SEQUENTIAL ENZYMATIC TREATMENT OF COTTON

BACKGROUND

[0001] Cotton is one of the largest crops world-wide. Cotton production is hampered by the increasing costs in water and energy. It is therefore highly desirable to convert raw coarse, immature, waste cottons to mature, fine industrially viable cotton suitable for re-use.

SUMMARY

[0002] The present technology provides an efficient and eco-friendly enzymatic treatment that produces high quality cotton from waste cotton. The cotton thus produced has improved length, maturity, fineness, strength and color, while preventing fiber damage and loss in tensile strength.

[0003] In some embodiments, the present application provides a method of treating cotton that comprises: (a) contacting untreated cotton with a pectino lytic composition comprising at least one pectinolytic enzyme to provide a first intermediate cotton; (b) contacting the first intermediate cotton with a cellulolytic composition comprising at least one cellulolytic enzyme to provide a second intermediate cotton; and (c) contacting the second intermediate cotton with a ligno lytic composition comprising at least one ligno lytic enzyme to provide treated cotton. In some embodiments, step (a) is performed before step (b), and step (b) is performed before step (c).

[0004] In some embodiments, the pectinolytic enzyme is one or more of pectinase, protopectinase, polygalacturonase, pectate lyase, pectin lyase and pectin esterase. In some embodiments, the cellulolytic enzyme is one or more of cellulase, endo-l,4-beta-D-glucanase, beta-l,4-glucanase, beta-l,4-endoglucan hydrolase, celluase A, cellulosin AP, endoglucanase D, alkali cellulase, cellulase A 3, celludextrinase, 9.5 cellulase, avicelase, pancellase SS and 1,4- (1,3, l,4)-beta-D-glucan 4-glucanohydrolase. In yet another embodiment, the lignolytic enzyme is one or more of laccase, lignin peroxidase, manganese peroxidase and multicopper oxidase.

[0005] In some embodiments, the pectinolytic enzymes are supplemented with hydroxyapatite nanoparticles. In some embodiments, the hydroxyapatite nanoparticles are present in the pectinolytic composition in a concentration of about 2 to about 11 μg/ml. In some embodiments, the cellulolytic enzymes are supplemented with hydroxyapatite nanoparticles. In some embodiments, the hydroxyapatite nanoparticles are present in the cellulolytic composition in a concentration of about 2 to about 12 μg/ml. In some embodiments, the lignolytic enzymes are supplemented with copper oxide nanoparticles. In some embodiments, the copper nanoxide particles are present in the lignolytic composition in a concentration of about 0.05 to about 0.25 mM. In some embodiments, the pectinolytic composition further comprises hydroxyapatite nanoparticles; the cellulolytic composition further comprises hydroxyapatite nanoparticles; and the lignolytic composition further comprises copper oxide nanoparticles.

[0006] In some embodiments, at least one of steps (a), (b), and (c) are performed at a temperature of about 50 °C to about 70 °C. In some embodiments, step (a) is performed at a temperature of about 50 °C to about 70 °C; step (b) is performed at a temperature of about 50 °C to about 70 °C; and step (c) is performed at a temperature of about 50 °C to about 70 °C.

[0007] In some embodiments, at least one of steps (a), (b), and (c) are performed at a pH of about 7 to about 10. In some embodiments, step (a) is performed at a pH of about 7 to about 10; step (b) is performed at a pH of about 7 to about 10; and step (c) is performed at a pH of about 7 to about 10.

[0008] In some embodiments, step (a) is performed for about 1 hour to about 6 hours; step (b) is performed for about 1 hour to about 6 hours; and step (c) is performed for about 1 hour to about 6 hours.

[0009] In some embodiments, the treated cotton has a percent maturity value of about 50% to about 90% . In some embodiments, the treated cotton has a micronaire value of about 3.8 to about 4.5. In yet another embodiment, the treated cotton has a fineness value of about 85 mtex to about 100 mtex. In some embodiments, the treated cotton has a percent maturity value of about 64% to about 82%; a micronaire value of about 3.8 to about 4.5; and a fineness value equal of about 88.6 mtex to about 100 mtex.

[0010] In some embodiments, the treated cotton has a percent maturity value that is higher than a percent maturity value of the untreated cotton. In some embodiments, the treated cotton has a micronaire value that is lower than a micronaire value of the untreated cotton. In yet another embodiment, the treated cotton has a fineness value that is lower than a fineness value of the untreated cotton. In some embodiments, the treated cotton has a percent maturity value that is higher than a percent maturity value of the untreated cotton; a micronaire value that is lower than a micronaire value of the untreated cotton; and a fineness value that is lower than a fineness value of the untreated cotton.

