content of b-sheet and random coil structure in cuticle samples. Because the shift of intensity from the higher frequency component of the a-carbon signal towards lower frequency suggests a shift from a-helical structure to a more extended, random coil or b-sheet structure.

[000109] The peak at 40 ppm is related to b-carbon in leucine and cross-linked cysteine residues. Intact hair and the three descaled hair samples show a similar resonance signal at 40 ppm, while that of cuticle occurred at 42 ppm. No literature expressed and explained this difference. Besides, the complex signals at 10-35 ppm are due to the alkyl components of the side chains. The intense peak centered at 25 ppm is ascribed b-carbons in glutamic acid and glutamine residues, with some contribution from arginine and cysteine. The methyl group side chain of alanine at 16 ppm (shoulder) and b-carbon of leucine around 20 ppm (shoulder). Cuticle shows obvious higher intensity at 33, 30 ppm than intact hair and three descaled hair samples, which may be due to the higher content of lipids in the cuticle.

[000110] Table 1 shows the amino acid composition (mole/ 100 mol of total amino acids) in hair, cuticle, and three descaled hair samples. Reference experimental results are included in the table for comparison purposes. The amino acid compositions of three descaled hair samples are the same as that of virgin hair, showing slight deviation which may be due to differences in natural diversity. The isolated cuticle contains significantly higher concentrations of serine, proline, valine, and lysine; and a lower concentration of aspartic acid, threonine, glutamic acid, proline, leucine, and arginine. It has also been reported that cuticle show higher content of cystine of about 19-20% in hair cuticle compared to the hair (16%-18%). Virtually, the cystine in the cuticle is mainly located in the exocuticle and A-layers which occupy about 65% of the total area of the cuticle. In this disclosure, the cystine contents in all five samples are lower (about 2%), which is due to the cysteines being derived or modified into cysteine acid during the acid hydrolyzing process.

Table 1 Amino acid composition of delipidised hair, isolated cuticle and three descaled samples (H-E, H-EL, H-SL) and related data from previous method.

Amino acid Reference

Hair Cuticle H-E H-EL H-SL _ (mol%) hair Cuticle

Tyrosine 2.36 1.06 2.29 2.21 2.60 2.04 1.4 Phenylalanine 2.04 0.77 2.24 2.29 2.07 1.6 1.2 Histidine 1.11 0.61 1.22 1.14 1.15 0.8 0.55 Methionine* 0.71 0.79 0.77 0.82 0.72 0.17 0.41 Glutamic Acid 14.89 11.36 15.61 15.98 16.12 11.85 8.87 Lysine 4.21 4.72 4.35 4.51 4.11 2.54 3.46 Glutamine 4.28 4.72 4.35 4.09 4.03 ND ND Aspartic Acid 7.37 1.81 8.49 8.05 7.86 5.16 3.14 Valine 5.66 8.61 6.01 6.29 5.90 5.24 6.74 Arginine 7.10 2.45 7.73 7.33 7.76 6.03 3.02 Asparagine 5.67 3.16 6.26 6.32 6.27 5.65 8.75 Threonine 1.85 1.24 2.17 2.15 2.15 7.17 4.31 Cysteine* 1.59 2.63 1.84 1.36 1.99 18.56 19.67 Proline 14.97 23.55 15.32 15.08 15.19 7.61 9.42 Serine 14.13 25.94 8.45 9.56 10.53 12.51 17.03 Alanine 1.34 0.51 1.35 1.25 1.15 4.49 5.23 Leucine 7.42 4.35 8.22 8.03 7.59 5.91 4.23 Isoleucine 3.31 1.72 3.32 3.53 3.40 2.25 1.95

* amino acid is modified or degraded into its derivatives. ND: not detected. Thermal Behaviour Characterisation of Hair Cuticle and Descaled Hair Samples

