TW202336316A - Process for dyeing a cellulosic material - Google Patents

Process for dyeing a cellulosic material Download PDF

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TW202336316A
TW202336316A TW111148843A TW111148843A TW202336316A TW 202336316 A TW202336316 A TW 202336316A TW 111148843 A TW111148843 A TW 111148843A TW 111148843 A TW111148843 A TW 111148843A TW 202336316 A TW202336316 A TW 202336316A
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group
formula
laccase
dye
borane
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TW111148843A
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艾維達 瑪考
古伊爾勞麥 波伊索納特
大衛 古伊西
席夢 瑪葛里 雷默德
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法商Ｐｉｌｉ公司
法國圖盧茲應用科學學院
法國國家農業食品與環境研究院
法國國家科學研究中心
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    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P1/00—General processes of dyeing or printing textiles, or general processes of dyeing leather, furs, or solid macromolecular substances in any form, classified according to the dyes, pigments, or auxiliary substances employed
    • D06P1/38—General processes of dyeing or printing textiles, or general processes of dyeing leather, furs, or solid macromolecular substances in any form, classified according to the dyes, pigments, or auxiliary substances employed using reactive dyes
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P1/00—General processes of dyeing or printing textiles, or general processes of dyeing leather, furs, or solid macromolecular substances in any form, classified according to the dyes, pigments, or auxiliary substances employed
    • D06P1/38—General processes of dyeing or printing textiles, or general processes of dyeing leather, furs, or solid macromolecular substances in any form, classified according to the dyes, pigments, or auxiliary substances employed using reactive dyes
    • D06P1/382—General processes of dyeing or printing textiles, or general processes of dyeing leather, furs, or solid macromolecular substances in any form, classified according to the dyes, pigments, or auxiliary substances employed using reactive dyes reactive group directly attached to heterocyclic group
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P1/00—General processes of dyeing or printing textiles, or general processes of dyeing leather, furs, or solid macromolecular substances in any form, classified according to the dyes, pigments, or auxiliary substances employed
    • D06P1/38—General processes of dyeing or printing textiles, or general processes of dyeing leather, furs, or solid macromolecular substances in any form, classified according to the dyes, pigments, or auxiliary substances employed using reactive dyes
    • D06P1/384—General processes of dyeing or printing textiles, or general processes of dyeing leather, furs, or solid macromolecular substances in any form, classified according to the dyes, pigments, or auxiliary substances employed using reactive dyes reactive group not directly attached to heterocyclic group
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P1/00—General processes of dyeing or printing textiles, or general processes of dyeing leather, furs, or solid macromolecular substances in any form, classified according to the dyes, pigments, or auxiliary substances employed
    • D06P1/41—General processes of dyeing or printing textiles, or general processes of dyeing leather, furs, or solid macromolecular substances in any form, classified according to the dyes, pigments, or auxiliary substances employed using basic dyes
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P3/00—Special processes of dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form, classified according to the material treated
    • D06P3/58—Material containing hydroxyl groups
    • D06P3/60—Natural or regenerated cellulose
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P3/00—Special processes of dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form, classified according to the material treated
    • D06P3/58—Material containing hydroxyl groups
    • D06P3/60—Natural or regenerated cellulose
    • D06P3/6016—Natural or regenerated cellulose using basic dyes
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P3/00—Special processes of dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form, classified according to the material treated
    • D06P3/58—Material containing hydroxyl groups
    • D06P3/60—Natural or regenerated cellulose
    • D06P3/66—Natural or regenerated cellulose using reactive dyes
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P3/00—Special processes of dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form, classified according to the material treated
    • D06P3/58—Material containing hydroxyl groups
    • D06P3/60—Natural or regenerated cellulose
    • D06P3/66—Natural or regenerated cellulose using reactive dyes
    • D06P3/663—Natural or regenerated cellulose using reactive dyes reactive group directly attached to heterocyclic group
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P3/00—Special processes of dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form, classified according to the material treated
    • D06P3/58—Material containing hydroxyl groups
    • D06P3/60—Natural or regenerated cellulose
    • D06P3/66—Natural or regenerated cellulose using reactive dyes
    • D06P3/666—Natural or regenerated cellulose using reactive dyes reactive group not directly attached to heterocyclic group
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P5/00—Other features in dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form
    • D06P5/002—Locally enhancing dye affinity of a textile material by chemical means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0061—Laccase (1.10.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
    • C12Y110/03002—Laccase (1.10.3.2)
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
    • D06P5/00—Other features in dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form
    • D06P5/22—Effecting variation of dye affinity on textile material by chemical means that react with the fibre

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  • Engineering & Computer Science (AREA)
  • Textile Engineering (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
  • Coloring (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The present invention relates to the field of dyeing processes. More particularly, it relates to a process for dyeing a cellulosic material, comprising an enzymatic oxidation of a cellulosic material followed by a reductive amination in the presence of an aminated dye.

Description

纖維素材料之染色方法Dyeing methods for cellulose materials

本發明係關於染色方法之領域。更明確而言，係關於一種纖維素材料之染色方法，該方法包含纖維素材料之酶促氧化，隨後在胺化染料存在下進行還原胺化。The present invention relates to the field of dyeing methods. More specifically, it relates to a method for dyeing cellulosic materials, which method involves enzymatic oxidation of cellulosic materials followed by reductive amination in the presence of an amined dye.

反應性染料具有較廣顏色範圍且常用於纖維素纖維，諸如棉及亞麻的染色。在反應性染料方法中，染料分子擴散至纖維中且經由連接至染料發色團之反應性官能基(諸如氯三嗪、乙烯碸或溴丙烯酸酯)與纖維建立化學鍵。由於與纖維形成此永久性鍵，因此反應性染料無法藉由在中性條件下用沸水反覆處理而輕易移除。因此，染料變為纖維之一部分，且從而具有出色的耐洗色牢度。Reactive dyes have a wide color range and are commonly used to dye cellulosic fibers such as cotton and linen. In the reactive dye approach, dye molecules diffuse into the fiber and establish chemical bonds with the fiber via reactive functional groups attached to the dye chromophore, such as chlorotriazine, vinylene, or bromoacrylate. Because of this permanent bond with the fiber, reactive dyes cannot be easily removed by repeated treatment with boiling water under neutral conditions. The dye therefore becomes part of the fiber and thus has excellent color fastness to washing.

然而，基於反應性染料之方法具有若干缺點。特定言之，反應性染料之製備並不簡單，因為必須根據發色團之性質及與其連接之反應性官能基來研發特定合成方法。此外，必須使用苛刻的染色條件，諸如高達14之高鹼性pH，及高達80℃之高溫，以便在染料與纖維素纖維之間產生鍵結。使用此類鹼性pH亦不理想，因為其在染色過程期間誘導反應性染料之高速率水解，且因此可在廢水中發現大量反應性染料。However, reactive dye-based approaches have several disadvantages. Specifically, the preparation of reactive dyes is not simple because specific synthetic methods must be developed based on the nature of the chromophore and the reactive functional groups attached to it. In addition, harsh dyeing conditions, such as a highly alkaline pH of up to 14, and high temperatures of up to 80°C, must be used to create bonds between the dye and cellulose fibers. The use of such an alkaline pH is also undesirable since it induces a high rate of hydrolysis of the reactive dyes during the dyeing process and therefore large amounts of reactive dyes can be found in the wastewater.

為克服上述缺點，已在過去幾年中進行替代性染料及染色方法的研發。特定言之，已經研究在染色方法中蟲漆酶將酚及芳族化合物轉化為有色物質之用途。例如，Hadzhiyska等人(Biotechnology Letters 2006, 28, 755)研發出一種染色方法，其中使棉與兒茶酚及2,5-二胺基苯磺酸在蟲漆酶存在下、在30℃至70℃之溫度下及在5之pH下接觸。然而，棉與染料之間的弱鍵結不允許獲得高耐洗色牢度。此外，Diaz Blanco等人(Enzyme and Microbial Technology 2009, 44, 380)已研發一種染色方法，其包含對棉材料進行甲苯磺醯化；將芳族胺接枝於甲苯磺醯化之棉上，且隨後使經接枝棉與蟲漆酶氧化之兒茶酚共聚合。然而，此類方法包含很多步驟，其使得難以實施，尤其難以在工業規模下實施。此外，該方法使用化學計量之甲苯磺醯化劑及吡啶，其為腐蝕性、有毒且極其昂貴的。To overcome the above disadvantages, the development of alternative dyes and dyeing methods has been carried out over the past few years. In particular, the use of laccase in converting phenolic and aromatic compounds into colored substances in dyeing processes has been studied. For example, Hadzhiyska et al. (Biotechnology Letters 2006, 28, 755) developed a dyeing method in which cotton was mixed with catechol and 2,5-diaminobenzenesulfonic acid in the presence of laccase at 30°C to 70°C. °C and in contact at a pH of 5. However, the weak bond between cotton and dye does not allow high wash fastness to be achieved. In addition, Diaz Blanco et al. (Enzyme and Microbial Technology 2009, 44, 380) have developed a dyeing method that includes toluenesulfonation of cotton materials; grafting aromatic amines onto the toluenesulfonation of cotton, and The grafted cotton was then copolymerized with laccase-oxidized catechol. However, such methods involve many steps, which make them difficult to implement, especially on an industrial scale. Additionally, this method uses stoichiometric amounts of toluene chelating agent and pyridine, which are corrosive, toxic and extremely expensive.

因此，仍需要提供一種藉由可在溫和及環境友好條件下實施的具有成本效益的方法獲得之染色纖維素材料。Therefore, there remains a need to provide a dyed cellulosic material obtained by a cost-effective process that can be carried out under mild and environmentally friendly conditions.

在此情形下，本發明人已研發出一種纖維素材料之兩步染色法。此方法包含使纖維素材料與氧化還原酶及氧化還原介質接觸，且隨後使所得纖維素材料與包含至少一個NH 2基團之染料在還原劑存在下接觸。此兩步法為具有成本效益且環境友好的。特定言之，該方法使用便宜且無毒之試劑，其有利地以催化量使用。此外，該方法可在溫和條件下實施，特定言之在低溫下及在中等酸性至中等鹼性pH條件下實施。此方法允許在胺基染料與纖維素材料之間建立強共價連接，從而獲得高色牢度。 Under this situation, the present inventors have developed a two-step dyeing method for cellulosic materials. This method involves contacting a cellulosic material with a redox enzyme and a redox mediator, and subsequently contacting the resulting cellulosic material with a dye comprising at least one NH group in the presence of a reducing agent. This two-step method is cost-effective and environmentally friendly. In particular, this method uses cheap and non-toxic reagents, which are advantageously used in catalytic amounts. Furthermore, the method can be carried out under mild conditions, in particular at low temperatures and under moderately acidic to moderately alkaline pH conditions. This method allows the establishment of strong covalent linkages between amine dyes and cellulosic materials, resulting in high color fastness.

因此，本發明係關於一種纖維素材料之染色方法，其包含以下步驟： (a)使纖維素材料與氧化還原酶及氧化還原介質接觸，以獲得氧化纖維素材料； (b)在還原劑存在下，使步驟(a)之氧化纖維素材料與包含至少一個NH 2基團之染料接觸，以獲得染色纖維素材料；及 (c)回收染色纖維素材料。 Therefore, the present invention relates to a method for dyeing cellulose materials, which includes the following steps: (a) contacting cellulose materials with oxidoreductase and redox media to obtain oxidized cellulose materials; (b) in the presence of a reducing agent Next, contacting the oxidized cellulose material of step (a) with a dye comprising at least one NH 2 group to obtain a dyed cellulose material; and (c) recovering the dyed cellulose material.

在一特定實施例中，步驟(b)中之該染料為式(I)化合物： [H 2N-(X-Y) n] m-Z   (I)， 其中： - n為0或1， - m為1至4之整數， - X為伸芳基或伸雜芳基，該基團視情況經選自由以下組成之群的至少一個取代基取代：(C 1-C 12)烷基、(C 2-C 12)烯基、(C 2-C 12)炔基、(C 3-C 12)環烷基、(C 2-C 12)雜環烷基、芳基、雜芳基、鹵素、-CN、-NO 2、-C(O)R 1、-C(O)OR 2、-C(O)NR 3R 4、-S(O) 2R 5、-S(O) 2OR 6、-NHS(O) 2R 7、-NHS(O) 2OR 8、-OR 9、-SR 10、及-NR 11R 12， R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R 11及R 12各自獨立地選自氫、(C 1-C 12)烷基、(C 2-C 12)烯基、(C 2-C 12)炔基、(C 3-C 12)環烷基、(C 2-C 12)雜環烷基、芳基及雜芳基， - Y為選自以下基團之間隔基：(C 1-C 12)伸烷基、(C 2-C 12)伸烯基、(C 2-C 12)伸炔基、(C 3-C 12)伸環烷基、(C 2-C 12)伸雜環烷基、伸芳基及伸雜芳基，該基團視情況間雜有選自以下之至少一個基團：-O-、-S-、-NH-、-C(O)-及-S(O) 2，及 - Z為發色團部分， 或其鹽。 In a specific embodiment, the dye in step (b) is a compound of formula (I): [H 2 N-(XY) n ] m -Z (I), where: - n is 0 or 1, - m is an integer from 1 to 4 , - 2 -C 12 )alkenyl, (C 2 -C 12 )alkynyl, (C 3 -C 12 )cycloalkyl, (C 2 -C 12 )heterocycloalkyl, aryl, heteroaryl, halogen, -CN, -NO 2 , -C(O)R 1 , -C(O)OR 2 , -C(O)NR 3 R 4 , -S(O) 2 R 5 , -S(O) 2 OR 6 , -NHS(O) 2 R 7 , -NHS(O) 2 OR 8 , -OR 9 , -SR 10 , and -NR 11 R 12 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6. R 7 , R 8 , R 9 , R 10 , R 11 and R 12 are each independently selected from hydrogen, (C 1 -C 12 ) alkyl, (C 2 -C 12 ) alkenyl, (C 2 - C 12 ) alkynyl, (C 3 -C 12 ) cycloalkyl, (C 2 -C 12 ) heterocycloalkyl, aryl and heteroaryl, - Y is a spacer selected from the following groups: (C 1 -C 12 )alkylene group, (C 2 -C 12 ) alkenylene group, (C 2 -C 12 ) alkynylene group, (C 3 -C 12 ) cycloalkylene group, (C 2 -C 12 ) Heterocycloalkyl, aryl and heteroaryl groups, the group is optionally mixed with at least one group selected from the following: -O-, -S-, -NH-, -C(O)- and -S(O) 2 , and -Z are chromophore moieties, or salts thereof.