[0011] In some embodiments, the method of treating cotton is performed in the absence of hydrogen peroxide. In some embodiments, the method of treating cotton does not comprise contacting the cotton with hydrogen peroxide.

[0012] In some embodiments, the untreated cotton is waste cotton, raw cotton, immature cotton, coarse cotton, or a combination thereof.

[0013] In some embodiments, kits are provided for the treatment of cotton. In some embodiments, the kits comprise: a first container comprising a pectinolytic composition comprising at least one pectinolytic enzyme; a second container comprising a cellulolytic composition comprising at least one cellulolytic enzyme; and a third container comprising a lignolytic composition comprising at least one lignolytic enzyme.

[0014] In some embodiments, the pectinolytic enzyme in the kit is one or more of a pectinase, protopectinase, polygalacturonase, pectate lyase, pectin lyase and pectin esterase. In some embodiments, the cellulolytic enzyme in the kit is one or more of a cellulase, endo-l,4-beta-D- glucanase, beta-l,4-glucanase, beta-l,4-endoglucan hydrolase, celluase A, cellulosin AP, endoglucanase D, alkali cellulase, cellulase A 3, celludextrinase, 9.5 cellulase, avicelase, pancellase SS and 1,4-(1,3, l,4)-beta-D-glucan 4-glucanohydrolase. In some embodiments, the lignolytic enzyme in the kit is one or more of a laccase, lignin peroxidase, manganese peroxidase and multicopper oxidase.

[0015] In some embodiments, the pectinolytic composition in the kit further comprises hydroxyapatite nanoparticles. In some embodiments, the hydroxyapatite nanoparticles are present in a concentration range of about 2 to about 12 μg/ml. In some embodiments, the cellulolytic composition further comprises hydroxyapatite nanoparticles. In some embodiments, the hydroxyapatite nanoparticles are present in a concentration range of about 2 to about 12 μg/ml. In some embodiments, the lignolytic composition further comprises copper oxide nanoparticles. In some embodiments, the copper nanoxide particles are in a concentration range of about 0.05 to about 0.25 mM. [0016] In some embodiments, the pectinolytic composition further comprises hydroxyapatite nanoparticles; the cellulolytic composition further comprises hydroxyapatite nanoparticles; and the lignolytic composition further comprises copper oxide nanoparticles.

[0017] In some embodiments, the kit further comprises instructions for the treatment of cotton.

[0018] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features will become apparent by reference to the following drawings and the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 shows the effect of temperature exposure of cotton during the sequential treatment with pectate lyase, cellulase and laccase treatment on subsequent weight loss of waste cotton. Cotton was treated in sequential fashion first with pectate lyase, then with cellulase and then with laccase. The three enzymes were either all in non-nanoparticulate form or all in nanoparticulate form. Cotton was not rinsed with water between treatments. Treatment was at different temperatures from 30° to 90° C.

[0020] Figure 2 shows the effect of pH of the enzymatic sequential reactions with nanoparticle pectate lyase, cellulase and laccase on subsequent weight loss of waste cotton. Cotton was treated in sequential fashion first with pectate lyase, then with cellulase and then with laccase in two treatments sets. In the first set, the three enzymes were each supplemented with nanoparticles, and in a second set none of the three enzymes was supplemented with nanoparticles. Cotton was not rinsed with water between treatments. Treatment was at different pH values from 3 to 10.

[0021] Figure 3 shows the effect of the length of time of the enzymatic sequential reactions with nanoparticle pectate lyase, cellulase and laccase on subsequent weight loss of waste cotton. Cotton was treated in sequential fashion first with pectate lyase, then with cellulase and then with laccase in two treatments sets. In the first set, the three enzymes were each supplemented with nanoparticles, and in a second set none of the three enzymes was supplemented with nanoparticles. Cotton was not rinsed with water between treatments. Treatment was for different periods of time from 30 minutes to six hours. [0022] Figure 4 shows the effect of enzyme concentration on subsequent weight loss of waste cotton. Column 1 : cotton was treated with pectate lyase; column 2: cotton was treated with cellulase; column 3: cotton was treated with laccase; column 4: cotton was first treated with pectate lyase, then with cellulase and then with laccase, wherein none of the enzymes was supplemented with nanoparticles; Column 5: cotton was first treated with pectate lyase, then with cellulase and then with laccase, wherein each of the enzymes was supplemented with nanoparticles. Cotton was not rinsed with water between treatments.

[0023] Figure 5 shows the effects of the various treatments on cotton texture. Left Panel: no treatment; center panel: cotton was first treated with pectate lyase, then with cellulase and then with laccase in a sequential fashion, wherein none of the enzymes was supplemented with nanoparticles, for a total of 3 hours; right panel: cotton was first treated with pectate lyase, then with cellulase and then with laccase, wherein each of the enzymes was supplemented with nanoparticles, for a total of 3 hours. Cotton was not rinsed with water between treatments.