[000111] Thermal behaviour of the hair, cuticle, and three descaled hair samples are examined by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). In Figure 8(a), (b), all the samples record two distinct stages of weight loss. The first weight loss occurs below 150 °C, which is attributed to the loss of adsorbed water in samples. While the second weight loss from 150 °C to 400 °C attributed to the lateral chain destruction of hair proteins. Upon exposure to elevated temperature, hydrogen sulfide and sulfur oxide are released, followed by the cleavage of disulfide bonds(-S-S-) at 230-250 °C. Three descaled hair samples show similar thermal stability to intact hair (Figure 8(b)), due to the cortex is the main structure of hair. Cuticle shows higher stability due to a higher proportion of b-sheet structure enhances more intermolecular interactions. Cuticle shows better thermal resistance at temperature £ 260 °C.

[000112] The differential scanning calorimetry (DSC) curves are shown in Figure 8(c). The endotherm peak which occurs at around 100°C is related to the evaporation of water. The endotherm peak for intact hair (96°C) and three descaled hair samples (HE: 97°C; HEL: 98°C; HSL: 84 °C) occurred slightly below 100°C, while above 100°C for cuticle (106°C). This may be due to impermeability of the cuticle therefore little bounding water within the cuticle sample. The second endotherm peak at around 220-250°C is related to the denaturation of a-helix structure, referred to as the crystalline melting peak. There are two characteristic peaks at 233°C and 245°C for intact human hair. While three descaled hair samples, H-E, H-EL, H-SL show an intense endotherm peak at 235°C, 241 °C, 242°C, respectively, while cuticle only shows slight endotherm peak at 262°C.

Extractability of Cuticle Cells from Hair

[000113] The intact hair, cuticle, and three descaled hair samples are extracted with the KAP and keratin extraction solutions to estimate the extractability by the percent weight loss. As shown in Figure 9(a), three descaled hair samples show a similar weight loss of 60% compared to intact hair after two extractions. The yield of KAP and keratin extraction from intact hair, with respect to original hair mass, is about 26% and 30%, respectively. This could be due to the overlapping cuticles around the cortex that limit the dissolubility of keratin in the cortex. The H-E shows a lower extractability than that of H- EL and H-SL due to the disrupted cortex structures of H-EL and H-SL from the removal of cortical cells (Figure 5(a) and Figure 5(b)). The corresponding SDS-PAGE results of extracted KAP and Keratin are shown in Figure 9(b). Keratins extracted from hair samples, H-E, H-EL, and H-SL show similar characteristic bands at about 45-50 kDa (type I keratin) and 50-60 kDa (type II keratin), respectively, suggests keratin structure is preserved. However, obvious bands of Type I and type II keratin could also be observed in KAP of H-EL and H-SL, indicating complete removal of cuticle leads to decrease of selectivity of KAP extraction solution.

[000114] The total extractability of cuticle (as labeled as “C”, in Figure 9(a)) is 26%, and about 24% is extracted with KAP solution, only 2% is extracted with keratin extraction solution, agreeing with published results that exocuticle is highly resistant to chemical attack and difficult to be dissolved. The proteins extracted from cuticle isolated with methods disclosed herein has low molecule mass due to part of the peptide bonds in cuticle proteins are cleaved during the long-term processing with serine protease, for instance, esperase. The disulfide bonds are cleaved in the subsequent KAP and keratin extraction process. Therefore, the corresponding SDS-PAGE of cuticle proteins extracted with KAP extraction solution almost show no band, while that of proteins extracted with keratin extraction solution contains bands near 10 kDa. It is reported that K31, K33, K86, and K81 only exist in the cortex; K32 and K82 only exist in the cuticle, while K35 and K85 exist in both the cuticle and cortex. Figure 9 (c) shows the result of western blotting for 4 type I keratins (K31, K33, K35, K32) and 4 type II keratins (K85, K86, K81, and K82) in keratin extracted from hair and three descaled hair samples. Obvious bands could be observed in keratin extracted from intact hair and three descaled hair samples for K31 , K32, K33, K35, K81, K85, K86, while no signal is observed for K82. As shown in figure 9 (d), the normalization result indicated relative content of K32 in the keratin of about 0.22, 0.09, and 0.37 for H- E, H-EL, and H-SL, respectively (keratin extracted from intact hair is the standard). The contents of K35, and K86 in keratins extracted from three descaled hair samples decrease slightly compared to that of virgin hair. K33, K31, K86 and K 81 in descaled hair samples are better retained especially in H-EL and H-SL than H-E, because long treatment time with esterase cause keratin degradation to some degree.