在一更特定實施例中，該式(I)化合物使得n為0。In a more specific embodiment, the compound of formula (I) is such that n is zero.

在另一更特定實施例中，該式(I)化合物使得m為1或2，較佳m為1。In another more specific embodiment, the compound of formula (I) is such that m is 1 or 2, preferably m is 1.

在一特定實施例中，該式(I)化合物使得Z為選自由以下組成之群的發色團部分：苯基、若丹明(rhodamine)、蒽醌、三芳基甲烷、酞菁、單偶氮染料、雙偶氮染料、三偶氮染料、聚偶氮染料、雙亞吲哚基二酮、茚二酮、喹啉茚二酮、吖啶、噻𠯤、噻唑、㗁𠯤、啡㗁𠯤、𠮿、氯、二酮吡咯并吡咯、喹吖啶酮、花青素、類黃酮及其衍生物。In a specific embodiment, the compound of formula (I) is such that Z is a chromophore moiety selected from the group consisting of phenyl, rhodamine, anthraquinone, triarylmethane, phthalocyanine, monocouple Nitrogen dyes, disazo dyes, triazo dyes, polyazo dyes, bisindolylidenedione, indenedione, quinoline indenedione, acridine, thiazole, thiazole, thiazole, phenylenedione ,𠮿 , chlorine, diketopyrrolopyrrole, quinacridone, anthocyanins, flavonoids and their derivatives.

在另一特定實施例中，該染料由下式中之任一者表示： 。 In another specific embodiment, the dye is represented by any of the following formulas: .

較佳地，該染料由下式中之任一者表示： 。 Preferably, the dye is represented by any one of the following formulas: .

更佳地，該染料由下式中之任一者表示： 。 More preferably, the dye is represented by any one of the following formulas: .

在另一特定實施例中，該氧化還原介質為TEMPO。In another specific embodiment, the redox mediator is TEMPO.

在另一特定實施例中，該氧化還原酶為蟲漆酶。更明確而言，該蟲漆酶可為來自以下之蟲漆酶：貝萊斯芽孢桿菌( Bacillus velezensis)、平流層芽孢桿菌 (Bacillus stratosphericus)、無氧芽孢桿菌( Anoxybacillus)、曲黴菌屬( Aspergillus)或變色栓菌( Trametes versicolor)，較佳為變色栓菌。較佳地，蟲漆酶包含選自由SEQ ID NO: 1、SEQ ID NO: 2、SEQ ID NO: 3、SEQ ID NO: 4、SEQ ID NO: 5及其變異體組成之群的胺基酸序列，其中該等變異體表現出蟲漆酶活性且具有至少60%之胺基酸序列一致性。 In another specific embodiment, the oxidoreductase is laccase. More specifically, the laccase may be a laccase from Bacillus velezensis, Bacillus stratosphericus , Anoxybacillus , Aspergillus ) or Trametes versicolor, preferably Trametes versicolor. Preferably, laccase comprises an amino acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and variants thereof Sequences, wherein the variants exhibit laccase activity and have at least 60% amino acid sequence identity.

在另一特定實施例中，步驟(a)在包含於10℃與60℃之間、較佳20℃與50℃之間、且更佳25℃與35℃之間的溫度下進行。In another specific embodiment, step (a) is performed at a temperature comprised between 10°C and 60°C, preferably between 20°C and 50°C, and more preferably between 25°C and 35°C.

在另一特定實施例中，步驟(a)在包含於2.5與12之間、較佳在4與10之間、更佳在4與8之間、甚至更佳在5與8之間的pH下進行。In another specific embodiment, step (a) is performed at a pH comprised between 2.5 and 12, preferably between 4 and 10, more preferably between 4 and 8, even more preferably between 5 and 8 proceed below.

在另一特定實施例中，步驟(b)在包含於10℃與60℃之間、較佳在30℃與50℃之間、更佳在35℃與45℃之間的溫度下進行。In another specific embodiment, step (b) is performed at a temperature comprised between 10°C and 60°C, preferably between 30°C and 50°C, more preferably between 35°C and 45°C.

在另一特定實施例中，步驟(b)在酸(諸如乙酸)存在下進行。In another specific embodiment, step (b) is performed in the presence of an acid, such as acetic acid.

在另一特定實施例中，步驟(a)在水中進行。In another specific embodiment, step (a) is performed in water.

在另一特定實施例中，步驟(b)在水中進行。In another specific embodiment, step (b) is performed in water.

在另一特定實施例中，該還原劑為硼烷化合物，該硼烷化合物較佳選自由以下組成之群：硼氫化鈉或硼氫化鉀、硼氫化鋅、氰基硼氫化鈉、硼烷(BH 3)、二硼烷(B 2H 6)、吡啶硼烷、甲基吡啶硼烷、5-乙基-2-甲基吡啶硼烷、𠰌啉硼烷、4-甲基𠰌啉硼烷、三乙胺硼烷、9-硼雙環[3.3.1]壬烷(9-BBN)、單異松蒎硼烷、二環己基硼烷、二基硼烷、二異戊基硼烷(disiamylborane)、兒茶酚硼烷、頻哪醇硼烷、三仲丁基硼氫化鋰(L-selectride)及其混合物，更佳為甲基吡啶硼烷。 In another specific embodiment, the reducing agent is a borane compound, and the borane compound is preferably selected from the group consisting of: sodium borohydride or potassium borohydride, zinc borohydride, sodium cyanoborohydride, borane ( BH 3 ), diborane (B 2 H 6 ), pyridine borane, methylpyridine borane, 5-ethyl-2-methylpyridine borane, 𠰌line borane, 4-methyl 𠰌line borane , triethylamine borane, 9-borane bicyclo[3.3.1]nonane (9-BBN), monoisopinepine borane, dicyclohexylborane, dicyclohexylborane borane, diisoamylborane (disiamylborane), catecholborane, pinacolborane, tri-sec-butyllithium borohydride (L-selectride) and mixtures thereof, more preferably methylpyridine borane .

在一特定實施例中，該纖維素材料係選自棉、亞麻、***、黃麻、人絲(viscose)、萊賽爾(lyocell)、螺縈(rayon)及莫代爾(modal)，較佳為棉。In a specific embodiment, the cellulosic material is selected from cotton, linen, hemp, jute, viscose, lyocell, rayon and modal, preferably cotton.

在一較佳實施例中，本發明方法包含以下連續步驟： (a)使纖維素材料與蟲漆酶及TEMPO或其衍生物接觸，以獲得氧化纖維素材料； (b)在硼烷化合物存在下，使步驟(a)之氧化纖維素材料與如本文所定義之式(I)染料接觸，以獲得染色纖維素材料；及 (c)回收染色纖維素材料。 In a preferred embodiment, the method of the present invention includes the following consecutive steps: (a) contacting cellulose materials with laccase and TEMPO or derivatives thereof to obtain oxidized cellulose materials; (b) contacting the oxidized cellulosic material of step (a) with a dye of formula (I) as defined herein in the presence of a borane compound to obtain a dyed cellulosic material; and (c) Recycling of dyed cellulosic materials.

本發明之另一目標為一種藉由如本文中所定義之方法獲得的染色纖維素材料。Another object of the invention is a dyed cellulosic material obtained by a method as defined herein.

定義根據本發明，以下術語具有以下含義： 本文所提及之帶有字首，諸如C 1-C 6之術語亦可與較低碳原子數，諸如C 1-C 2一起使用。若例如使用術語C 1-C 12，則其意謂對應烴鏈可包含1至12個碳原子，尤其1、2、3、4、5、6、7、8、9、10、11或12個碳原子。例如，若使用術語C 1-C 6，則其意謂對應烴鏈可包含1至6個碳原子，尤其1、2、3、4、5或6個碳原子。 Definitions According to the present invention, the following terms have the following meanings: Terms referred to herein with a prefix such as C 1 -C 6 may also be used with lower numbers of carbon atoms, such as C 1 -C 2 . If for example the term C 1 -C 12 is used, this means that the corresponding hydrocarbon chain may contain from 1 to 12 carbon atoms, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. For example, if the term C 1 -C 6 is used, this means that the corresponding hydrocarbon chain may contain 1 to 6 carbon atoms, especially 1, 2, 3, 4, 5 or 6 carbon atoms.

術語「烷基」係指飽和直鏈或分支鏈脂族基團。術語「(C 1-C 12)烷基」包括例如甲基、乙基、丙基、異丙基、丁基、異丁基、戊基、異戊基、己基、庚基、辛基、壬基、癸基、十一烷基或十二烷基。 The term "alkyl" refers to a saturated straight or branched aliphatic group. The term "(C 1 -C 12 )alkyl" includes, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl base, decyl, undecyl or dodecyl.

術語「烯基」係指具有至少一個碳-碳雙鍵之不飽和直鏈或分支鏈脂族基團。術語「(C 2-C 12)烯基」包括例如乙烯基、丙烯基、丁烯基、戊烯基、己烯基、庚烯基、辛烯基、壬烯基、癸烯基、十一烯基、十二烯基。 The term "alkenyl" refers to an unsaturated straight or branched aliphatic group having at least one carbon-carbon double bond. The term "(C 2 -C 12 )alkenyl" includes, for example, vinyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecanyl Alkenyl, dodecenyl.

術語「炔基」係指具有至少一個碳-碳參鍵之不飽和直鏈或分支鏈脂族基團。術語「(C 2-C 12)炔基」包括例如乙炔基、丙炔基、丁炔基、戊炔基、己炔基、庚烯基、辛炔基、壬炔基、癸炔基、十一炔基、十二炔基。 The term "alkynyl" refers to an unsaturated linear or branched aliphatic group having at least one carbon-carbon bond. The term "(C 2 -C 12 )alkynyl" includes, for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptenyl, octynyl, nonynyl, decynyl, deca Monoalkynyl, dodecynyl.

術語「環烷基」對應於飽和或不飽和單環、雙環或三環烷基。其亦包括稠合、橋接或螺接之環烷基。術語「(C 3-C 12)環烷基」包括例如環丙基、環丁基、環戊基及環己基。 The term "cycloalkyl" corresponds to saturated or unsaturated monocyclic, bicyclic or tricyclic alkyl groups. It also includes fused, bridged or spiro cycloalkyl groups. The term "(C 3 -C 12 )cycloalkyl" includes, for example, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

術語「雜環烷基」對應於如上文所定義之飽和或不飽和環烷基，其進一步包含至少一個雜原子，諸如氮、氧或硫原子。其亦包括稠合、橋接或螺接之雜環烷基。術語「(C 2-C 12)雜環烷基」包括例如二氧戊環基、苯并[1,3]間二氧雜環戊烯基、氮雜環丁烷基、氧雜環丁烷基、吡唑啉基、哌喃基、硫代𠰌啉基、吡唑啶基、哌啶基、哌𠯤基、1,4-二氧雜環己烷基、咪唑啉基、吡咯啉基、吡咯啶基、哌啶基、咪唑啶基、𠰌啉基、1,4-二噻唑基、吡咯啶基、㗁唑啉基、㗁唑啶基、異㗁唑啉基、異㗁唑啶基、噻唑啉基、噻唑啶基、異噻唑啉基、異噻唑啶基、二氫哌喃基、四氫哌喃基、四氫呋喃基及四氫噻吩基。 The term "heterocycloalkyl" corresponds to a saturated or unsaturated cycloalkyl group as defined above, which further contains at least one heteroatom, such as a nitrogen, oxygen or sulfur atom. It also includes fused, bridged or spiro-linked heterocycloalkyl groups. The term "(C 2 -C 12 )heterocycloalkyl" includes, for example, dioxolanyl, benzo[1,3]dioxolyl, azetidinyl, oxetane base, pyrazolinyl, piperanyl, thio-𠰌linyl, pyrazolinyl, piperidinyl, piperanyl, 1,4-dioxanyl, imidazolinyl, pyrrolinyl, Pyrrolidinyl, piperidinyl, imidazolidinyl, 𠰌linyl, 1,4-dithiazolyl, pyrrolidinyl, tetrazolinyl, tetrazolinyl, isothiazolinyl, isothiazolinyl, Thiazolinyl, thiazolidinyl, isothiazolinyl, isothiazodinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrofuranyl and tetrahydrothienyl.

「環烷基」及「雜環烷基」亦包括環烯基及雜環烯基，其分別對應於具有至少一個碳-碳雙鍵之環烷基及具有至少一個碳-碳雙鍵之雜環烷基，諸如環己烯基及二氫哌喃基。"Cycloalkyl" and "heterocycloalkyl" also include cycloalkenyl and heterocycloalkenyl, which correspond to cycloalkyl having at least one carbon-carbon double bond and heterocycloalkyl having at least one carbon-carbon double bond, respectively. Cycloalkyl groups such as cyclohexenyl and dihydropyranyl.

術語「芳基」對應於具有6至12個碳原子之單環或雙環芳族烴。例如，術語「芳基」包括苯基、萘基或蒽基。在一較佳實施例中，芳基為苯基或萘基，更佳苯基。The term "aryl" corresponds to a monocyclic or bicyclic aromatic hydrocarbon having 6 to 12 carbon atoms. For example, the term "aryl" includes phenyl, naphthyl, or anthracenyl. In a preferred embodiment, the aryl group is phenyl or naphthyl, more preferably phenyl.