[0024] Figure 6 shows the effects of the various treatments on micronaire (Figure 6A) and fineness (Figure 6B). Cotton was untreated, treated with pectate lyase, treated sequentially first with pectate lyase, then with cellulase and then with laccase, wherein none of the enzymes was supplemented with nanoparticles, or cotton was treated sequentially first with pectate lyase, then with cellulase and then with laccase, wherein each of the enzymes was supplemented with nanoparticles. Cotton was not rinsed with water between treatments.

[0025] Figure 7 shows the effects of the length of time of the various treatments on the fineness of the final product. Cotton was treated with pectate lyase alone, or treated in sequential fashion first pectate lyase, then with cellulase and then with laccase, wherein either none of the enzymes was supplemented with nanoparticles, or each of the enzymes was supplemented with nanoparticles. Cotton was not rinsed with water between treatments.

[0026] Figure 8 shows changes in cotton maturity in relation to the fineness. Cotton was treated in sequential fashion first with pectate lyase, then with cellulase and then with laccase, wherein either each of the enzymes was supplemented with nanoparticles (Figure 8A), or none of the enzymes was supplemented with nanoparticles (Figure 8B), or it was untreated (Figure 8C). Cotton was not rinsed with water between treatments. DETAILED DESCRIPTION

[0027] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be used, and other changes may be made, without departing from the spirit or scope of the subject matter presented here.

[0028] Disclosed herein are economical and environmental-friendly methods and kit for the conversion of waste, coarse or immature cotton into high quality mature and fine cotton. The methods and kits of the present technology are directed to a sequential enzymatic treatment of cotton, first with the enzyme pectate lyase, then with the enzyme cellulase and then with the enzyme laccase, wherein at least one, at least two or all three enzymes are supplemented with nanoparticles.

[0029] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

[0030] As used herein, "about" will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, "about" will mean up to plus or minus 10% of the particular term.

[0031] As used herein, the term "pectinase" indicates an enzyme that hydrolyzes pectin, and includes without limitation, the enzymes pectolyase, pectozyme and polygalacturonase. In some embodiments, the pectinase disclosed herein is isolated from pectinolytic bacterial strains obtained from soils of various sources. In some embodiments, the pectinase is identified using Ruthedium red staining and then partially purified in two consecutive steps by ion exchange chromatography (CM Sepharose) and gel filtration chromatography (Sephadex G-75). In some embodiments, pectinase activity is measured by reacting pectinase with poly-galactouronic acid (PGA) in 25mM Tris-Cl buffer (pH-8.5) for 2 hours at 200°C and measuring absorbance at 550 nm. [0032] As used herein, the term "cellulolytic enzyme" indicates an enzyme that catalyzes the hydrolysis of cellulose and includes, without limitation, endo-l,4-beta-D-glucanase, endo-1,4- beta-glucanase, carboxymethyl cellulase (CMCase), beta-l,4-glucanase, beta-l,4-endoglucan hydrolase, celludextrinase, celluase A, cellulosin AP, endoglucanase D, alkali cellulase, cellulase A 3, celludextrinase, 9.5 cellulase and 1,4-(1,3, l,4)-beta-D-glucan 4-glucanohydrolase. In some embodiments, the cellulolytic enzymes described herein are isolated from cellulolytic bacterial strains obtained from soils of various sources. In some embodiments, the cellulolytic enzyme is identified using Congo red staining and then partially purified in two consecutive steps by ion exchange chromatography (DEAE cellulosa) and by gel filtration chromatography (Sephadex G- 100). In some embodiments, cellulase activity is determined by reacting the enzyme with dinitrosalicylic and measuring absorbance at 510 nm.

[0033] As used herein, the term "lignolytic enzyme" indicates an enzyme that hydrolyzes lignin and includes laccase, lignin peroxidase, manganese peroxidase and multicopper oxidase. In some embodiments, the lignolytic enzyme is laccase, which oxidizes phenolic compounds and amines. In some embodiments, the laccase is isolated from bacterial strains obtained from soils of various sources. In some embodiments, the enzyme is identified using syringaldazine and then partially purified in three consecutive steps by precipitation with 30-80% ammonium sulphate, ion exchange chromatography (CM Sepharose) and gel filtration chromatography (Sephadex G-75). In some embodiments, laccase activity is determined by reacting the enzyme in 25mM tris-Cl buffer at pH: 8.5 with syringaldazine at 20°C and measuring absorbance at 525 nm.