Isolation of Keratin-associated proteins (KAP) and Keratin

Keratin-associated proteins (KAP) and keratin were extracted from human hair following a known extraction protocol. For example, delipidised hair was first incubated in a KAP extraction solution made up of 25mM Tris-HCl buffer, pH 9.5 (Sigma), 8 M urea (Chem-Impex), 200 mM DTT (GoldBiotechnology), and 25% ethanol at 50 °C for about 72 hours [hair / KAP extraction buffer = 2.5g/50 mL). The extracted KAP fraction was filtered and used for other application while the remaining KAP-free hair residues were wash thoroughly with DI water and left to air-dry prior to a subsequent keratin extraction. KAP-free hair (10 g) was added to a pH 8.5 Tris-HCl buffer (200 mL) consisting of 5 M urea, 2.6 M thiourea (Sigma), and 200m M DTT for 24 hours at 50 °C. The extracted keratin fraction was then filtered and used for other applications, while the residue was collected and immediately used to isolate melanosomes using protease treatment. Enzymatic Isolation of Melanosome

[000115] The hair residue generated from 10 g KAP-free hair after keratin extraction was collected and immersed immediately in 25 mM Tris-HCl, pH 9.5, and the total volume was adjusted to 100 mL before 200 m L esperase (enzyme activity: 8 Anson Unit /pL) was added into the system. After the incubation at 50°C for 4 hours, the hydrolysate was filtered through a nylon cloth (800 mesh). The collected filtrate was centrifuged at 8586 g (8000 rpm) for 10 minutes twice. Then, the sediment was resuspended in DI water and centrifuged again at 8586 g (8000 rpm) for 10 minutes, and the supernatant was removed. This procedure was repeated six times to completely remove soluble contaminants like soluble products of the hydrolysis, enzyme and buffer. Finally, the sediment was resuspended in deionised water and freeze-dried into melanosome powder. The schematic of melanosome isolation procedures is shown in Figure 10.

Characterization Of Isolated Melanosomes

[000116] The experimental data as disclosed herein shows that intact hair melanosomes were isolated successfully using the esperase treatment described. Pure, dipersed melanosomes with yields of about 1.3% (by weight of original hair) could be obtained with centrifugation procedures as all protein components in the hair residues could be easily degraded into soluble products, while the melanosomes maintain their structural integrity. Morphological observations of the melanosomes showed that the isolated melanosomes are separate from one another, meaning that the melanosomes were individually isolated. The melanosomes were well defined rod-shaped and ellopsoidal particles with lengths of 904+171 nm and diameters of 341+75 nm (Figure l l(a)(c)). High magnification scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images showed rough surfaces of the hair melanosomes, and melanosome containing 10-60 nm secondary nanoparticles (Figure 1 l(b)(d)). Those results are consistent with published work showing nanoparticles in sepia and human hair mealanosomes, indicating the intergity of the melanosomes was well preserved.

[000117] The transmission electron microscopy (TEM) images of longitudinal and cross sections of the melanosomes were shown in Figure 12. In the membrane-like outer structure of the melanosome, spherical vesicles with diameters of 10-20 nm were evident, along with lamella structures aligned with the long axis in the longitudinal section (Figure 12(a)). In a cross sectional view, a series of concentric ring structures was observed (Figure 12(b)). All these morphological observations in isolated melanosomes corresponded to published results of intact melanosomes in human hair, indicating that the structure of the melanosome isolated using the methods as decribed herein was maintained. [000118] Comparing to the melanosomes isolated using conventional methods, for example, the enzymetic method published by Fiu et al using fungal serine protease as shown in Figure 13(d) or the acid/base method published by Fiu et al (Pigment Cell Res. 16:355-365.2003) shown in Figure 13 (e) and Figure 13(f), the melanosomes isolated using the method disclosed herein are characterised by the intact, non-aggragative appearance. The individual melanosomes do not adhere to one another after being isolated, indicating the efficient removal of hair matrix that binds to the melanosomes. In contrast, the aggregates observed in the published results from Liu et al. indicate the presence of remaining hair matrix proteins, which would have erroneously contributed to the mass of isolated melanosomes in the conventional method.