如本文所使用之術語「雜芳基」對應於包含5個與14個之間的原子且包含至少一個雜原子(諸如氮、氧或硫原子)之芳族單環或多環基團。如本文所使用，術語「雜芳基」進一步包括「稠合芳基雜環烷基」及「稠合雜芳基環烷基」。術語「稠合芳基雜環烷基」及「稠合雜芳基環烷基」對應於雙環基團，其中如上所定義之芳基或雜芳基分別與如上所定義之雜環烷基或環烷基藉由至少兩個碳形成邊界。換言之，芳基或雜芳基與雜環烷基或環烷基共用碳鍵。此類單環及多環雜芳基、稠合芳基雜環烷基及稠合芳基環烷基之實例可為：吡啶基、苯硫基、呋喃基、吡咯基、吡唑基、咪唑基、㗁唑基、異㗁唑基、噻唑基、異噻唑基、***基、四唑基、㗁二唑基、呋呫基、噻二唑基、四唑基、苯并呋喃基、噻萘基、吲哚基、吲哚啉基、二氫茚基、喹啉基、異喹啉基、苯并咪唑基、四氫喹啉基、四氫異喹啉基、三𠯤基、噻嗯基、苯并呋喃基、二氫苯并呋喃基、異苯并呋喃基、苯并噻吩基、異苯并噻吩基、𠳭烯基、𠮿基、苯氧蒽基、吡𠯤基、嗒𠯤基、吲哚𠯤基、異吲哚基、吲唑基、嘌呤基、喹𠯤基、酞𠯤基、㖠啶基、喹喏啉基、喹唑啉基、㖕啉基、喋啶基、咔唑基、β-咔啉基、啡啶基、吖啶基、嘧啶基、啡啉基、啡𠯤基、啡噻𠯤基、呋呫基、啡㗁𠯤基、異𠳭烷基、𠳭烷基、咪唑啶基、咪唑啉基、吡唑啶基、吡唑啉基、吲哚啉基、異吲哚啉基、㗁唑啶基、苯并***基、苯并異㗁唑基、羥吲哚基、苯并㗁唑基、苯并㗁唑啉基、苯并㗁𠯤基、苯并噻吩基、苯并噻唑基、苯并二氮雜卓基、苯并氮呯基、苯并㗁氮呯基、靛藍、二氫苯并二氧呯基、二氫吡啶基、嘧啶基、s-三𠯤基、㗁唑基或硫代呋喃基。稠合芳基雜環烷基為例如吲哚啉基(與吡咯啶基稠合之苯基)及二氫苯并呋喃基(與二氫呋喃基稠合之苯基)。The term "heteroaryl" as used herein corresponds to an aromatic monocyclic or polycyclic group containing between 5 and 14 atoms and containing at least one heteroatom, such as a nitrogen, oxygen or sulfur atom. As used herein, the term "heteroaryl" further includes "fused arylheterocycloalkyl" and "fused heteroarylcycloalkyl." The terms "fused arylheterocycloalkyl" and "fused heteroarylcycloalkyl" correspond to bicyclic groups, where aryl or heteroaryl as defined above is associated with heterocycloalkyl or heteroaryl as defined above, respectively. Cycloalkyl groups are bounded by at least two carbons. In other words, the aryl or heteroaryl group shares a carbon bond with the heterocycloalkyl or cycloalkyl group. Examples of such monocyclic and polycyclic heteroaryl groups, fused arylheterocycloalkyl groups and fused arylcycloalkyl groups may be: pyridyl, phenylthio, furyl, pyrrolyl, pyrazolyl, imidazole base, ethazolyl, isothiazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, thiadiazolyl, furazolyl, thiadiazolyl, tetrazolyl, benzofuranyl, thiazolyl Naphthyl, indolyl, indolinyl, indenyl, quinolyl, isoquinolinyl, benzimidazolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, trisyl, thienyl Base, benzofuranyl, dihydrobenzofuranyl, isobenzofuranyl, benzothienyl, isobenzothienyl, alkenyl, phenyl, phenoxyanthracenyl, pyridyl, phenyl , indolinyl, isoindolyl, indazolyl, purinyl, quinoyl, phthaloyl, quinolinyl, quinolinyl, quinazolinyl, eulinyl, pyridinyl, carbazole Base, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenanthrolinyl, phenanthioyl, furyl, phenanthroline, iso-alkyl, phenanthyl alkyl, Imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, indolinyl, isoindolinyl, oxazolidinyl, benzotriazolyl, benzisothiazolyl, oxindole Benzodiazepine, benzodiazepine, benzodiazepine, benzodiazepine, benzodiazepine, benzodiazepine base, indigo, dihydrobenzodioxanol, dihydropyridyl, pyrimidinyl, s-tris-trihydroxy, ethazolyl or thiofuranyl. Fused arylheterocycloalkyl groups are, for example, indolinyl (phenyl fused to pyrrolidinyl) and dihydrobenzofuranyl (phenyl fused to dihydrofuranyl).

術語「鹵素」對應於氟、氯、溴或碘原子，較佳氟或氯。The term "halogen" corresponds to a fluorine, chlorine, bromine or iodine atom, preferably fluorine or chlorine.

術語「伸烷基」係指二價烷基，其中「烷基」如本文所定義。術語「(C 1-C 12)伸烷基」尤其係指式-(CH 2) q-之基團，其中q為1至12之整數。「(C 1-C 12)伸烷基」包括例如亞甲基、伸乙基、伸丙基、伸丁基、伸異丁基、伸戊基、伸異戊基、伸己基、伸庚基、伸辛基、伸壬基、伸癸基、伸十一烷基或伸十二烷基。 The term "alkylene" refers to a divalent alkyl group, where "alkyl" is as defined herein. The term "(C 1 -C 12 )alkylene" refers in particular to a group of the formula -(CH 2 ) q -, where q is an integer from 1 to 12. "(C 1 -C 12 )alkylene" includes, for example, methylene, ethylene, propylene, butylene, isobutyl, pentylene, isopentyl, hexylene, and heptyl , octyl, nonyl, decyl, undecyl or dodecyl.

術語「伸烯基」係指二價烯基，其中「烯基」如本文所定義。「(C 2-C 12)伸烯基」包括例如伸乙烯基(-CH=CH-)、伸丙烯基、伸丁烯基、伸戊烯基、伸己烯基、伸庚烯基、伸辛烯基、伸壬烯基、伸癸烯基、伸十一烷烯基或伸十二烷烯基。 The term "alkenylene" refers to a divalent alkenyl group, where "alkenyl" is as defined herein. "(C 2 -C 12 ) alkenylene" includes, for example, vinylene (-CH=CH-), propenylene, butenylene, pentenylene, hexenylene, heptenylene, and vinylidene. Octenyl, nonenyl, decenenyl, undecenyl or dodecenyl.

術語「伸炔基」係指二價炔基，其中「炔基」如本文所定義。「(C 2-C 12)伸炔基」包括例如伸乙炔基(-C≡C-)、伸丙炔基、伸丁炔基、伸戊炔基、伸己炔基、伸庚炔基、伸辛炔基、伸壬炔基、伸癸炔基、伸十一炔基或伸十二炔基。 The term "alkynyl" refers to a divalent alkynyl group, where "alkynyl" is as defined herein. "(C 2 -C 12 )alkynylene" includes, for example, ethynyl (-C≡C-), propynyl, butynyl, pentynyl, hexynyl, heptynyl, Octynyl, nonynyl, decynyl, undecynyl or dodecynyl.

術語「伸環烷基」係指二價環烷基，其中「環烷基」如本文所定義。例如，(C 3-C 12)伸環烷基包括伸環丙基、伸環戊基或伸環己基。 The term "cycloalkyl" refers to a divalent cycloalkyl group, where "cycloalkyl" is as defined herein. For example, (C 3 -C 12 )cycloalkylene includes cyclopropylene, cyclopentylene or cyclohexylene.

術語「伸雜環烷基」係指二價雜環烷基，其中「雜環烷基」如本文中所定義。例如，(C 3-C 12)伸環烷基包括伸硫代𠰌啉基、伸吡唑啶基、伸哌啶基或伸哌𠯤基。 The term "heterocycloalkyl" refers to a divalent heterocycloalkyl group, where "heterocycloalkyl" is as defined herein. For example, (C 3 -C 12 ) cycloalkylene groups include thiopyridinyl, pyrazolinyl, piperidinyl or piperidinyl.

術語「伸芳基」係指二價芳基，其中「芳基」如本文所定義。例如，伸芳基包括伸苯基。The term "arylene" refers to a divalent aryl group, where "aryl" is as defined herein. For example, aryl groups include phenyl groups.

術語「伸雜芳基」係指二價雜芳基，其中「雜芳基」如本文所定義。例如，伸雜芳基包括伸吡啶基、伸噻吩基或伸呋喃基。The term "heteroaryl" refers to a divalent heteroaryl group, where "heteroaryl" is as defined herein. For example, heteroaryl groups include pyridyl, thienyl, or furyl.

表述「經至少一個取代基取代」意謂經給定清單之「一個或若干個取代基取代」。表述「視情況經取代」意謂「未經取代或經取代」。The expression "substituted with at least one substituent" means "substituted with one or several substituents" from the given list. The expression "superseded as the case may be" means "unsubstituted or substituted".

根據本發明，術語「包含(comprise(s))」或「包含(comprising)」(及其他類似術語，例如「含有(containing)」及「包括(including)」)為「開端式」且通常可解釋為包括特別提及的特徵及任何視情況存在、其他及未指定的特徵。根據特定實施例，其亦可解釋為片語「基本上由……組成」，其中包括指定的特徵及任何視情況存在、其他及未指定的特徵，該等特徵對所主張發明的基本及新穎特徵無實質影響，或片語「由……組成」，其中僅包括指定的特徵，除非另有說明。According to the present invention, the terms "comprise(s)" or "comprising" (and other similar terms such as "containing" and "including") are "open-ended" and generally can Construed to include the features specifically mentioned and any other and unspecified features as the case may be. According to certain embodiments, it may also be interpreted as the phrase "consisting essentially of" including the specified features and any optional, other and unspecified features that are essential and novel to the claimed invention. Characteristics are insubstantial, or the phrase "consisting of" includes only the specified characteristics unless otherwise stated.

「鹽」包括無機以及有機酸鹽。適合無機酸之代表性實例包括鹽酸、氫溴酸、氫碘酸、磷酸及其類似物。適合有機酸之代表性實例包括甲酸、乙酸、三氯乙酸、三氟乙酸、丙酸、苯甲酸、肉桂酸、檸檬酸、反丁烯二酸、順丁烯二酸、甲磺酸及其類似物。「鹽」亦包括無機以及有機鹼鹽。適合無機鹼之代表性實例包括鈉鹽或鉀鹽、鹼土金屬鹽(諸如鈣鹽或鎂鹽)或銨鹽。"Salt" includes inorganic and organic acid salts. Representative examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid and the like. Representative examples of suitable organic acids include formic acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, propionic acid, benzoic acid, cinnamic acid, citric acid, fumaric acid, maleic acid, methanesulfonic acid, and the like. things. "Salt" also includes inorganic and organic alkali salts. Representative examples of suitable inorganic bases include sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts, or ammonium salts.

如本文所使用，術語「序列一致性」或「一致性」係指在兩個多肽序列比對之位置中匹配(相同胺基酸殘基)的數目(%)。序列一致性係藉由在比對以使重疊及一致性達到最大，同時使序列間隙減至最小時比較該等序列來判定。特定言之，視兩個序列之長度而定，可使用多種數學全域或局域比對演算法中之任一者來判定序列一致性。類似長度之序列較佳使用最佳地在整個長度上比對序列之全局比對演算法(例如，Needleman及Wunsch演算法；Needleman and Wunsch, 1970)比對，而實質上不同長度之序列較佳使用局部比對演算法(例如，Smith及Waterman演算法(Smith and Waterman, 1981)或Altschul演算法(Altschul等人, 1997；Altschul等人, 2005))比對。用於判定胺基酸序列一致性百分比之目的之比對可以此項技術中之技能內的各種方式達成，例如使用可在諸如http://blast.ncbi.nlm.nih.gov/或http://www.ebi.ac.uk/Tools/emboss/)之網際網路網站上獲得的公開可用的電腦軟體。熟習此項技術者可測定用於量測比對之適當參數，包括用於達成所比較序列之全長內之最大比對所需的任何演算法。較佳地，出於本文之目的，胺基酸序列一致性%係指使用局部比對演算法，較佳基於局部比對算法的搜尋工具(BLAST，Basic Local Alignment Search Tool)產生之值，其發現序列之間的局部相似性區域且計算匹配統計顯著性，其中所有搜尋參數設定為既定值，亦即blastp演算法，期望臨限值=0.05，字語大小=3，評分矩陣=BLOSUM62，間隙成本：存在=11、擴展=1，條件組合評分矩陣調節。As used herein, the term "sequence identity" or "identity" refers to the number (%) of matches (identical amino acid residues) in the aligned positions of two polypeptide sequences. Sequence identity is determined by comparing the sequences when aligned to maximize overlap and identity while minimizing sequence gaps. In particular, depending on the length of the two sequences, sequence identity can be determined using any of a variety of mathematical global or local alignment algorithms. Sequences of similar lengths are better aligned using global alignment algorithms that optimally align sequences over their entire length (e.g., the Needleman and Wunsch algorithm; Needleman and Wunsch, 1970), whereas sequences of substantially different lengths are better Align using local alignment algorithms (eg, Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignments for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example using information available at sites such as http://blast.ncbi.nlm.nih.gov/ or http: Publicly available computer software available on the Internet at http://www.ebi.ac.uk/Tools/emboss/). One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximal alignment over the full length of the sequences being compared. Preferably, for the purposes of this article, the % amino acid sequence identity refers to the value generated using a local alignment algorithm, preferably a search tool based on a local alignment algorithm (BLAST, Basic Local Alignment Search Tool), in which Discover local similarity areas between sequences and calculate matching statistical significance, where all search parameters are set to predetermined values, that is, the blastp algorithm, expectation threshold = 0.05, word size = 3, scoring matrix = BLOSUM62, gap Cost: existence=11, expansion=1, condition combination scoring matrix adjustment.

本發明提供一種簡單且環保的纖維素材料染色兩步法。本發明方法包含以下步驟： (a)使纖維素材料與氧化還原酶及氧化還原介質接觸，以獲得氧化纖維素材料； (b)在還原劑存在下，使步驟(a)之氧化纖維素材料與包含至少一個NH 2基團之染料接觸，以獲得染色纖維素材料；及 (c)回收染色纖維素材料。 The present invention provides a simple and environmentally friendly two-step method for dyeing cellulose materials. The method of the present invention includes the following steps: (a) contacting the cellulose material with an oxidoreductase and a redox medium to obtain an oxidized cellulose material; (b) in the presence of a reducing agent, oxidizing the cellulose material of step (a) contacting with a dye comprising at least one NH 2 group to obtain a dyed cellulosic material; and (c) recovering the dyed cellulosic material.

如本文所使用，「纖維素材料」係指包含纖維素或由纖維素組成的任何材料。纖維素材料可具有任何尺寸及任何形式，且可來自任何來源。例如，纖維素可來自天然或合成來源。在一特定實施例中，纖維素材料包含至少20重量%、30重量%或40重量%、較佳至少50重量%、60重量%或70重量%、更佳至少80重量%、90重量%、95重量%、98重量%、99重量%或100重量%之纖維素。As used herein, "cellulosic material" refers to any material that contains or consists of cellulose. Cellulosic material can be of any size and in any form, and can come from any source. For example, cellulose can be from natural or synthetic sources. In a specific embodiment, the cellulosic material comprises at least 20%, 30% or 40% by weight, preferably at least 50%, 60% or 70% by weight, more preferably at least 80%, 90% by weight, 95%, 98%, 99% or 100% cellulose by weight.