[0034] As used herein, the term "fiber length" indicates the average length of the longer half of the fibers (upperhalf mean length). In some embodiments, fiber length is expressed in both lOOths and 32nds of an inch, and is measured by passing a "beard" of parallel fibers through an optical sensing point. In some embodiments, the beard is formed when fibers from a sample of cotton are automatically grasped by a clamp, then combed and brushed into parallel orientation. Typically, fiber length affects yarn strength, yarn evenness, and the efficiency of the spinning process. The fineness of the yarn that can be successfully produced from given fibers also is influenced by fiber length. [0035] As used herein, the term "length uniformity" indicates the ratio between the mean length and the upperhalf mean length of the fibers, expressed as a percentage. Length uniformity affects yarn evenness and strength and the efficiency of the spinning process. Cotton with a high uniformity index has a low percentage of short fibers, is easy to process and produces high- quality yarn.

[0036] As used herein, the term "fiber strength" indicates the force in grams required to break a bundle of fibers one tex unit in size, and is measured in grams per tex. A tex unit is equal to the weight in grams of 1,000 meters of fiber. Cotton with high fiber strength can typically withstand breakage during the manufacturing process.

[0037] As used herein, the term "fineness" indicates the mean fiber weight in micrograms per inch. In some embodiments, fineness is determined by measuring the rate of airflow through a porous plug in which a sample of known weight is compressed in a cylinder to known volume. Finer fibres offer a greater resistance to air than coarser fibres because of the greater total surface area. Fineness is generally expressed as gravimetric fineness or linear density (wall area times a constant) .

[0038] As used herein, the term "maturity" indicates the thickness of the cotton cell wall expressed as the ratio between the wall area and the total surface. Maturity affects spinning performance and yarn and fabric quality. In some embodiments, maturity is measured by double compression airflow tests.

[0039] As used herein, the term "micronaire" indicates the weight of one inch of the fibre in micrograms, and refers to a fiber quality trait that measures fiber fineness and maturity. Typically, yarns made from finer fiber have more fibers per cross-section, which results in stronger yarns. Dye absorbency and retention are affected by the maturity of the fibers; the greater the maturity, the better the absorbency and retention. A high micronaire (> 4.5) may indicate that the fiber is coarse and unsuted for spinning, whereas a low micronaire (< 3.8) may indicate that the fibers are immature and may break during textile processing.

[0040] As used herein, the term "trash" indicates the amount of non-lint materials in cotton, such as leaf and bark from the cotton plant. [0041] As used herein, the term "hydroxyapatite nanoparticles" refers to nanoparticles made of hydroxyapatite of the formula Ca ₅ (P0 ₄ )3(OH) and having a size of about 10 nm to about 1000 nm. In some embodiments, the nanoparticles have a size of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 nm. In some embodiments, the nanoparticles have a size of about 20 nm to about 40 nm.

[0042] As used herein, the term "copper oxide nanoparticles" refers to nanoparticles made of copper of the formula Cu ₂ 0(OH) and having a size of about 10 nm to about 1000 nm. In some embodiments, the nanoparticles have a size of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 nm. In some embodiments, the nanoparticles have a size of about 20 nm to about 40 nm.

[0043] The sequential treatment of cotton with pectate lyase, cellulase and laccase in nanoparticulate form achieves effective bio-scouring, bio-polishing, bio-desizing and bio- bleaching of cotton and dramatically improves the micronaire value, maturity and fineness of cotton. The present technology provides economical, environment-friendly methods and kits for the production of high quality cotton (for example, fine and mature cotton) from waste, coarse or hard cotton in a short period of time. In some embodiments, the method can be performed from about 1 hour to about 20 hours. In some embodiments, the methods is performed in about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 hours. In some embodiments the method can be performed from about 3 hours to about 10 hours, from about 4 hours to about 8 hours, or from about 5 hours to about 6 hours. In some embodiments, high quality cotton can be produced from waste, coarse or hard cotton in about 5 hours to about 6 hours. By way of example, but not by way of limitation, in some embodiments, cotton is sequentially treated first with a pectionolytic enzyme, then with a cellulosyc enzyme and then with a lignolytic enzyme.

[0044] The enzymes used in the present technology may be isolated from natural sources such as bacteria. Pectinolytic enzymes including pectinase, protopectinase, polygalacturonase, pectate lyase, pectin lyase and pectin esterase, effectively perform bio-scouring of the fibers. Cellulolytic enzymes, including cellulose and endoglucanase, effectively perform bio-polishing and bio-desizing of the cotton. Lignolytic enzymes, including laccase, lignin peroxidase, manganese peroxidase and multicopper oxidase, perform bio-bleaching of the cotton. The method and kits of the present technology are non-toxic and water and energy savers as they avoid the use of sodium hydroxide and hydrogen peroxide, which are conventionally used for scouring and bleaching.