[000119] The FTIR spectra of human hair and isolated melanosomes are shown in Figure 14. Since hair mainly consists of keratins, while melanosome mainly contains indole structures, the FTIR spectra of hair shows chracteristic absorption bands asscribed to peptide bonds (CONF1-) including the amide A (3000-3600 cm⁴), the amide B (3056-3075 cm⁴), the amide I (1700-1600 cm ), II (1580-1480 cm ) and the amide III (1300-1220 cm ) absorption peaks. The absorpion peak at 3268 cm ¹ and 3073 cm ¹ is assigned to the stretching vibration of N-Fl and O-Fl (amide A), and aromatic C-Fl (amide B), respectively. A strong peak at 1629 cm ¹ should be ascribed to the C=0 stretching vibration (amide I), a medium peak at 1523 cm ¹ is assigned to N-Fl in-plane bending and C-N stretching vibrations (amide II), while the weak band in the range of 1238-1240 cm ¹ is related to the C-N and C-0 stretching vibrations (amide III). Finally, the intense peaks at the range of 1195 to 1021 cm ¹ are related to the asymmetric and symmetric S-0 stretching vibrations of the Bunte salts residues.

[000120] For isolated hair melanosome, the absorption peak of indolic and pyrrolic groups can be observed at 3000-3600 cm⁴, 2950-2980, 1610-1690 cm⁴, 1520-1540 cm⁴, 1400-1500 cm⁴, 1360- 1380 cm⁴, 1200-1240 cm ¹ and 1050-1100 cm⁴. The strong broad absorption peak observed at 3248 cm ¹ (3000-3600 cm⁴) is assigned to the stretching vibrations of the -OF! and -NH groups belonging to the amine, amide, carboxylic acid, phenolic and aromatic amino functions present in the indolic and pyrrolic systems. The spectra of melanin also recored absorption peaks between 2950-2850 cm⁴, which were ascribed to the strethching vibration of aliphatic C-H groups. In this range, very weak absorptions can be observed at 2967, 2940, 2885 cm⁴. In the region of 1700-1480 cm⁴, various vibrational modes overlap and a broad absorption plateau was observed. The absorption at this region are attributed to aromatic C=C, C=N bending (1500-1700 cm¹), amide C=0 asymmetical stretching (1630-1690), and N-H bending from indole, pyrrole in melanin and amino acids. The absorption at 1446 cm ¹ and 1375 cm ¹ are ascribed as aliphatic C-C bending and C-N strethcing, respectively. The weak absorption peaks observed at region of 1200-1240 cm ¹ (1240 cm⁴) are ascribed as phenolic C-OH stretching form both DHICA and DHI while the absorption peaks at 1050-1100 cm ¹ (1081 cm ¹) are ascribed as alcoholic O-H from amino acids. Since hair mainly consists of keratin while melanosome mainly contains indole structure, the spectra of hair shows higher intensities in the regions of 2850-2950 cm⁴, 1455 cm⁴, 1360- 1380 cm ¹ and 1050-1100 cm⁴, which can be explained sufficiently by the different amino acid contents based on the above peak assignment.