在另一特定實施例中，纖維素材料包含纖維或由纖維組成，該纖維包含纖維素或由纖維素組成。更明確而言，纖維素材料可為織物，例如非織物或編織物。In another specific embodiment, the cellulosic material comprises or consists of fibers comprising or consisting of cellulose. More specifically, the cellulosic material may be a woven fabric, such as a non-woven fabric or a woven fabric.

在一較佳實施例中，纖維素材料係選自棉、亞麻、***、黃麻、人絲、萊賽爾、螺縈及莫代爾。更佳地，纖維素材料為棉。In a preferred embodiment, the cellulosic material is selected from cotton, linen, hemp, jute, rayon, lyocell, rayon and modal. More preferably, the cellulosic material is cotton.

在本發明方法之步驟(a)中，使纖維素材料與氧化還原酶及氧化還原介質接觸，從而獲得氧化纖維素材料。In step (a) of the method of the present invention, the cellulose material is contacted with the oxidoreductase and the redox mediator, thereby obtaining the oxidized cellulose material.

如本文所使用，「氧化還原酶」係指能夠觸發或催化氧化還原反應的酶。氧化還原酶可尤其能夠氧化氧化還原介質。氧化還原酶可來自任何來源，諸如細菌、真菌、苔癬、昆蟲或植物。氧化還原酶可尤其為任何已知氧化還原酶及其變異體。更明確而言，氧化還原酶可選自已知細菌、真菌、苔癬、昆蟲或植物氧化還原酶及其變異體。較佳地，該等變異體表現出氧化還原酶活性且與該氧化還原酶中之任一者具有至少60%、較佳至少70%或80%、更佳至少90%、95%、96%、97%、98%或99%胺基酸序列一致性。As used herein, "oxidoreductase" refers to an enzyme capable of triggering or catalyzing an oxidation-reduction reaction. Oxidoreductases may be particularly capable of oxidizing redox mediators. Oxidoreductases can come from any source, such as bacteria, fungi, lichen, insects or plants. The oxidoreductase may in particular be any known oxidoreductase and variants thereof. More specifically, the oxidoreductase may be selected from known bacterial, fungal, lichen, insect or plant oxidoreductases and variants thereof. Preferably, the variants exhibit oxidoreductase activity and are at least 60%, preferably at least 70% or 80%, more preferably at least 90%, 95%, 96% identical to any one of the oxidoreductases , 97%, 98% or 99% amino acid sequence identity.

鑒於本發明方法可在溫和溫度下進行，氧化還原酶可為熱穩定的或不為熱穩定的。Given that the methods of the present invention can be performed at mild temperatures, the oxidoreductase enzyme may or may not be thermostable.

在一特定實施例中，氧化還原酶係選自由以下組成之群：蟲漆酶、對二酚：氧氧化還原酶、亞鐵氧化酶(諸如EC 1.10.3.2)、蟲漆酶樣多銅氧化酶(諸如EC 1.10.3.-)、纖維二糖脫氫酶(諸如EC 1.1.99.18)、葡萄糖1-氧化酶(諸如EC 1.1.3.4)、芳基醇氧化酶(諸如EC 1.1.3.7)、醇氧化酶(諸如EC 1.1.3.13)、哌喃醣氧化酶(諸如EC 1.1.3.10)、受體-氧氧化酶(諸如EC 1.1.3.-)、半乳糖氧化酶(諸如EC 1.1.3.9)、乙二醛氧化酶(諸如EC 1.2.3.15)、醇氧化酶(諸如EC 1.1.3.13)、棉子糖氧化酶(諸如EC 1.1.3.-)及裂解多醣單加氧酶(LPMO) (諸如EC 1.14.99.-)，及其組合。In a specific embodiment, the oxidoreductase is selected from the group consisting of laccase, diphenol:oxygen oxidoreductase, ferrous oxidase (such as EC 1.10.3.2), laccase-like polycopper oxidase Enzymes (such as EC 1.10.3.-), cellobiose dehydrogenase (such as EC 1.1.99.18), glucose 1-oxidase (such as EC 1.1.3.4), aryl alcohol oxidase (such as EC 1.1.3.7) , alcohol oxidase (such as EC 1.1.3.13), perranose oxidase (such as EC 1.1.3.10), acceptor-oxygen oxidase (such as EC 1.1.3.-), galactose oxidase (such as EC 1.1. 3.9), glyoxal oxidase (such as EC 1.2.3.15), alcohol oxidase (such as EC 1.1.3.13), raffinose oxidase (such as EC 1.1.3.-) and lytic polysaccharide monooxygenase (LPMO) ) (such as EC 1.14.99.-), and combinations thereof.

如本文所使用，EC編號係指酶委員會(Enzyme Commission)編號，該編號係基於酶催化之化學反應的酶的數值分類方案。As used herein, the EC number refers to the Enzyme Commission number, which is a numerical classification scheme for enzymes based on the chemical reactions they catalyze.

在一個較佳實施例中，氧化還原酶係蟲漆酶。在此類實施例中，蟲漆酶可來自任何來源，諸如細菌、真菌、苔癬、昆蟲或植物。該蟲漆酶可為任何已知蟲漆酶，尤其可選自已知細菌、真菌、苔癬、昆蟲或植物蟲漆酶及其變異體。較佳地，該等變異體表現出蟲漆酶活性且與該蟲漆酶中之任一者具有至少60%、較佳至少70%或80%、更佳至少90%、95%、96%、97%、98%或99%胺基酸序列一致性。蟲漆酶活性可藉由使用酚類受質之光度測試且藉由監測氧化產物來確定。用於蟲漆酶分析之酚類受質之實例包括但不限於愈創木酚、2,6-二甲氧基苯酚、丁香醛連氮、2,2-次偶氮基-雙-(3-乙基苯并噻唑啉-6-磺酸、苯二酚及3,4-二甲氧基苯甲醇。蟲漆酶活性之測定方法具體描述於以下文章中：Johannes及Maicherczyk, Journal of Biotechnology, 2000, 78, 193-199；Harkin及Obst, Ex-perientia 1973, 37, 381-387；Prillinger及Esser Molec. Gen. Genet. 1975, 156, 333-345。In a preferred embodiment, the oxidoreductase is laccase. In such embodiments, the laccase can be derived from any source, such as bacteria, fungi, lichen, insects, or plants. The laccase may be any known laccase, and may in particular be selected from known bacterial, fungal, lichen, insect or plant laccases and variants thereof. Preferably, the variants exhibit laccase activity and are at least 60%, preferably at least 70% or 80%, more preferably at least 90%, 95%, 96% identical to any one of the laccases , 97%, 98% or 99% amino acid sequence identity. Laccase activity can be determined by photometric testing using phenolic substrates and by monitoring oxidation products. Examples of phenolic substrates for laccase analysis include, but are not limited to, guaiacol, 2,6-dimethoxyphenol, syringaldazine, 2,2-azo-bis-(3 -Ethylbenzothiazoline-6-sulfonic acid, diphenol and 3,4-dimethoxybenzyl alcohol. The method for measuring laccase activity is described in detail in the following article: Johannes and Maicherczyk, Journal of Biotechnology, 2000, 78, 193-199; Harkin and Obst, Ex-perientia 1973, 37, 381-387; Prillinger and Esser Molec. Gen. Genet. 1975, 156, 333-345.

在一特定實施例中，蟲漆酶為來自細菌或真菌之蟲漆酶。In a specific embodiment, the laccase is a laccase enzyme from a bacterium or fungus.

產生氧化還原酶(諸如蟲漆酶)之細菌的實例包括但不限於： - 芽孢桿菌，諸如貝萊斯芽孢桿菌(例如，貝萊斯芽孢桿菌TCCC 111904)、平流層芽孢桿菌 (例如，平流層芽孢桿菌BCMC2)、枯草芽孢桿菌( Bacillus subtilis)、短小芽孢桿菌( Bacillus pumilus)、克勞氏芽孢桿菌( Bacillus clausii)、耐鹽芽孢桿菌( Bacillus halodurans) 或龍舌蘭芽孢桿菌( Bacillus tequilensis) (例如，龍舌蘭芽孢桿菌 SN4)， - 無氧芽孢桿菌 ， 例如無氧芽孢桿菌屬UARK-01， - 假單胞菌，諸如極端假單胞菌， - 鏈黴菌，諸如綠色產色鏈黴菌、蕃薯鏈黴菌或深藍鏈黴菌， - 海單胞菌，諸如地中海海單胞菌， - 埃希氏菌，諸如大腸桿菌， - 克雷伯氏菌，諸如肺炎克雷伯氏桿菌， 及- 耶氏桿菌，諸如小腸大腸炎耶氏桿菌。 Examples of bacteria that produce oxidoreductases such as laccase include, but are not limited to: - Bacillus species, such as Bacillus veleis (e.g., B. veleis TCCC 111904), Bacillus stratospora ( e.g., B. Bacillus BCMC2), Bacillus subtilis , Bacillus pumilus, Bacillus clausii , Bacillus halodurans or Bacillus tequilensis ( For example, Bacillus agave SN4 ), - anaerobic Bacilli , such as anaerobic Bacillus UARK-01, - Pseudomonas, such as Pseudomonas extremum, - Streptomyces, such as Streptomyces viridis, Streptomyces tuberosum or Streptomyces cyanogens, - Seamonas species, such as Seamonas mediterranea, - Escherichia species, such as Escherichia coli, - Klebsiella species, such as Klebsiella pneumoniae, and - J. coli, such as Yersinia enterocolitica.

來自短小芽孢桿菌之蟲漆酶之實例為短小芽孢桿菌CotA。來自枯草芽孢桿菌之蟲漆酶之實例為枯草芽孢桿菌CotA。來自克勞氏芽孢桿菌之蟲漆酶之實例為克勞氏芽孢桿菌CotA。來自大腸桿菌之氧化還原酶之實例為大腸桿菌CueO。An example of a laccase enzyme from B. pumilus is B. pumilus CotA. An example of a laccase enzyme from Bacillus subtilis is Bacillus subtilis CotA. An example of a laccase from Bacillus clausii is Bacillus clausii CotA. An example of an oxidoreductase from E. coli is E. coli CueO.

產生氧化還原酶(諸如蟲漆酶)之真菌的實例包括但不限於： - 栓菌屬，諸如變色栓菌(例如變色栓菌 52J或變色栓菌 ATCC 32745)、絨毛栓菌( Trametes pubescens)、長絨毛栓菌( Trametes villosa)、毛栓菌( Trametes hirsuta)或栓菌( Trametes trogii)， - 小薄孔菌屬( Antrodiella)，諸如山毛櫸小薄孔菌( Antrodiella faginea)， - 曲黴菌屬，諸如米曲黴菌( Aspergillus oryzae)或黑麴菌( Aspergillus niger) (例如黑麴菌 CBS 513.88)， - 齒毛菌屬( Cerrena)，諸如最大齒毛菌( Cerrena maxima)或齒毛菌屬 RSD1， - 密孔菌屬( Pycnoporus)，諸如朱紅密孔菌( Pycnoporus cinnabarinus)或血紅密孔菌( Pycnoporus sanguineus)， - 韌傘屬( Lentinus)，諸如虎皮韌傘( Lentinus tigrinus)或韌傘屬 WR2， - 革孔菌屬，諸如粗毛革孔菌( Coriolopsis gallica) (例如粗毛革孔菌 SAH-12)、灰革孔菌( Coriolopsis cinerea) (例如灰革孔菌 A3387)或硬毛革孔菌( Coriolopsis caperata)， - 革蓋菌屬，諸如環紋革蓋菌( Coriolus zonatus)， - 射脈菌屬( Phlebia)，諸如短孢射脈菌( Phlebia brevispora)， - 齒耳菌屬( Steccherinum)，諸如赭黃齒耳( Steccherinum ochraceum)或穆氏齒耳菌( Steccherinum murashkinski)， - 硬孔菌屬( Rigidoporus)諸如小孔硬孔菌( Rigidoporous microporus)， - 毀絲黴屬( Myceliophtora)諸如嗜熱毀絲黴( Myceliophtora thermophila)， - 黑果( Melanocarpus)諸如黑果白黴菌( Melanocarpus albomyces)， - 蠟蘑屬( Laccaria)諸如雙色蠟蘑( Laccaria bicolor)， - 擬鬼傘屬( Coprinopsis)，諸如灰擬鬼傘( Coprinopsis cinerea)， - 殘孔菌屬( Abortiporus)，諸如粉殘孔菌( Abortiporus biennis)， - 梭孢殼屬( Thielavia)，諸如沙生梭孢殼( Thielavia arenaria)， - 黃囊菇屬( Deconica)，諸如栗色黃囊菇( Deconica castanella)， 及- 木黴菌屬( Trichoderma)，諸如棘孢木黴( Trichoderma asperellum) (例如棘孢木黴 BPLMBT1)。 Examples of fungi that produce oxidoreductases such as laccase include, but are not limited to: Trametes, such as Trametes versicolor (eg Trametes versicolor 52J or Trametes versicolor ATCC 32745 ), Trametes pubescens , Trametes villosa, Trametes hirsuta or Trametes trogii , - Antrodiella , such as Antrodiella faginea, - Aspergillus, Such as Aspergillus oryzae or Aspergillus niger (for example, Aspergillus niger CBS 513.88 ), - Cerrena , such as Cerrena maxima or Cerrena RSD1 , - Pycnoporus , such as Pycnoporus cinnabarinus or Pycnoporus sanguineus , - Lentinus , such as Lentinus tigrinus or Lentinus WR2 , - Coriolopsis gallica, such as Coriolopsis gallica (e.g. Coriolopsis gallica SAH-12 ), Coriolopsis cinerea (e.g. Coriolopsis cinerea A3387 ) or Coriolopsis gallica (e.g. Coriolopsis cinerea) Coriolopsis caperata , - Coriolopsis genus, such as Coriolus zonatus , - Phlebia , such as Phlebia brevispora , - Steccherinum , Such as Steccherinum ochraceum or Steccherinum murashkinski , - Rigidoporus such as Rigidoporous microporus , - Myceliophtora such as Thermophilus Myceliophtora thermophila , - Melanocarpus such as Melanocarpus albomyces , - Laccaria such as Laccaria bicolor , - Coprinopsis , such as Coprinopsis cinerea , - Abortiporus , such as Abortiporus biennis, - Thielavia , such as Thielavia arenaria , - Yellow Deconica , such as Deconica castanella , and Trichoderma , such as Trichoderma asperellum (eg Trichoderma BPLMBT1 ).