[0045] In one embodiment, the enzymes are supplemented with nanoparticles In some embodiments, this is accomplished by combining the enzymes with nanoparticles, e.g., nanoparticles having a size of about 20 nm to about 40 nm. In some embodiments, the pectinolytic enzyme is supplemented with hydroxyapatite nanoparticles. In some embodiments, the cellulolytic enzyme is supplemented with hydroxyapatite nanoparticles. In some embodiments, the lignolytic enzyme is supplemented with copper oxide nanoparticles.

[0046] In some embodiments, the pectinolytic enzyme is supplemented with hydroxyapatite nanoparticles in a concentration from about 1 μg/ml to about 100 μg/ml. In some embodiments, the concentration of hydroxyapatite nanoparticles is about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg/ml. In some embodiments, the concentration of hydroxyapatite nanoparticles is about 1 to about 90, about 10 to about 80, about 20 to about 70, about 30 to about 60 or about 40 to about 50 μg/ml. In some embodiments, the concentration is about 1-15, 1 -14, 1-13, 1 -12, 1-1 1, 1 -10, 1-9, 1 -8, 1-7, 1-6 or about 1 -5 μg/ml. In some embodiments, the concentration is about 2-15, 2- 14, 2-13, 2-12, 2-11 , 2-10, 2-9, 2-8, 2-7, 2-6 or about 2-5 μg/ml. In some embodiments, the concentration is about 2 to about 1 1 μg/ml.

[0047] In some embodiments, the cellulolytic enzyme is supplemented with hydroxyapatite nanoparticles in a concentration of about 1 μg/ml to about 100 μg/ml. In some embodiments, the concentration of nanoparticles is about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg/ml. In some embodiments, the concentration of nanoparticles is from about 1 to about 90, about 10 to about 80, about 20 to about 70, about 30 to about 60 or about 40 to about 50 μg/ml. In some embodiments, the concentration is about 1 -15, 1-14, 1 -13, 1-12, 1 -11, 1-10, 1-9, 1-8, 1 -7, 1 -6 or about 1 -5 μg/ml. In some embodiments, the concentration is about 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6 or about 2-5 μg/ml. In some embodiments, the cellulolytic enzyme is supplemented with hydroxyapatite nanoparticles in a concentration from about 2 μg/ml to about 12 μg/ml.

[0048] In some embodiments, the lignolytic enzyme is supplermented with copper oxide nanoparticles in a concentration of about 0.01 mM to about 1 mM. In some embodiments, the concentration range of nanoparticles is about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8 0.85, 0.9, 0.95 or 1 mM. In some embodiments, the concentration range of nanoparticles is about 0.01 to 0.9, about 0.02 to 0.8, about 0.03 to 0.7, about 0.04 to 0.6, about 0.05 to 0.5, about 0.06 to 0.4, about 0.07 to 0.3, about 0.08 to 0.2, about 0.09 to 0.1 mM. In some embodiments, the lignolytic enzyme is supplemented with copper oxide nanoparticles in a concentration of about 0.05 mM to about 0.25 mM. One or more of the enzymes may be supplemented with nanoparticles.

[0049] In some embodiments, the treatment of cotton according to the technology disclosed herein is performed at a temperature of about 30° to about 90° C. In some embodiments, the temperature is about 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85° or 90° C. In some embodiments, the temperature is about 50° to about 70° C.

[0050] In some embodiments, the pH is about 5, 6, 7, 8, 9 or 10. In some embodiments, the pH is about 7 to about 10.

[0051] In some embodiments, the treatment of cotton according to the technology disclosed herein is performed at a temperature of about 50° C to about 70° C, at a pH of about 7 to about 10, for a period of time of about 1 hour to about 6 hours.

[0052] The methods and kits of the present technology produce high quality cotton, as measured by micronaire, maturity and fineness. Cotton treated according to the methods and/or kits of the present technology can have a micronaire value that is lower than untreated cotton, a maturity value that is higher than untreated cotton and a fineness value that is lower than untreated cotton.