[000121] The solid-state ¹³C NMR spectra of intact hair, used as the starting material for the extraction of melanosome and the melanosomes isolated with esperase method, is shown in Figure 15 Hair fibres are mainly composed of proteins (90%) and minor components including lipids (5%) and melanin (1-3%). Therefore, the solid state ¹³C NMR spectrum of hair is dominated by the signal of the most abundant proteins including keratins and keratin-associated proteins (KAPs). In general, KAPs result in ¹³C NMR signals that can be grouped into four characteristic spectral ranges: (1) intense signal in the range of 165-180 ppm mainly due to backbone carbonyls belonging to peptide bonds and to side chain carboxyl and amino groups; (2) weak signal in the aromatic region at 110-160 ppm, due to the bulk of the aromatic residues of pheylalanine tyrosine, tryptophan and hisitidine. A chatacteristic peak centered at 157 ppm was found due to the imine carbon of arginine side chains and the phenolic aroumatic carbon of tyrosine; (3) intense signal in the 45-80 ppm range due to backbone a-carbons and Thr/Ser b-carbons; (4) intense signal in the range of 10-45 ppm due to the side chain methylene, methyl and methyne carbon. To isolate melanosome (from hair) from the keratin matrix in which they are embedded, esperase was used to digest the hard keratin after keratin extration with DTT-urea solution. The solid state ¹³C NMR spectra of the esperase isolated melanosome is also shown in Figure 15. Generally, isolated human hair melanosomes are tightly associated with variable amounts of proteins, giving characteristic spectra. The intense signals found in the carbonyl region (ranged from 160-180 ppm, and centered at 172 ppm) and in the aliphatic region (10-90 ppm) are mostly due to residual proteinous components, whereas the signals in 90-160 ppm are due to both the melanosome and the proteinous components. As proteins mainly contribute to the carboxyl and aliphatic signals, while melanosomes mainly contribute to the aromatic signal, the comparison of the aromatic-to carbonyl signal area ratio (93-160 ppm and 160-190 ppm, repectively) could be used to qualitatively evaluate the relative content of melanin and proteins in the melanosomes extracted from hair. As shown in Table 2 disclosed herein, a previous study has reported the aromatic-to-carbonyl signal area ratio for intact hair to be 0.15, while those of melanosomes isolated with concentrated HC1 and enzymes (papain and DTT) were 2.63 and 0.75, respectively. These values were 2.54 and 0.46 for the isolated melanosomes and intact hair samples using the method disclosed herein, respectively. This suggests a higher melanin content in melanosomes due to more complete removal of hair proteins. In addition, the abundance of the protein matrix in the isolated melanosome has decreased such that the signal at 157 ppm can now be clearly seen which was mostly contributed by the side-chain imine carbon of arginine (Figure 15). This result is in line with the previously published observation that melanosomes from human hair have a high content of arginine. Table 2. Normalized signal areas in ¹³C-NMR spectra of human hair and isolated melanosomes.

a CH-MGs: melanin granules isolated from human hair using concentrated HC1. b CP-MGs: melanin granules isolated from human hair using protease (Papain). c sMGs: synthetic melanin granules synthesized by enzymatic (tyrosinase) oxidation of L-dopa.

Thermal Behaviour Characterisation Of Isolated Melanosomes

[000122] The thermal behaviour of isolated melanosomes was tested using thermogravimetric analysis (TGA, Figure 16 (a)), and corresponding derivative thermogravimetric (DTG) curves (Figure 16(b)). The curves showed the weight loss process could be mainly divided into two stages (30°C - 153°C , 153°C -500°C ), and weight loss rates were larger at the temperature of 65. °C , 346°C . The first stage with endothermic peak at near 65.5°C was mainly due to the evaporation of weakly and /or strongly bound water. An exothermic peak appeared immediately after that at 346°C , which was mainly due to the evolvement of carbon dioxide, water and ammonia during the heating of melanosomes at elevated temperatures. Up to 500°C , the melanosomes only lost about 48.84% of their total mass, which was mainly due to the decarboxylation of carboxylic groups of melanosomes under high temperature conditions. This suggests that the hair melanosomes have significant thermal stability and can potentially be used in polymer blends that are processed at high temperatures in processes such as melt extrusion and injection molding.