在一特定實施例中，蟲漆酶為來自貝萊斯芽孢桿菌、平流層芽孢桿菌、無氧芽孢桿菌、曲黴菌屬(諸如黑麴菌)或變色栓菌之蟲漆酶，較佳來自變色栓菌。In a specific embodiment, the laccase is a laccase from Bacillus veleisi, Bacillus stratospora, Bacillus anaerobicus, Aspergillus (such as Kojima) or Trametes versicolor, preferably from Bacillus versicolor Trametes.

在一特定實施例中，蟲漆酶包含選自由SEQ ID NO: 1至5組成之群的胺基酸序列、或由其組成，及其變異體，該等變異體表現出蟲漆酶活性且與SEQ ID NO: 1至5中之任一者具有至少60%、較佳至少61%、62%、63%、64%、65%、66%、67%、68%、69%、75%、更佳至少80%、85%、90%、95%、96%、97%、98%或99%，且甚至更佳至少90%、95%、96%、97%、98%或99%胺基酸序列一致性。In a specific embodiment, laccase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 5, and variants thereof, which variants exhibit laccase activity and At least 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 75% with any one of SEQ ID NO: 1 to 5 , better at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, and even better at least 90%, 95%, 96%, 97%, 98% or 99% Amino acid sequence identity.

在一更特定實施例中，蟲漆酶為來自貝萊斯芽孢桿菌，且更特定言之來自貝萊斯芽孢桿菌TCCC 111904之蟲漆酶。更明確而言，蟲漆酶較佳為包含以下胺基酸序列：SEQ ID NO: 1，或由其組成之蟲漆酶或其變異體，該變異體表現出蟲漆酶活性且與SEQ ID NO: 1具有至少60%、較佳至少75%、更佳至少80%、85%、90%、95%、96%、97%、98%或99%且甚至更佳至少90%、95%、96%、97%、98%或99%胺基酸序列一致性。具有胺基酸序列SEQ ID NO: 1之蟲漆酶描述於Li等人Sci. Total. Environ. 2020, 713, 136713中。In a more specific embodiment, the laccase is a laccase from Bacillus veleisi, and more specifically from B. veleisis TCCC 111904. More specifically, laccase preferably contains the following amino acid sequence: SEQ ID NO: 1, or a laccase or a variant thereof, which variant exhibits laccase activity and is consistent with SEQ ID NO. NO: 1 has at least 60%, preferably at least 75%, better at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% and even better at least 90%, 95% , 96%, 97%, 98% or 99% amino acid sequence identity. The laccase enzyme having the amino acid sequence SEQ ID NO: 1 is described in Li et al. Sci. Total. Environ. 2020, 713, 136713.

在另一更特定實施例中，蟲漆酶為來自無氧芽孢桿菌，且更特定言之來自無氧芽孢桿菌屬UARK-01之蟲漆酶。更明確而言，蟲漆酶較佳為包含以下胺基酸序列：SEQ ID NO: 2，或由其組成之蟲漆酶或其變異體，該變異體表現出蟲漆酶活性且與SEQ ID NO: 2具有至少60%、較佳至少75%、更佳至少80%、85%、90%、95%、96%、97%、98%或99%且甚至更佳至少90%、95%、96%、97%、98%或99%胺基酸序列一致性。具有胺基酸序列SEQ ID NO: 2之蟲漆酶描述於Al-Kahem Al-balawi等人Curr Microbiol. 2017,74, 762-771中。In another more specific embodiment, the laccase is a laccase from anaerobic Bacillus, and more specifically from anaerobic Bacillus UARK-01. More specifically, the laccase preferably contains the following amino acid sequence: SEQ ID NO: 2, or a laccase or a variant thereof, which variant exhibits laccase activity and is consistent with SEQ ID NO. NO: 2 has at least 60%, preferably at least 75%, better at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% and even better at least 90%, 95% , 96%, 97%, 98% or 99% amino acid sequence identity. The laccase enzyme having the amino acid sequence SEQ ID NO: 2 is described in Al-Kahem Al-balawi et al. Curr Microbiol. 2017, 74, 762-771.

在另一更特定實施例中，蟲漆酶為來自平流層芽孢桿菌，且更特定言之來自平流層芽孢桿菌BCMC2之蟲漆酶。更明確而言，蟲漆酶較佳為包含以下胺基酸序列：SEQ ID NO: 3，或由其組成之蟲漆酶或其變異體，該變異體表現出蟲漆酶活性且與SEQ ID NO: 3具有至少60%、較佳至少75%、更佳至少80%、85%、90%、95%、96%、97%、98%或99%且甚至更佳至少90%、95%、96%、97%、98%或99%胺基酸序列一致性。具有胺基酸序列SEQ ID NO: 3之蟲漆酶描述於Xiao等人Int J Biol Macromol. 2021, 179, 270-278中。In another more specific embodiment, the laccase is a laccase from Bacillus stratosphericus, and more specifically from Bacillus stratosphericus BCMC2. More specifically, the laccase preferably contains the following amino acid sequence: SEQ ID NO: 3, or a laccase or a variant thereof, which variant exhibits laccase activity and is consistent with SEQ ID NO. NO: 3 has at least 60%, preferably at least 75%, better at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% and even better at least 90%, 95% , 96%, 97%, 98% or 99% amino acid sequence identity. The laccase enzyme having the amino acid sequence SEQ ID NO: 3 is described in Xiao et al. Int J Biol Macromol. 2021, 179, 270-278.

在一特定實施例中，蟲漆酶為來自曲黴菌屬諸如黑麴菌之蟲漆酶。更明確而言，蟲漆酶較佳為包含以下胺基酸序列：SEQ ID NO: 5，或由其組成之蟲漆酶或其變異體，該變異體表現出蟲漆酶活性且與SEQ ID NO: 5具有至少60%、較佳至少61%、62%、63%、64%、65%、66%、67%、68%、69%或75%、更佳至少80%、85%、90%、95%、96%、97%、98%或99%且甚至更佳至少90%、95%、96%、97%、98%或99%胺基酸序列一致性。In a specific embodiment, the laccase is a laccase from the genus Aspergillus, such as Kojima. More specifically, the laccase preferably contains the following amino acid sequence: SEQ ID NO: 5, or a laccase or a variant thereof, which variant exhibits laccase activity and is consistent with SEQ ID NO. NO: 5 has at least 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 75%, better at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% and even better at least 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity.

在一較佳實施例中，蟲漆酶為來自變色栓菌之蟲漆酶。更明確而言，蟲漆酶較佳為包含以下胺基酸序列：SEQ ID NO: 4，或由其組成之蟲漆酶或其變異體，該變異體表現出蟲漆酶活性且與SEQ ID NO: 4具有至少60%、較佳至少61%、62%、63%、64%、65%、66%、67%、68%、69%或75%、更佳至少80%、85%、90%、95%、96%、97%、98%或99%且甚至更佳至少90%、95%、96%、97%、98%或99%胺基酸序列一致性。In a preferred embodiment, laccase is laccase from Trametes versicolor. More specifically, the laccase preferably contains the following amino acid sequence: SEQ ID NO: 4, or a laccase or a variant thereof, which variant exhibits laccase activity and is consistent with SEQ ID NO. NO: 4 has at least 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 75%, better at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% and even better at least 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity.

如本文所使用，「氧化還原介質」係指能夠交換(特定言之，提供且隨後恢復)至少一個電子之化合物。特定言之，氧化還原介質可能能夠經氧化還原酶氧化以形成經氧化之氧化還原介質，且該經氧化之氧化還原介質可能能夠氧化纖維素。As used herein, "redox mediator" refers to a compound capable of exchanging (specifically, donating and subsequently recovering) at least one electron. In particular, the redox mediator may be able to be oxidized by an oxidoreductase to form an oxidized redox mediator, and the oxidized redox mediator may be capable of oxidizing cellulose.

在一特定實施例中，氧化還原介質為烷氧基，較佳為硝醯基。如本文所使用，「烷氧基」表示在氧原子上具有一個自由基(亦即一個單一不成對電子)之有機化合物。如本文所使用，「硝醯基」指示如本文所定義之氧基，其中具有一個自由基之該氧原子與氮原子結合。In a specific embodiment, the redox mediator is an alkoxy group, preferably a nitroxyl group. As used herein, "alkoxy" refers to an organic compound having a free radical (ie, a single unpaired electron) on the oxygen atom. As used herein, "nitrogenyl" refers to an oxygen group, as defined herein, in which the oxygen atom having a free radical is bonded to a nitrogen atom.

在一更特定實施例中，氧化還原介質為選自由以下組成之群的硝醯基：2,2,6,6-四甲基哌啶-1-基)醯基(TEMPO)、2,2,5,5-四甲基-1-吡咯啶基醯基(PROXYL)、胺醯基、亞胺基硝醯基、硝基硝醯基、亞胺醯基、其衍生物及其混合物。In a more specific embodiment, the redox mediator is a nitroyl group selected from the group consisting of: 2,2,6,6-tetramethylpiperidin-1-yl) nitroyl (TEMPO), 2,2 ,5,5-Tetramethyl-1-pyrrolidinyl acyl (PROXYL), amine acyl, imino nitroyl, nitro nitro acyl, imino acyl, their derivatives and their mixtures.

在一特定實施例中，胺醯基為式(II-a)化合物： (II-a)， 其中R 13及R 14各自獨立地選自(C 1-C 12)烷基、(C 2-C 12)烯基、(C 2-C 12)炔基、(C 3-C 12)環烷基、(C 2-C 12)雜環烷基、芳基及雜芳基。 In a specific embodiment, the amine acyl group is a compound of formula (II-a): (II-a), wherein R 13 and R 14 are each independently selected from (C 1 -C 12 ) alkyl, (C 2 -C 12 ) alkenyl, (C 2 -C 12 ) alkynyl, (C 3 -C 12 )cycloalkyl, (C 2 -C 12 )heterocycloalkyl, aryl and heteroaryl.

在另一特定實施例中，亞胺基硝醯基為式(II-b)化合物： (II-b)， 其中R 15、R 16及R 17各自獨立地選自(C 1-C 12)烷基、(C 2-C 12)烯基、(C 2-C 12)炔基、(C 3-C 12)環烷基、(C 2-C 12)雜環烷基、芳基及雜芳基， 或R 15及R 16與其所連接之原子一起形成C 5-C 12含氮環。 In another specific embodiment, the iminonitrogenyl group is a compound of formula (II-b): (II-b), wherein R 15 , R 16 and R 17 are each independently selected from (C 1 -C 12 ) alkyl, (C 2 -C 12 ) alkenyl, (C 2 -C 12 ) alkynyl, (C 3 -C 12 ) cycloalkyl, (C 2 -C 12 ) heterocycloalkyl, aryl and heteroaryl, or R 15 and R 16 together with the atoms to which they are connected form C 5 -C 12 nitrogen-containing ring.

在另一特定實施例中，硝基硝醯基為式(II-c)化合物： (II-c)， 其中R 18、R 19及R 20各自獨立地選自(C 1-C 12)烷基、(C 2-C 12)烯基、(C 2-C 12)炔基、(C 3-C 12)環烷基、(C 2-C 12)雜環烷基、芳基及雜芳基， 或R 18及R 19與其所連接之原子一起形成C 5-C 12含氮環。 In another specific embodiment, the nitronitrogenyl group is a compound of formula (II-c): (II-c), wherein R 18 , R 19 and R 20 are each independently selected from (C 1 -C 12 ) alkyl, (C 2 -C 12 ) alkenyl, (C 2 -C 12 ) alkynyl, (C 3 -C 12 ) cycloalkyl, (C 2 -C 12 ) heterocycloalkyl, aryl and heteroaryl, or R 18 and R 19 together with the atoms to which they are connected form C 5 -C 12 nitrogen-containing ring.

在一特定實施例中，亞胺醯基為式(II-d)化合物： (II-d)， 其中R 21及R 22各自獨立地選自(C 1-C 12)烷基、(C 2-C 12)烯基、(C 2-C 12)炔基、(C 3-C 12)環烷基、(C 2-C 12)雜環烷基、芳基及雜芳基。 In a specific embodiment, the iminyl group is a compound of formula (II-d): (II-d), wherein R 21 and R 22 are each independently selected from (C 1 -C 12 ) alkyl, (C 2 -C 12 ) alkenyl, (C 2 -C 12 ) alkynyl, (C 3 -C 12 )cycloalkyl, (C 2 -C 12 )heterocycloalkyl, aryl and heteroaryl.

較佳地，氧化還原介質為TEMPO或其衍生物。TEMPO可表示如下： Preferably, the redox mediator is TEMPO or its derivatives. TEMPO can be expressed as follows:

如本文所使用，「TEMPO之衍生物」係指經至少一個(例如一個、兩個或三個，較佳一個)取代基取代之TEMPO分子。例如，TEMPO之衍生物可由下式(II-1)表示： (II-1)， 其中R 23選自由以下組成之群：OH、NH 2、CN、C(O)OH、側氧基、(C 1-C 6)烷基、-O-(C 1-C 6)烷基。 As used herein, "derivatives of TEMPO" refers to TEMPO molecules substituted with at least one (eg, one, two or three, preferably one) substituent. For example, the derivative of TEMPO can be represented by the following formula (II-1): (II-1), wherein R 23 is selected from the group consisting of: OH, NH 2 , CN, C(O)OH, pendant oxy, (C 1 -C 6 ) alkyl, -O-(C 1 - C 6 )alkyl.

更佳地，氧化還原介質為TEMPO。More preferably, the redox medium is TEMPO.

在步驟(a)中，接觸可藉由在適合溶劑(較佳水)中混合纖維素材料、氧化還原酶及氧化還原介質來進行。更明確而言，此類接觸可藉由將纖維素材料浸漬至包含氧化還原酶及氧化還原介質之溶劑(較佳水)中來進行。In step (a), contacting can be carried out by mixing the cellulosic material, the oxidoreductase enzyme and the redox mediator in a suitable solvent, preferably water. More specifically, such contacting can be carried out by impregnating the cellulosic material into a solvent (preferably water) containing an oxidoreductase enzyme and a redox mediator.

本發明方法之步驟(a)可在溫和條件，特定言之溫和溫度及pH下進行。Step (a) of the method of the present invention can be carried out under mild conditions, in particular mild temperature and pH.