[0053] Micronaire is an indicator of air permeability and expresses the relation between fineness (linear density) and maturity (degree of cell- wall development). Maturity is a measure of primary and secondary wall thickness and expresses the relative degree of thickening of the fibre wall. Fineness is the mean fiber diameter which is usually expresses in microns. Fineness is expressed as gravimetric fineness or linear density (wall area times a constant). In one embodiment, the methods and kits of the present technology produce a cotton with a micronaire value of about 3.8 to about 4.5. Additionally or alternatively, in some embodiments, the methods and kits of the present technology produce a cotton with a maturity value of about 64% to about 82%. Additionally or alternatively in some embodiments, the methods and kits of the present technology produce a cotton with a fineness value of about 88.6 mtex to about 100 mtex. [0054] In some embodiments, the technology provides a kit for the treatment of cotton. In one embodiment, the kit comprises a first container comprising a pectinolytic composition comprising at least one pectinolytic enzyme; a second container comprising a cellulolytic composition comprising at least one cellulolytic enzyme; and a third container comprising a lignolytic composition comprising at least one lignolytic enzyme, and instructions for the treatment of cotton.

[0055] The pectinolytic enzyme may be a pectinase, protopectinase, polygalacturonase, pectate lyase, pectin lyase or pectin esterase. The cellulolytic enzyme may be a cellulase, endo-l,4-beta- D-glucanase, beta-l,4-glucanase, beta-l,4-endoglucan hydrolase, celluase A, cellulosin AP, endoglucanase D, alkali cellulase, cellulase A 3, celludextrinase, 9.5 cellulase, avicelase, pancellase SS or 1,4-(1,3, 1 ,4)-beta-D-glucan 4-glucanohydrolase. The lignolytic enzyme may be a laccase, lignin peroxidase, manganese peroxidase or multicopper oxidase.

[0056] In some embodiments, the enzymes are supplemented with nanoparticles and the nanoparticles have a size of about 20 nm to about 40 nm. In some embodiments, the pectinolytic enzyme is supplemented with hydroxyapatite nanoparticles in a concentration of about 2 μg/ml to about 11 μg/ml; the cellulolytic enzyme is supplemented with hydroxyapatite nanoparticles in a concentration of about 2 μg/ml to about 12 μg/ml; and the lignolytic enzyme is supplemented with copper oxide nanoparticles in a concentration of about 0.05 mM to about 0.25 mM. In some embodiments, one or more of the enzymes in the kit may be supplemented with nanoparticles. By way of example, but not by way of limitation, in one embodiment, the pectinolytic enzyme is supplemented with hydroxyapatite nanoparticles; the cellulolytic enzyme is supplemented with hydroxyapatite nanoparticles; and the lignolytic enzyme is supplemented with copper oxide nanoparticles.

[0057] The environmentally friendly method and kit of the present technology can be used for a variety of purposes. Typically, the methods and kits disclosed herein do not use hydrogen peroxide or other harsh chemicals; that is, the cotton is not contacted with hydrogen peroxide or other harsh chemicals during processing. In some embodiments, the present technology may be used for the recycling of waste cotton into fine cotton for industristal applications. In some embodiments, the present technology may be used for the recycling of waste cotton into bioethanol. In other embodiments, the present technology may be used for the recycling of waste cotton into animal feed.

[0058] The following examples are set forth to aid the reader in understanding the present technology. These examples are not to be construed as limiting the scope of the technology in any manner. It should be understood that many variations and modifications can be made while remaining within the spirit and scope of the technology.

EXAMPLES

[0059] The present technolgy is further illustrated by the following examples, which should not be construed as limiting in any way.

Example 1: Enzyme Isolation, Purification and Activity

[0060] Pectinase enzymes were isolated from pectinolytic bacterial strains which were obtained from soils of various sources. Bacterial colonies were grown on YP agar plates at 37°C, stained with ruthedium red and washed. Pectinases were then partially purified from the stained bacterial colonies in two consecutive steps, first by ion exchange chromatography (CM Sepharose) and then by gel filtration chromatography (Sephadex G-75). Pectinase activity was determined using poly-galactouronic acid (PGA) as substrate in a 25mM Tris-Cl buffer solution (pH-8.5). The reaction was performed for 2 hours at 200°C, and the solution absorbance at 550 nm was then measured.

[0061] Cellulolytic enzymes were isolated from cellulolytic bacterial strains which were obtained from soils of various sources. Bacterial colonies were grown on CMC-agar plates at 37°C, stained with Congo red and washed. Cellulolytic enzymes were then partially purified in two consecutive steps, first by ion exchange chromatography (DEAE cellulosa) and by gel filtration chromatography (Sephadex G-100). Cellulase activity was determined using dinitrosalicylic (DNS) as substrate. A 1 ml culture filtrate was diluted with 1 ml of distilled water, 3 ml of DNS reagent was added, and the solution was heated in a boiling water bath for 5 minutes. The solution was then allowed to cool at room temperature and 7 ml of freshly prepared 40% sodium potassium tartarate was added. After cooling, the solution absorbance at 510 nm was measured using a U.V. spectrophotometer and the amount of reducing sugar was determined. [0062] Lignolytic enzymes (laccase, lignin peroxidase, manganese peroxidase and multicopper oxidase) were isolated from bacterial strains obtained from soils of various sources. Bacterial colonies were grown on agar plates at 37°C, stained with syringaldazine and washed. Lignolytic enzymes were then partially purified in three consecutive steps by precipitation with 30-80% ammonium sulphate, ion exchange chromatography (CM Sepharose) and gel filtration chromatography (Sephadex G-75). Laccase activity was determined by reacting the enzyme in 25mM tris-Cl buffer at pH: 8.5 with syringaldazine at 20°C. A 1ml solution was added to 3 ml of 25mM tris-Cl buffer, pH: 8.5, and 2 ml syringaldazine in methanol (1 :2 dilution by Dioxan) was added. Absorbance was determined at 525 nm.