[000123] UV-visible absorbance and transmittance spectra of human hair melanosomes suspended in deionised water are shown in Figure 17. Melanosomes at different concentrations showed broad absorption peaks in the UV (280-400 nm) and visible-light (400-800 nm) regions. The absorption intensities in the UV region are higher than that in the visible light region, and the absorption intensity increased with increasing concentrations of melanosomes. Such spectra in transmittance has frequently been observed in various melanin solutions. When the concentration of melanosome was over 0.25 mg/mL, the absorbance curves were almost completely overlapping over the region 200 nm-420 nm, indicating the excellent UV-shielding property of hair melanosomes at concentrations from 0.25 mg/mL (Figure 17(a)). The UV-vis transmittance curves were used to caclulate the percentage blocking for UV including UVA, UVB and UVC and evaluate the transparency of melanosome suspensions (T660), and the results are presented in Table 3.

Table 3. Percentage of blocking from UVA, UVB and UVC of melanosome suspensions at various concentations.

Blocking (%) Transparency

Melanosome (Transmittance % at 660 Concentration (mg/mL)

UV-A UV-B UV-C nm)

0 0 0 0 100

0.025 55.27 57.21 59.79 64.71

0.05 85.85 87.09 88.70 34.20

0.1 97.29 97.73 98.13 12.82

0.25 99.58 99.69 99.69 0.83

0.5 99.59 99.70 99.70 0.20

[000124] The percentage blocking for UV-A(320-400 nm), UV-B(280-320 nm), UV-B(220-280 nm) were caculated by equations 1, 2 and 3, respectively. While the transparency of melanin suspension or films was assessed using the percent transmitance at 660 nm.

UV-A blocking ( 100 (equation 1)

UV-A blocking ( 100 (equation 2)

UV-C blocking 100 (equation 3)

[000125] Where T(l) is average spectral transmittance of fabric, dk is bandwidth, and l is wavelength. The low UV light transmittance of melanin was presumably due to the complex conjugated molecules in the melanin which absorb and scatter photons of UV and blue solar light. The blocking percentage of melanosome suspensions increased with increasing concentration, while the transparency showed a direct opposite trend. At concentrations up to 0.1 mg/mL, the blocking precentages of all UVA, UVB and UVC were above 99%, with transparency of 12.82%. After that, increasing the concentration of melanosome suspensions did not increase the UV blocking precentages further, while transparency decreased sharply. Therefore, human hair melanosomes isolated based on this protocol retains highly effective UV shielding properties, although transparency is compromised. The UV barrier property of melanosomes can be exploited as a UV and photo-blocking agent in various applications.

Claims

CLAIMS What is claimed is:

1. A method of isolating melanosomes from hair, wherein the method comprises:

• exposing hair which has undergone a process of removal of keratin and keratin-associated proteins (KAP) to a solution comprising a bacterial serine protease for at least 0.5 hours to release melanosomes into the solution; and

• isolating melanosomes from the solution.

2. A method of isolating melanosome, keratin, and keratin-associated proteins (KAP) from hair, wherein the method comprises: a) mixing hair in a KAP extraction solution to release the KAP into the KAP extraction solution; b) separating hair and the solution of a); and isolating the KAP from the solution; c) mixing the hair from b) with a keratin extraction solution to release keratin into the keratin extraction solution; d) separating hair and the solution of c); and isolating the keratin from the solution; e) exposing the hair from d) to a solution comprising a bacterial serine protease for at least 0.5 hours to release melanosomes; and f) isolating melanosomes obtained under e).

3. The method of claim 1, further comprising a process of removal of keratin and keratin- associated proteins (KAP) prior to isolating melanosomes from hair, wherein the process comprises: a) mixing hair in a KAP extraction solution to release the KAP into the KAP extraction solution; b) separating hair and the solution of a); and removing the solution containing KAP; c) mixing the hair from b) with a keratin extraction solution to release keratin into the keratin extraction solution; and d) separating hair and the solution of c); and removing the solution containing keratin.