在一特定實施例中，步驟(a)在包含於10℃與60℃之間、較佳20℃與50℃之間、更佳25℃與35℃之間的溫度下進行。在另一特定實施例中，步驟(a)在包含於2.5與12之間、較佳在4與10之間、更佳在4與8之間、甚至更佳在5與8之間的pH下進行。步驟(a)可在水性介質中進行。步驟(a)之pH可尤其藉由使用pH統計系統及/或緩衝液來控制。緩衝液之實例包括但不限於磷酸鈉緩衝液、乙酸鹽緩衝液、檸檬酸鹽緩衝液或Tri-CuCl 2緩衝液。 In a specific embodiment, step (a) is performed at a temperature comprised between 10°C and 60°C, preferably between 20°C and 50°C, more preferably between 25°C and 35°C. In another specific embodiment, step (a) is performed at a pH comprised between 2.5 and 12, preferably between 4 and 10, more preferably between 4 and 8, even more preferably between 5 and 8 proceed below. Step (a) can be carried out in an aqueous medium. The pH of step (a) can be controlled, inter alia, by using pH statistical systems and/or buffers. Examples of buffers include, but are not limited to, sodium phosphate buffer, acetate buffer, citrate buffer, or Tri -CuCl buffer.

在另一特定實施例中，步驟(a)進行的持續時間包含於1小時與30小時之間，例如5小時與15小時之間。步驟(a)可在空氣下進行。In another specific embodiment, step (a) is performed for a duration comprised between 1 hour and 30 hours, such as between 5 hours and 15 hours. Step (a) can be carried out under air.

氧化還原酶及氧化還原介質可以催化量使用。步驟(a)中之氧化還原介質之濃度可例如包含於0.1 mM與100 mM之間。步驟(a)中氧化還原酶之濃度可例如包含於0.001 U/mL與100 U/mL之間。Oxidoreductases and redox mediators can be used in catalytic amounts. The concentration of the redox mediator in step (a) may, for example, be comprised between 0.1 mM and 100 mM. The concentration of the oxidoreductase in step (a) may, for example, be comprised between 0.001 U/mL and 100 U/mL.

本發明方法之步驟(a)允許獲得經氧化之纖維素材料。「經氧化之纖維素材料」可尤其係指纖維素材料，其中纖維素之醇官能基之全部或部分呈氧化形式，較佳呈醛且視情況呈酮及/或羧酸。在一特定實施例中，包含氧化還原酶及氧化還原介質之溶劑(較佳水)可在回收經氧化之纖維素材料後再使用。Step (a) of the method of the invention allows to obtain oxidized cellulosic material. "Oxidized cellulosic material" may in particular mean cellulosic material in which all or part of the alcohol functional groups of the cellulose are in oxidized form, preferably as aldehydes and optionally as ketones and/or carboxylic acids. In a specific embodiment, a solvent (preferably water) containing an oxidoreductase enzyme and a redox mediator can be used after recovering the oxidized cellulosic material.

本發明方法之步驟(b)包含在還原劑存在下使步驟(a)之經氧化之纖維素材料與包含至少一個NH 2基團之染料接觸，以獲得染色纖維素材料。 Step (b) of the method of the invention comprises contacting the oxidized cellulosic material of step (a) with a dye comprising at least one NH 2 group in the presence of a reducing agent to obtain a dyed cellulosic material.

如本文所使用，「包含至少一個NH 2基團之染料」係指包含發色團部分及至少一個NH 2基團的任何無機或有機化合物。如本文所使用，術語「發色團部分」指示包含共軛雙鍵(例如，芳族鍵)且賦予染料顏色之化學部分，通常藉由吸收可見光之一部分且藉由反射、透射或漫射該可見光之互補部分。發色團部分及至少一個NH 2基團可直接或間接(亦即經由間隔基)，較佳直接彼此共價連接。 As used herein, "dye containing at least one NH group" refers to any inorganic or organic compound that contains a chromophore moiety and at least one NH group. As used herein, the term "chromophore moiety" refers to a chemical moiety that contains conjugated double bonds (eg, aromatic bonds) and that imparts color to a dye, typically by absorbing a portion of visible light and by reflecting, transmitting, or diffusing that Complementary parts of visible light. The chromophore moiety and the at least one NH 2 group may be covalently linked to each other directly or indirectly (ie via a spacer), preferably directly.

發色團部分可尤其選自通常用於染色過程中之發色團部分及其衍生物。此類發色團部分尤其描述於以下文章中：Klaus Hunger, Industrial dyes, chemistry, properties, applications, 2003, Wiley；Benkhaya等人Inorganic Chemistry Communications, 2020, 115, 107891。發色團部分之「衍生物」尤其包括經一或多個取代基(諸如-CF 3、-CN、-NO 2、-OH、C 1-C 6烷基、鹵素、-S(O) 2ONa或-C(O)O(C 1-C 6烷基))取代之衍生物，且使其發色團保留活性。 The chromophore moieties may especially be selected from chromophore moieties commonly used in dyeing processes and their derivatives. Such chromophore moieties are described inter alia in Klaus Hunger, Industrial dyes, chemistry, properties, applications, 2003, Wiley; Benkhaya et al. Inorganic Chemistry Communications, 2020, 115, 107891. "Derivatives" of chromophore moieties particularly include those substituted with one or more substituents such as -CF 3 , -CN, -NO 2 , -OH, C 1 -C 6 alkyl, halogen, -S(O) 2 Derivatives substituted by ONa or -C(O)O(C 1 -C 6 alkyl)), and the chromophore retains activity.

發色團部分之實例包括但不限於：苯基、若丹明、蒽醌、三芳基甲烷、酞菁、單偶氮染料、雙偶氮染料、三偶氮染料、聚偶氮染料、雙亞吲哚基二酮、茚二酮、喹啉茚二酮、吖啶、噻𠯤、噻唑、㗁𠯤、啡㗁𠯤、𠮿、氯、二酮吡咯并吡咯、喹吖啶酮、花青素、類黃酮及其衍生物。Examples of chromophore moieties include, but are not limited to: phenyl, rhodamine, anthraquinone, triarylmethane, phthalocyanine, monoazo dyes, disazo dyes, triazo dyes, polyazo dyes, bis-azo dyes, Indolyldione, indenedione, quinoline indenedione, acridine, thiazole, thiazole, thiazole, phenanthrene, thiazole , chlorine, diketopyrrolopyrrole, quinacridone, anthocyanins, flavonoids and their derivatives.

在一特定實施例中，該染料為包括至少一個pKa等於或小於11、較佳等於或小於9、更佳等於或小於7、甚至更佳等於或小於5之NH 2基團的染料。例如，該pKa可包含於-5與11之間、-5與9之間、-5與7之間、0與7之間、3與7之間、-5與5之間、0與5之間或3與5之間。 In a specific embodiment, the dye is a dye that includes at least one NH 2 group with a pKa of 11 or less, preferably 9 or less, more preferably 7 or less, and even more preferably 5 or less. For example, the pKa may be between -5 and 11, between -5 and 9, between -5 and 7, between 0 and 7, between 3 and 7, between -5 and 5, between 0 and 5 between 3 and 5.

如本文所使用，「該至少一個NH 2基團之pKa」係指-log(Ka)，其中Ka為該至少一個NH 2基團與對應酸性形式NH 3 +之間的酸解離常數。pKa可藉由熟習此項技術者已知之任何技術，例如藉由電位滴定或電導測定來測定。 As used herein, "pKa of the at least one NH 2 group" refers to -log(Ka), where Ka is the acid dissociation constant between the at least one NH 2 group and the corresponding acidic form NH 3+ . pKa may be determined by any technique known to those skilled in the art, such as by potentiometric titration or conductometric measurement.

在另一特定實施例中，該NH 2基團為直接連接至芳族基(諸如伸芳基或伸雜芳基)之NH 2基團。在此類實施例中，染料因此為芳族胺。 In another specific embodiment, the NH 2 group is an NH 2 group directly linked to an aromatic group, such as an aryl or heteroarylene group. In such embodiments, the dye is therefore an aromatic amine.

在一更特定實施例中，該染料為式(I)化合物： [H 2N-(X-Y) n] m-Z   (I)， 其中： - n為0或1， - m為1至4之整數， - X為伸芳基或伸雜芳基，該基團視情況經選自由以下組成之群的至少一個取代基取代：(C 1-C 12)烷基、(C 2-C 12)烯基、(C 2-C 12)炔基、(C 3-C 12)環烷基、(C 2-C 12)雜環烷基、芳基、雜芳基、鹵素、-CN、-NO 2、-C(O)R 1、-C(O)OR 2、-C(O)NR 3R 4、-S(O) 2R 5、-S(O) 2OR 6、-NHS(O) 2R 7、-NHS(O) 2OR 8、-OR 9、-SR 10、及-NR 11R 12， R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R 11及R 12各自獨立地選自氫、(C 1-C 12)烷基、(C 2-C 12)烯基、(C 2-C 12)炔基、(C 3-C 12)環烷基、(C 2-C 12)雜環烷基、芳基及雜芳基， - Y為選自以下基團之間隔基：(C 1-C 12)伸烷基、(C 2-C 12)伸烯基、(C 2-C 12)伸炔基、(C 3-C 12)伸環烷基、(C 2-C 12)伸雜環烷基、伸芳基及伸雜芳基，該基團視情況間雜有選自以下之至少一個基團：-O-、-S-、-NH-、-C(O)-及-S(O) 2，及 - Z為發色團部分， 或其鹽。 In a more specific embodiment, the dye is a compound of formula (I): [H 2 N-(XY) n ] m -Z (I), where: - n is 0 or 1, - m is between 1 and 4 Integer , - _ Alkenyl, (C 2 -C 12 ) alkynyl, (C 3 -C 12 ) cycloalkyl, (C 2 -C 12 ) heterocycloalkyl, aryl, heteroaryl, halogen, -CN, -NO 2 , -C(O)R 1 , -C(O)OR 2 , -C(O)NR 3 R 4 , -S(O) 2 R 5 , -S(O) 2 OR 6 , -NHS(O ) 2 R 7 , -NHS(O) 2 OR 8 , -OR 9 , -SR 10 , and -NR 11 R 12 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 are each independently selected from hydrogen, (C 1 -C 12 )alkyl, (C 2 -C 12 )alkenyl, (C 2 -C 12 )alkynyl , (C 3 -C 12 ) cycloalkyl, (C 2 -C 12 ) heterocycloalkyl, aryl and heteroaryl, - Y is a spacer selected from the following groups: (C 1 -C 12 ) Alkylene group, (C 2 -C 12 ) alkenylene group, (C 2 -C 12 ) alkynylene group, (C 3 -C 12 ) cycloalkylene group, (C 2 -C 12 ) heterocycloalkyl group , aryl and heteroaryl groups, the group is optionally mixed with at least one group selected from the following: -O-, -S-, -NH-, -C(O)- and -S(O) 2 , and -Z is the chromophore part, or its salt.

因此，根據本發明之式(I)化合物包含連接至m個H 2N-(X-Y) n-基團之發色團部分Z。 Compounds of formula (I) according to the invention therefore comprise a chromophore moiety Z linked to m H2N- (XY) n -groups.

如本文所使用，m表示共價連接至發色團部分Z之[H 2N-(X-Y) n]-基團之數目。根據本發明，m為1至4之整數，較佳地m為1或2，更佳地m為1。 As used herein, m represents the number of [ H2N- (XY) n ]-groups covalently attached to the chromophore moiety Z. According to the present invention, m is an integer from 1 to 4, preferably m is 1 or 2, and more preferably m is 1.

如本文所使用，n表示[H 2N-(X-Y) n]-基團中之(X-Y)基團之數目，其中(X-Y)基團共價連接至NH 2及發色團部分。根據本發明，n為0或1。換言之，根據本發明，式(I)化合物之各NH 2可直接(當n為0時)或經由(X-Y)基團(當n為1時)連接至發色團。 As used herein, n represents the number of (XY) groups in the [ H2N- (XY) n ]-group, where the (XY) group is covalently linked to the NH2 and chromophore moieties. According to the invention, n is 0 or 1. In other words, according to the present invention, each NH of the compound of formula (I) can be linked to the chromophore directly (when n is 0) or via an (XY) group (when n is 1).

當m高於1，即2、3或4時，應理解各n獨立地為0或1。When m is higher than 1, ie 2, 3 or 4, it is understood that each n is independently 0 or 1.

在一特定實施例中，該式(I)化合物使得n為1。在此類實施例中，當m高於1時，應理解各n為1。In a specific embodiment, the compound of formula (I) is such that n is 1. In such embodiments, when m is higher than 1, it will be understood that each n is 1.

在一特定實施例中，X係伸芳基，較佳伸苯基，該基團視情況經選自由以下組成之群的至少一個取代基取代：(C 1-C 12)烷基、(C 2-C 12)烯基、(C 2-C 12)炔基、(C 3-C 12)環烷基、(C 2-C 12)雜環烷基、芳基、雜芳基、鹵素、-CN、-NO 2、-C(O)R 1、-C(O)OR 2、-C(O)NR 3R 4、-S(O) 2R 5、-S(O) 2OR 6、-NHS(O) 2R 7、-NHS(O) 2OR 8、-OR 9、-SR 10、及-NR 11R 12， R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R 11及R 12各自獨立地選自氫、(C 1-C 12)烷基、(C 2-C 12)烯基、(C 2-C 12)炔基、(C 3-C 12)環烷基、(C 2-C 12)雜環烷基、芳基及雜芳基。 In a specific embodiment , -C 12 )alkenyl, (C 2 -C 12 )alkynyl, (C 3 -C 12 )cycloalkyl, (C 2 -C 12 )heterocycloalkyl, aryl, heteroaryl, halogen, - CN, -NO 2 , -C(O)R 1 , -C(O)OR 2 , -C(O)NR 3 R 4 , -S(O) 2 R 5 , -S(O) 2 OR 6 , -NHS(O) 2 R 7 , -NHS(O) 2 OR 8 , -OR 9 , -SR 10 , and -NR 11 R 12 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 are each independently selected from hydrogen, (C 1 -C 12 ) alkyl, (C 2 -C 12 ) alkenyl, (C 2 -C 12 ) Alkynyl, (C 3 -C 12 ) cycloalkyl, (C 2 -C 12 ) heterocycloalkyl, aryl and heteroaryl.

在另一特定實施例中，Y為選自以下基團之間隔基：(C 1-C 12)伸烷基及伸芳基，該基團視情況間雜有選自以下之至少一個基團：-O-、-S-、-NH-、-C(O)-及-S(O) 2。 In another specific embodiment, Y is a spacer group selected from the following groups: (C 1 -C 12 ) alkylene and aryl groups, optionally interspersed with at least one group selected from the following: -O-, -S-, -NH-, -C(O)- and -S(O) 2 .