[0063] Protease was purified by three consecutive processes, first by 30-80% ammonium sulphate precipitation, then by ion exchange chromatography (CM Sepharose), and then by gel filtration chromatography (Sephadex G-50).

[0064] Xylanase was partially purified by two consecutive processes, first by ion exchange chromatography (CM Sepharose) and then by gel filtration chromatography (Sephadex G-50). Nanoparticle (NP) Supplementation

[0065] For nanoparticle supplementation, each enzyme was supplemented with nanoparticles as follows: pectate lyase was supplemented with 8.8 μg/ml hydroxyapatite nanoparticle (Hap NP); cellulase was supplemented with 10 μg/ml Hap NP; and laccase was supplemented with 0.1 mM copper oxide nanoparticles (Cu ₂ 0 NP). All enzymes and nanoparticles were in 25 rriM Tris-HCl, pH 8.5 buffer. The enzyme concentration was as follows: pectate lyase: 852 unit/mg; laccase: 2327.23 unit/mg; and cellulase: 156.8 unit/mg.

Example 2: Treatment of Waste Cotton

[0066] A 4 g sample of waste cotton was washed with distilled water several times, dried at room temperature and autoclaved. The autoclaved waste cottons were then sequentially treated first with purified pectate lyase, then with purified cellulase, and then with laccase, for a total of three hours. The three enzymes were either all supplemented with nanoparticles or none of the enzymes was supplemented with nanoparticles. Cotton was not rinsed with water between treatments. The effect of temperature, pH, and length of time of the reaction, as well as the effect of enzyme concentration on weight loss of cotton were then determined. (Figure 5). Temperature

[0067] To determine the effect of temperature, sequential treatment of cotton with the enzymes supplemented with nanoparticles or with the enzymes that were not supplemented with nanoparticles was performed at temperatures from 30°C to 90°C, and the weight loss of treated cotton was then determined.

[0068] Weight loss of cotton was not detectable at lower temperatures (30-40°C) and at temperatures higher than 70°C after sequential enzymatic treatment, independent on whether the enzymes were supplemented with nanoparticles or not. The weight loss was 9.2% at 50°C, 14.7% at 60°C and 8.7% at 70°C for cotton sequentially treated with pectate lyase, cellulase and laccase when the enzymes were not supplemented with nanoparticles, and 12.2% at 50°C, 16.8% at 60°C and 10.7% at 70°C for cotton sequentially treated with pectate lyase, cellulase and laccase when the enzymes were supplemented with nanoparticles (Figure 1).

pH

[0069] To determine the effect of pH, sequential treatment of cotton with the enzymes supplemented with nanoparticles or with the enzymes that were not supplemented with nanoparticles was performed at pH values from 3 to 10, and the weight loss of the treated cotton was then determined.

[0070] Weight loss was not substantial at acidic pH (pH 3-6) and at pH above 9 in cotton after sequential enzymatic treatment, independent on whether the enzymes were supplemented with nanoparticles or not. The maximum weight loss was 2.5% at pH 6. At basic pH (pH 7-9), the weight loss was 4.8% at pH 7, 9.5% at pH 8 and 7.6% at pH 9 for cotton sequentially treated with pectate lyase, cellulase and laccase when the enzymes were not supplemented with nanoparticles, and 6% at pH 7, 11.2% at pH 8 and 8.5% at pH 9 for cotton sequentially treated with pectate lyase, cellulase and laccase when the enzymes were supplemented with nanoparticles (Figure 2).

Length of Reaction

[0071] To determine the effect of the length of the reaction, sequential treatment of cotton with the enzymes supplemented with nanoparticles or with the enzymes that were not supplemented with nanoparticles was performed for a period of time from 30 minutes to six hours, and the weight loss of the treated cotton was then determined.