4. The method of claims 1-3, wherein the bacterial serine protease is isolated from a bacterium of Bacillus genus.

5. The method of any one of the preceding claims, wherein the bacterial serine protease is a subtilisin.

6. The method of any one of the preceding claims, wherein the bacterial serine protease is subtilisin 147.

7. The method of any one of the preceding claims, wherein the bacterial serine protease used in the solution comprising the bacterial serine protease is about 125 Anson Unit or more per gram of hair.

8. The method of any one of the preceding claims, wherein the exposure of hair to the solution comprising the bacterial serine protease is at a temperature of 30-70 °C.

9. The method of any one of the preceding claims, wherein the solution comprising the bacterial serine protease can be reused for exposure of a different batch of hair.

10. The method of any one of the preceding claims, wherein the hair is delipidised hair.

11. Isolated melanosomes produced by the method according to any one of the preceding claims wherein the melanosomes are characterized by non-aggregated appearance.

12. A method of isolating cuticle cells from hair, wherein the method comprises: a) exposing hair to a solution comprising a serine protease for 2 to 4 days; b) collecting a foam layer formed on the top of the solution of a); and c) isolating cuticle cells from the collected foam layer of b).

13. The method of claim 12, wherein the serine protease is isolated from a bacterium.

14. The method of claim 13, wherein the bacterium is of Bacillus genus.

15. The method of any one of claims 12-14, wherein the serine protease is a subtilisin.

16. The method of any one of claims 12-15, wherein the serine protease is subtilisin 147.

17. The method of claim 16, wherein the subtilisin 147 used is 320-1280 Anson Unit per gram of hair.

18. The method of any one of claims 12-17, wherein the exposure of hair to the solution comprising the serine protease is at a temperature of 50-60°C.

19. The method of claims 13, 16 and 17, wherein the solution comprises subtilisin 147 as sole bacterial serine protease and exposure of hair to the solution is for about 3 days.

20. The method of any one of claims 12-19, wherein the solution comprising the serine protease can be reused for exposure of a different batch of hair.

21. The method of any one of claims 12-20, wherein the hair is delipidised hair.

22. An isolated intact cuticle cell produced by the method according to any one of claims 12-21, wherein the cuticle cells are characterized by intact flaky appearance.

23. A film prepared from the cuticle cells isolated in the method of claims 12-21, or the isolated intact cuticle cells of claim 22.

24. A method of removing cuticle cells from hair, wherein the method comprises: a) exposing hair to a solution comprising L-cysteine, and at least one or more serine proteases for about 4 hours; and b) removing cuticle cells from the solution of a).

25. The method of claim 24, wherein the at least one or more serine proteases is isolated from a bacterium.

26. The method of claim 25, wherein the bacterium is of Bacillus genus.

27. The method of any one of claims 24-26, wherein the at least one or more serine proteases is a subtilisin.

28. The method of any one of claims 24-27, wherein the at least one or more serine proteases is selected from subtilisin 147 and /or subtilisin 309.

29. The method of claim 28, wherein the concentration of subtilisin 147 used is 320-1280 Anson Unit per gram of hair.

30. The method of claims 28 or 29, wherein the concentration of subtilisin 309 used is about at least 320 Anson Unit per gram of hair.

31. The method of any one of claims 24-30, wherein the concentration of L-cysteine is about 1- 5% (w/v).

32. The method of any one of claims 24-31, wherein the exposure of hair to the solution is at a temperature of 50-60°C.

33. The method of any one of claims 24-32, wherein the solution comprising L-cysteine and at least one or more serine proteases can be reused for exposure of a different batch of hair.

34. The method of any one of claims 24-33, wherein the hair is delipidised hair.

35. The method of any one of claims 1 to 10, 12 to 21 and 24 to 34, wherein the hair is an animal hair with scaly structure.

36. The method of any one of claims 1 to 10, 12 to 21 and 24 to 35, wherein the hair is keratinous hair.

37. The method of any one of claims 1 to 10, 12 to 21 and 24 to 36, wherein the hair is a human hair.

38. The method of any one of claims 1 to 10, 12 to 21 and 24 to 36, wherein the hair is wool.