在一較佳實施例中，該式(I)化合物使得n為0。在此類實施例中，當m高於1時，應理解各n為0。其中n為0之式(I)化合物可由下式(I-0)表示： (H 2N) m-Z   (I-0)， 其中m及Z如本文所定義。 In a preferred embodiment, the compound of formula (I) is such that n is 0. In such embodiments, when m is higher than 1, it will be understood that each n is 0. Compounds of formula (I) wherein n is 0 may be represented by the following formula (I-0): (H 2 N) m -Z (I-0), wherein m and Z are as defined herein.

在一特定實施例中，該式(I)化合物(或(I-0))使得Z為選自由以下組成之群的發色團部分：苯基、若丹明(rhodamine)、蒽醌、三芳基甲烷、酞菁、單偶氮染料、雙偶氮染料、三偶氮染料、聚偶氮染料、雙亞吲哚基二酮、茚二酮、喹啉茚二酮、吖啶、噻𠯤、噻唑、㗁𠯤、啡㗁𠯤、𠮿、氯、二酮吡咯并吡咯、喹吖啶酮、花青素及類黃酮。In a specific embodiment, the compound of formula (I) (or (I-0)) is such that Z is a chromophore moiety selected from the group consisting of: phenyl, rhodamine, anthraquinone, triaryl Methyl methane, phthalocyanine, monoazo dyes, disazo dyes, triazo dyes, polyazo dyes, bisindolylidenedione, indenedione, quinoline indenedione, acridine, thienedione, Thiazole, thiazole, thiazole, thiazole, thiazole, thiazole , chlorine, diketopyrrolopyrrole, quinacridone, anthocyanins and flavonoids.

在一更特定實施例中，該染料(或式(I)化合物)由下式中之任一者表示： 。 In a more specific embodiment, the dye (or compound of formula (I)) is represented by any of the following formulas: .

在另一特定實施例中，該染料(或式(I)化合物)由下式中之任一者表示： 。 In another specific embodiment, the dye (or compound of formula (I)) is represented by any of the following formulas: .

在一較佳實施例中，該染料(或式(I)化合物)由下式中之任一者表示： 。 In a preferred embodiment, the dye (or compound of formula (I)) is represented by any one of the following formulas: .

在另一較佳實施例中，該染料(或式(I)化合物)由下式中之任一者表示： 。 In another preferred embodiment, the dye (or compound of formula (I)) is represented by any one of the following formulas: .

在一更佳實施例中，該染料(或式(I)化合物)由下式中之任一者表示： 。 In a more preferred embodiment, the dye (or compound of formula (I)) is represented by any one of the following formulas: .

在一甚至更佳實施例中，該染料(或式(I)化合物)由下式中之任一者表示： 。 In an even more preferred embodiment, the dye (or compound of formula (I)) is represented by any one of the following formulas: .

如上所示之式(I-a)化合物在本文中亦稱為「 對-甲苯胺」。如上所示之式(I-b)化合物在本文中亦稱為「若丹明123」。如上所示之式(I-f)化合物在本文中亦稱為「酸性紅266」。如上所示之式(I-h)化合物在本文中亦稱為「酸性紅33」。如上所示之式(I-p)化合物在本文中亦稱為「酸性紫19」。如上所示之式(I-r)化合物在本文中亦稱為「酸性藍25」。如上所示之式(I-s)化合物在本文中亦稱為「酸性黑41」。 The compound of formula (Ia) shown above is also referred to herein as " p -toluidine". The compound of formula (Ib) shown above is also referred to herein as "rhodamine 123". The compound of formula (If) shown above is also referred to herein as "Acid Red 266". The compound of formula (Ih) shown above is also referred to herein as "acid red 33". The compound of formula (Ip) shown above is also referred to herein as "acid violet 19". The compound of formula (Ir) shown above is also referred to herein as "acid blue 25". The compound of formula (Is) shown above is also referred to herein as "Acid Black 41".

如本文所使用，「還原劑」係關於能夠將亞胺基還原為胺基之一或多種化合物。還原劑尤其可為還原胺化反應中通常使用之任何還原劑。更明確而言，該還原劑可為氫化物-型還原劑。如本文所使用，氫化物-型還原劑為具有一或多種氫化物(正式地：「H -」)的化合物，其可轉移至另一化合物，引起該另一化合物之還原。 As used herein, "reducing agent" refers to one or more compounds capable of reducing an imine group to an amine group. The reducing agent may in particular be any reducing agent customarily used in reductive amination reactions. More specifically, the reducing agent may be a hydride-type reducing agent. As used herein, a hydride-type reducing agent is a compound that has one or more hydrides (formally: "H - ") that can be transferred to another compound, causing the reduction of the other compound.

在一特定實施例中，該還原劑為硼烷化合物。如本文所使用，「硼烷化合物」係指具有至少一個B-H鍵的任何有機化合物。在一更特定實施例中，還原劑為選自由以下組成之群的硼烷化合物：硼氫化鈉或硼氫化鉀、硼氫化鋅、氰基硼氫化鈉、硼烷(BH 3)、二硼烷(B 2H 6)、吡啶硼烷、甲基吡啶硼烷、5-乙基-2-甲基吡啶硼烷、𠰌啉硼烷、4-甲基𠰌啉硼烷、三乙胺硼烷、9-硼雙環[3.3.1]壬烷(9-BBN)、單異松蒎硼烷、二環己基硼烷、二基硼烷、二異戊基硼烷、兒茶酚硼烷、頻哪醇硼烷、三仲丁基硼氫化鋰(L-selectride)及其混合物。 In a specific embodiment, the reducing agent is a borane compound. As used herein, "borane compound" refers to any organic compound having at least one BH bond. In a more specific embodiment, the reducing agent is a borane compound selected from the group consisting of sodium or potassium borohydride, zinc borohydride, sodium cyanoborohydride, borane (BH 3 ), diborane (B 2 H 6 ), pyridine borane, methylpyridine borane, 5-ethyl-2-methylpyridine borane, 𠰌line borane, 4-methyl𠰌line borane, triethylamine borane, 9-Boron bicyclo[3.3.1]nonane (9-BBN), monoisopinepineborane, dicyclohexylborane, dicyclohexylborane borane, diisoamylborane, catecholborane, pinacolborane, tri-sec-butyl lithium borohydride (L-selectride) and their mixtures.

在一較佳實施例中，該還原劑為甲基吡啶硼烷。In a preferred embodiment, the reducing agent is picoline borane.

在步驟(b)中，接觸可藉由在適合溶劑(較佳水)中混合步驟(a)中獲得之經氧化之纖維素材料(該染料包含至少一個NH 2基團)與該還原劑進行。更明確而言，此類接觸可藉由將該經氧化之纖維素材料浸漬至包含染料及還原劑之溶劑(較佳水)中來進行。 In step (b), contacting may be carried out by mixing the oxidized cellulosic material obtained in step (a) (the dye comprising at least one NH 2 group) with the reducing agent in a suitable solvent, preferably water. . More specifically, such contacting may be carried out by impregnating the oxidized cellulosic material into a solvent (preferably water) containing dye and reducing agent.

本發明方法之步驟(b)可在溫和條件，特定言之溫和溫度下進行。在一特定實施例中，步驟(b)在包含於10℃與60℃之間、較佳在30℃與50℃之間、更佳在35℃與45℃之間的溫度下進行。在另一特定實施例中，步驟(b)進行的持續時間包含於30分鐘與30小時之間，例如2小時與5小時之間。步驟(b)可在空氣下進行。Step (b) of the method of the present invention can be carried out under mild conditions, in particular at mild temperatures. In a particular embodiment, step (b) is performed at a temperature comprised between 10°C and 60°C, preferably between 30°C and 50°C, more preferably between 35°C and 45°C. In another specific embodiment, step (b) is performed for a duration comprised between 30 minutes and 30 hours, such as between 2 hours and 5 hours. Step (b) can be carried out under air.

在另一特定實施例中，步驟(b)在酸存在下進行。酸之實例包括但不限於鹽酸、硫酸、羧酸(諸如甲酸、乙酸、丙酸或苯甲酸)。較佳地，該酸為乙酸。步驟(b)中之該酸之濃度可有利地包含於2%與30% (v/v)之間。In another specific embodiment, step (b) is performed in the presence of acid. Examples of acids include, but are not limited to, hydrochloric acid, sulfuric acid, carboxylic acids such as formic acid, acetic acid, propionic acid, or benzoic acid. Preferably, the acid is acetic acid. The concentration of the acid in step (b) may advantageously be comprised between 2% and 30% (v/v).

就經氧化之纖維素材料而言，染料及還原劑可過量使用。步驟(b)中之染料(例如式(I)化合物)之濃度可例如包含於0.01 mM與10 M之間。步驟(b)中之還原劑之濃度可例如包含於0.1 mM與1 M之間。For oxidized cellulosic materials, dyes and reducing agents can be used in excess. The concentration of the dye (eg compound of formula (I)) in step (b) may, for example, be comprised between 0.01 mM and 10 M. The concentration of the reducing agent in step (b) may, for example, be comprised between 0.1 mM and 1 M.

在一特定實施例中，經氧化之纖維素材料之羰基與染料之莫耳比包含於1/20與1/1之間，較佳包含於1/10與1/1之間。在一特定實施例中，經氧化之纖維素材料之羰基與還原劑之莫耳比包含於1/2與1/1之間，較佳包含於1/1.5與1/1之間。經氧化之纖維素材料之羰基的莫耳量可尤其根據如下文在實例中所描述之方法藉由分光光度法測定。In a specific embodiment, the molar ratio of carbonyl groups to dye of the oxidized cellulose material is comprised between 1/20 and 1/1, preferably comprised between 1/10 and 1/1. In a specific embodiment, the molar ratio of the carbonyl group of the oxidized cellulose material to the reducing agent is comprised between 1/2 and 1/1, preferably comprised between 1/1.5 and 1/1. The molar amount of carbonyl groups of the oxidized cellulosic material can be determined spectrophotometrically, inter alia, according to the method as described below in the Examples.

在其中將經氧化之纖維素材料浸漬至包含染料及還原劑之溶劑(較佳水)中的一實施例中，可在回收經氧化之纖維素材料後再使用包含染料及還原劑之該溶劑。In one embodiment in which the oxidized cellulosic material is impregnated into a solvent (preferably water) containing a dye and a reducing agent, the solvent containing the dye and the reducing agent can be used after recovering the oxidized cellulosic material. .

本發明方法之步驟(b)允許獲得染色纖維素材料。在本發明方法之步驟(c)中，染色纖維素材料可藉由熟習此項技術者已知之任何適合技術回收，例如藉由自包含染料及還原劑之溶劑收集染色纖維素材料。Step (b) of the method of the invention allows obtaining dyed cellulosic material. In step (c) of the process of the invention, the dyed cellulosic material may be recovered by any suitable technique known to those skilled in the art, for example by collecting the dyed cellulosic material from a solvent containing dye and reducing agent.

有利地，本發明方法之步驟(a)、(b)及(c)連續進行。Advantageously, steps (a), (b) and (c) of the method of the invention are carried out continuously.

在一特定實施例中，該方法包含以下連續步驟： (a)使纖維素材料與蟲漆酶及TEMPO或其衍生物接觸，以獲得氧化纖維素材料； (b)使步驟(a)之氧化纖維素材料與如本文所定義之式(I)染料(較佳如上所示之式(I-a)、式(I-b)、式(I-c)、式(I-d)、式(I-e)、式(I-f)、式(I-g)、式(I-h)、式(I-i)、式(I-j)、式(I-k)、式(I-l)、式(I-m)、式(I-n)、式(I-o)、式(I-p)、式(I-q)、式(I-r)或式(I-s)，更佳式(I-a)、式(I-b)或式(I-h)之染料)在硼烷化合物存在下接觸，從而獲得染色纖維素材料；及 (c)回收染色纖維素材料。 In a specific embodiment, the method includes the following consecutive steps: (a) contacting cellulose materials with laccase and TEMPO or derivatives thereof to obtain oxidized cellulose materials; (b) combining the oxidized cellulose material of step (a) with a dye of formula (I) as defined herein (preferably formula (I-a), formula (I-b), formula (I-c), formula (I-d) as shown above , formula (I-e), formula (I-f), formula (I-g), formula (I-h), formula (I-i), formula (I-j), formula (I-k), formula (I-l), formula (I-m), formula (I-n) , formula (I-o), formula (I-p), formula (I-q), formula (I-r) or formula (I-s), more preferably formula (I-a), formula (I-b) or formula (I-h) dye) in the presence of borane compounds contact, thereby obtaining dyed cellulosic material; and (c) Recycling of dyed cellulosic materials.

在一特定實施例中，該方法包含以下連續步驟： (a)使纖維素材料與蟲漆酶及TEMPO或其衍生物接觸，以獲得氧化纖維素材料； (b)使步驟(a)之氧化纖維素材料與如本文所定義之式(I)染料(較佳如上所示之式(I-a)、式(I-b)、式(I-c)、式(I-d)、式(I-e)、式(I-f)、式(I-g)、式(I-h)、式(I-i)、式(I-j)、式(I-k)、式(I-l)、式(I-m)、式(I-n)、式(I-o)、式(I-p)、式(I-q)、式(I-r)或式(I-s)，更佳式(I-a)、式(I-b)或式(I-h)之染料)在硼烷化合物存在下接觸，從而獲得染色纖維素材料；及 (c)回收染色纖維素材料， 其中步驟(a)在包含於10℃與60℃之間(較佳在20與50℃之間)的溫度下進行，且步驟(b)在包含於10℃與60℃之間(較佳在30與50℃之間)的溫度下進行。 In a specific embodiment, the method includes the following consecutive steps: (a) contacting cellulose materials with laccase and TEMPO or derivatives thereof to obtain oxidized cellulose materials; (b) combining the oxidized cellulose material of step (a) with a dye of formula (I) as defined herein (preferably formula (I-a), formula (I-b), formula (I-c), formula (I-d) as shown above , formula (I-e), formula (I-f), formula (I-g), formula (I-h), formula (I-i), formula (I-j), formula (I-k), formula (I-l), formula (I-m), formula (I-n) , formula (I-o), formula (I-p), formula (I-q), formula (I-r) or formula (I-s), more preferably formula (I-a), formula (I-b) or formula (I-h) dye) in the presence of borane compounds contact, thereby obtaining dyed cellulosic material; and (c) Recycling of dyed cellulosic materials, Wherein step (a) is carried out at a temperature comprised between 10°C and 60°C (preferably between 20 and 50°C), and step (b) is carried out at a temperature comprised between 10°C and 60°C (preferably between 20°C and 50°C). between 30 and 50°C).