[0072] Weight loss was not detectable after 30 minute incubation, independent on whether the enzymes were supplemented with nanoparticles or not. After longer periods of incubation, the weight loss was 4.3% after 1 hour, 6.6% after 2 hours, 9.2% after 3 hours and 13.5% after 4 hours for cotton sequentially treated with pectate lyase, cellulase and laccase when the enzymes were not supplemented with nanoparticles, and 5.8% after 1 hour, 8.7% after 2 hours, 12.8% after 3 hours and 15.8% after 4 hours for cotton sequentially treated with pectate lyase, cellulase and laccase when the enzymes were supplemented with nanoparticles. Weight loss reached a plateau after longer period of times (Figure 3).

Enzyme Concentration

[0073] To determine the effect of enzyme concentration, sequential treatment of cotton with the enzymes supplemented with nanoparticles or with the enzymes that were not supplemented with nanoparticles was performed using an enzyme concentration from 1 mg/ml to 5 mg/ml, and the weight loss of the treated cotton was then determined.

[0074] Weight loss increased with increased enzyme concentrations, with the optimal concentration being 4 mg/ml. The weight loss was 3.4% at an enzyme concentration of 1 mg/ml, 4.1 at 2 mg/ml, 10.35% at 3 mg/ml, 14.5% at 4 mg/ml and 11.1% at 5 mg/ml for cotton sequentially treated with pectate lyase, cellulase and laccase when the enzymes were not supplemented with nanoparticles, and 17.3% at 4 mg/ml for cotton sequentially treated with pectate lyase, cellulase and laccase when the enzymes were supplemented with nanoparticles. Weight loss reached a plateau after longer period of times. (Results not shown).

Estimation of Cotton fiber quality

[0075] Cotton fiber quality before and after sequential enzymatic treatment was estimated using micronaire, maturity and fineness as parameters using Altra Fiber Fineness and Maturity Tester.

[0076] The micronaire value of the treated cotton decreased with the different enzymatic treatments. Treatment with pectate lyase that was not supplemented with nanoparticles alone resulted in a micronaire value that was lower than the micronaire value of untreated cotton. The micronaire value further decreased after sequential treatment with cellulase and laccase that were not supplemented with nanoparticles. Sequential treatment with the three enzymes resulted in the lowest micronaire value when each of the three enzymes were supplemented with nanoparticles. (Table 1 and Figure 6A). A micronaire value from 3.8 to 4.5 indicates that the fibers had adequate fineness and good maturity (Table 1). Higher micronaire (> 4.5) is indicative of a coarse fiber, which is undesirable for spinners, because it yields few fibers in yarn cross section and thus reduces its strength. Low micronaire (< 3.8) is indicative of immature fibers that break easily and have poor dye uptake during textile processing.

[0077] The maturity of the treated cotton increased with the different enzymatic treatments. Treatment with pectate lyase that was not supplemented with nanoparticles alone resulted in a maturity value that was much higher (64.02) than the maturity value of untreated cotton (39.14). The maturity value further increased after sequential treatment with cellulase and laccase that were not supplemented with nanoparticles (73.41). Sequential treatment with the three enzymes resulted in the highest maturity value (81.07) when each of the three enzymes were supplemented with nanoparticles (Table 1 and Figure 6B).

[0078] The fineness value of the treated cotton decreased with the different enzymatic treatments. Treatment with pectate lyase that was not supplemented with nanoparticles alone resulted in a fineness value (98.78 mtex) that was lower than the fineness value of untreated cotton (467.93 mtex). The fineness value slightly decreased after sequential treatment with cellulase and laccase that were not supplemented with nanoparticles (95.64 mtex). Sequential treatment with the three enzymes resulted in the lowest micronaire value (3.78) when each of the three enzymes were supplemented with nanoparticles. (Table 1 and Figure 6C).

Table 1: Parameters for the estimation of cotton fiber quality following sequential enzyme treatment of waste cotton

Fineness 467.93 98.78 95.64 88.43

(mtex)

[0079] Changes in cotton maturity and fineness as a function of the length of time of the enzymatic treatments are shown in Figures 7 and 8A-C. The results overall show that sequential treatment of cotton with pectate lyase, cellulase, and laccase dramatically improves the micronaire value, maturity and fineness of cotton when each of the three enzymes are supplemented with nanoparticles.

[0080] The embodiments, 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 claimed technology. Additionally, the phrase "consisting essentially of will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase "consisting of excludes any element not specified.

[0081] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of various aspects. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent compositions, apparatuses, and methods within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. [0082] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0083] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to," "at least," "greater than," "less than," and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

[0084] While certain embodiments have been illustrated and described, it should be understood that changes and modifications can be made therein in accordance with ordinary skill in the art without departing from the technology in its broader aspects as defined in the following claims.