在另一特定實施例中，該方法包含以下連續步驟： (a)使選自棉、亞麻、***、黃麻、人絲、萊賽爾、螺縈或莫代爾(較佳棉)之纖維素材料與來自貝萊斯芽孢桿菌、平流層芽孢桿菌、無氧芽孢桿菌、曲黴菌屬或變色栓菌之蟲漆酶、及TEMPO或其衍生物接觸，從而獲得氧化纖維素材料； (b)使步驟(a)之氧化纖維素材料與如本文所定義之式(I)染料(較佳如上所示之式(I-a)、式(I-b)、式(I-c)、式(I-d)、式(I-e)、式(I-f)、式(I-g)、式(I-h)、式(I-i)、式(I-j)、式(I-k)、式(I-l)、式(I-m)、式(I-n)、式(I-o)、式(I-p)、式(I-q)、式(I-r)或式(I-s)，更佳式(I-a)、式(I-b)或式(I-h)之染料)在甲基吡啶硼烷存在下接觸，從而獲得染色纖維素材料；及 (c)回收染色纖維素材料， 其中步驟(a)在包含於10℃與60℃之間(較佳在20與50℃之間)的溫度下進行，且步驟(b)在包含於10℃與60℃之間(較佳在30與50℃之間)的溫度下進行。 In another specific embodiment, the method includes the following consecutive steps: (a) Cellulosic materials selected from cotton, linen, hemp, jute, rayon, lyocell, spirone or modal (preferably cotton) are mixed with bacteria from Bacillus veleis, Bacillus stratospherica, anaerobic The laccase of Bacillus, Aspergillus or Trametes versicolor comes into contact with TEMPO or its derivatives to obtain oxidized cellulose materials; (b) combining the oxidized cellulose material of step (a) with a dye of formula (I) as defined herein (preferably formula (I-a), formula (I-b), formula (I-c), formula (I-d) as shown above , formula (I-e), formula (I-f), formula (I-g), formula (I-h), formula (I-i), formula (I-j), formula (I-k), formula (I-l), formula (I-m), formula (I-n) , formula (I-o), formula (I-p), formula (I-q), formula (I-r) or formula (I-s), more preferably formula (I-a), formula (I-b) or formula (I-h) dye) in methylpyridine boron contact in the presence of an alkane, thereby obtaining a dyed cellulosic material; and (c) Recycling of dyed cellulosic materials, Wherein step (a) is carried out at a temperature comprised between 10°C and 60°C (preferably between 20 and 50°C), and step (b) is carried out at a temperature comprised between 10°C and 60°C (preferably between 20°C and 50°C). between 30 and 50°C).

本發明之另一目標為一種藉由如本文中所定義之方法獲得的染色纖維素材料。Another object of the invention is a dyed cellulosic material obtained by a method as defined herein.

本發明亦將在以下實例中進一步詳細描述，該等實例不意欲限制如由隨附申請專利範圍所定義之本發明之範疇。The invention will also be described in further detail in the following examples, which are not intended to limit the scope of the invention as defined by the appended claims.

實例 材料及方法來自變色栓菌之蟲漆酶係購自Sigma Aldrich。 Example Materials and Methods Laccase enzyme from Trametes versicolor was purchased from Sigma Aldrich.

自變色栓菌純化蟲漆酶。使用帶有Pellicon XL過濾器PXC030C50 (30 KDa) (Merck)再生纖維素膜(過濾面積1.14 m 2)的Labscale TM系統(Millipore，Merck)在2.1巴之固定跨膜壓力下在4℃下進行不連續透濾。進料室裝填有50 mL 30 g/L之TvL溶液且用500 mL水或pH 6.0之20 mM乙酸鹽緩衝液透濾兩次，且隨後濃縮至15 mL。在透濾前，膜預先用對應緩衝液平衡15分鐘。在各次試驗後，膜用蒸餾水沖洗且儲存於50 mM NaOH中。經透濾且濃縮的酶製劑藉由HPAEC-PAD分析來分析。 Purification of laccase from Trametes versicolor. The experiments were carried out at a fixed transmembrane pressure of 2.1 bar at 4°C using a Labscale TM system (Millipore, Merck) with a Pellicon Continuous diafiltration. The feed chamber was filled with 50 mL of 30 g/L TvL solution and diafiltered twice with 500 mL of water or 20 mM acetate buffer, pH 6.0, and then concentrated to 15 mL. Before diafiltration, the membrane was pre-equilibrated with the corresponding buffer for 15 minutes. After each experiment, the membranes were rinsed with distilled water and stored in 50 mM NaOH. Diafiltered and concentrated enzyme preparations were analyzed by HPAEC-PAD analysis.

測定蟲漆酶活性 。 使用比色檢定用50 μL 1 mM ABTS (2,2-次偶氮基-雙(3-乙基苯并噻唑啉-6-磺酸)、10 μL蟲漆酶溶液及440 μL乙酸鹽緩衝液(20 mM，pH 3.5)測定蟲漆酶活性。藉由使用UV-Vis分光光度計(Cary 100 Bio)量測在30℃下在420 nm下之吸光度增加(ε₄₂₀= 3.6 × 10⁴ M -1cm -1)來監測ABTS之氧化。蟲漆酶活性單位(U)定義為每分鐘氧化1 μmol ABTS之蟲漆酶的量。 Determination of laccase activity . Use 50 μL of 1 mM ABTS (2,2-azo-bis(3-ethylbenzothiazoline-6-sulfonic acid)), 10 μL of laccase solution and 440 μL of acetate buffer for colorimetric assay. (20 mM, pH 3.5) to measure laccase activity. By measuring the absorbance increase at 420 nm at 30°C using a UV-Vis spectrophotometer (Cary 100 Bio) (ε₄₂₀= 3.6 × 10⁴ M -1 cm -1 ) to monitor the oxidation of ABTS. Laccase activity unit (U) is defined as the amount of laccase that oxidizes 1 μmol ABTS per minute.

氧化 Glc5 之 IC-HRMS 。Glc5氧化產物之質量係在與Thermo Scientific線性阱四極(LTQ，linear trap quadrupole)軌道阱質譜儀(Orbitrap Velos)混合傅里葉變換(FT，Fourier-transform)質譜儀(FTMS；Thermo Fisher Scientific, San Jose, CA, USA)耦聯的Dionex ICS-5000+無試劑HPIC (Thermo Fisher Scientific, Sunnyvale, CA, USA)系統上，使用液體陰離子交換層析測定。樣品在50分鐘內，在0.38 mL/min之流動速率下，使用應用於配備有AS11 Thermo Scientific TMDionex TMIonPac TM保護管柱(50×2 mm)之IC Dionex TMIonPac TMAS11-HC管柱(250×2 mm)的7 mM至100 mM KOH線性梯度溶離而分離。管柱及自動進樣器溫度分別為25℃及4℃。注入之樣品體積為5 µL。負模式下電噴霧電離(ESI)之條件如下：噴霧電壓為2.7 kV，毛細管及去溶劑化溫度分別為350及350℃，且最大注入時間為50 ms。氮氣用作鞘氣(壓力，50單位)及輔助氣體(壓力，5單位)。全掃描模式之自動增益控制(AGC)設定為1e6，質量解析度為60.000 (在400 m/z下)。對於全掃描MS分析，光譜在m/z 80.0至1000.0範圍內記錄。最後，使用Thermo Scientific Xcalibur軟體進行資料獲取。 IC-HRMS of oxidized Glc5 . The mass of the Glc5 oxidation product was measured with a Thermo Scientific linear trap quadrupole (LTQ) orbitrap mass spectrometer (Orbitrap Velos) hybrid Fourier transform (FT) mass spectrometer (FTMS; Thermo Fisher Scientific, San Francisco Jose, CA, USA) coupled to a Dionex ICS-5000+ reagent-free HPIC (Thermo Fisher Scientific, Sunnyvale, CA, USA) system, liquid anion exchange chromatography was used. The sample was applied to an IC Dionex TM IonPac TM AS11 - HC column (50 × 2 mm) equipped with an AS11 Thermo Scientific TM Dionex TM IonPac TM guard column (50 × 2 mm) at a flow rate of 0.38 mL/min. Separate by linear gradient elution from 7 mM to 100 mM KOH (250 × 2 mm). The column and autosampler temperatures are 25°C and 4°C respectively. The injected sample volume is 5 µL. The conditions for electrospray ionization (ESI) in negative mode are as follows: the spray voltage is 2.7 kV, the capillary and desolvation temperatures are 350 and 350°C, respectively, and the maximum injection time is 50 ms. Nitrogen was used as sheath gas (pressure, 50 units) and auxiliary gas (pressure, 5 units). The automatic gain control (AGC) of the full scan mode was set to 1e6, and the mass resolution was 60.000 (at 400 m/z). For full scan MS analysis, spectra were recorded in the range m/z 80.0 to 1000.0. Finally, Thermo Scientific Xcalibur software was used for data acquisition.

對甲苯胺或若丹明接枝之 Glc5 的 LC-HRMS/UV 分析。分析係在與Thermo Sceintific Q Exactive TMPlus混合四極桿-Orbitrap TM質譜儀(Thermo Fisher)耦聯之vanquish TM系統上進行。樣品在95分鐘內，以0.7 mL/min之流動速率，用應用於配備有Shodex™ Asahipak NH2P-50 4A保護管柱(4.6 mm × 10 mm)之Shodex™ Asahipak NH2P-50 4E (4.6mm × 250 mm)的含有10% ACN/40% H2O/50% 20 mM乙酸銨之混合物等度溶離而分離。管柱及自動進樣器溫度分別為40℃及4℃。注入之樣品體積為10 µL。根據接枝化合物在不同波長下監測LC溶離液：對甲苯胺為286 nm且若丹明123為540 nm。負模式下電噴霧電離(ESI)之條件如下：噴霧電壓為2.75 kV，毛細管及去溶劑化溫度分別為400及400℃，且最大注入時間為100 ms。氮氣用作鞘氣(壓力，75單位)及輔助氣體(壓力，20單位)。全掃描模式之自動增益控制(AGC)設定為1e6，質量解析度為70.000 (在800 m/z下)。對於全掃描MS分析，光譜在m/z 80.0至1000.0範圍內記錄。最後，使用Thermo Scientific Xcalibur軟體進行資料獲取。 LC-HRMS/UV analysis of p-toluidine or rhodamine-grafted Glc5 . Analyzes were performed on a vanquish TM system coupled to a Thermo Sceintific Q Exactive TM Plus hybrid quadrupole-Orbitrap TM mass spectrometer (Thermo Fisher). The sample was applied to a Shodex™ Asahipak NH2P-50 4E (4.6mm × 250 mm) mixture containing 10% ACN/40% H2O/50% 20 mM ammonium acetate was separated by isocratic dissolution. The column and autosampler temperatures are 40°C and 4°C respectively. The injected sample volume is 10 µL. The LC eluate was monitored at different wavelengths depending on the grafted compound: 286 nm for p-toluidine and 540 nm for rhodamine 123. The conditions for electrospray ionization (ESI) in negative mode are as follows: the spray voltage is 2.75 kV, the capillary and desolvation temperatures are 400 and 400°C, respectively, and the maximum injection time is 100 ms. Nitrogen was used as sheath gas (pressure, 75 units) and auxiliary gas (pressure, 20 units). The automatic gain control (AGC) of the full scan mode was set to 1e6, and the mass resolution was 70.000 (at 800 m/z). For full scan MS analysis, spectra were recorded in the range m/z 80.0 to 1000.0. Finally, Thermo Scientific Xcalibur software was used for data acquisition.

水解棉纖維之 HPLC-UV-MS 分析 ：分析係用配備有Dionex 340 UV/VIS偵測器且與簡單四極桿質譜儀(MSQ Plus，Thermo Scientific)耦聯之Ultimate 3000系列層析儀，且由維持在40℃之Phenomenex PFP C18管柱(Luna 5 μm，100 A，250 × 4.6 mm，USA)分離進行。根據染料之最大吸收波長，分別在286 nm、500 nm及532 nm下對接枝有對甲苯胺(「 pT」)、若丹明123 (「RHO123」)或酸性紅33 (「AR33」)之低聚醣進行UV偵測。使用A=H 2O/甲酸0.05% (v/v)及B=ACN/甲酸0.05% (v/v)作為溶離劑溶離所注入之樣品(50 µL)。接枝有RHO123之樣品所用的梯度如下：對於接枝有RHO123之樣品，0-20分鐘自10% B至30% B，或對於接枝有AR33之樣品，0-50分鐘自10% B至30% B，隨後對於所有樣品，在10分鐘內30% B增加至40% B，隨後在5分鐘內40% B增加至100% B，且在100% B保持5分鐘，最後恢復至起始條件，且使管柱平衡5分鐘。在兩種情況下，使用0.5 mL/min之流動速率。質譜儀係在正模式下使用，其中電壓錐在75 V下，電噴霧電離(ESI)離子源之溫度為450℃且載氣為氮氣。噴霧電壓設定為3.5 kV。質譜儀自 m/z100至1500掃描。使用Chromeleon™ 7.2資料系統進行資料獲取及處理。 HPLC-UV-MS analysis of hydrolyzed cotton fibers : An Ultimate 3000 series chromatograph equipped with a Dionex 340 UV/VIS detector coupled to a simple quadrupole mass spectrometer (MSQ Plus, Thermo Scientific) was used. The separation was performed on a Phenomenex PFP C18 column (Luna 5 μm, 100 A, 250 × 4.6 mm, USA) maintained at 40°C. According to the maximum absorption wavelength of the dye, p-toluidine (" p T"), rhodamine 123 ("RHO123") or acid red 33 ("AR33") are grafted at 286 nm, 500 nm and 532 nm respectively. of oligosaccharides for UV detection. Use A=H 2 O/formic acid 0.05% (v/v) and B=ACN/formic acid 0.05% (v/v) as eluants to elute the injected sample (50 µL). The gradient used for samples grafted with RHO123 was as follows: 0-20 minutes from 10% B to 30% B for samples grafted with RHO123, or 0-50 minutes from 10% B to 30% B for samples grafted with AR33. 30% B, then for all samples, 30% B increased to 40% B over 10 minutes, then 40% B increased to 100% B over 5 minutes, and maintained at 100% B for 5 minutes, finally returning to starting conditions and allow the column to equilibrate for 5 minutes. In both cases, a flow rate of 0.5 mL/min was used. The mass spectrometer was used in positive mode with the voltage cone at 75 V, the electrospray ionization (ESI) ion source temperature at 450°C, and the carrier gas was nitrogen. The spray voltage was set to 3.5 kV. The mass spectrometer scans from m/z 100 to 1500. Use Chromeleon™ 7.2 data system for data acquisition and processing.

冷凍乾燥之氧化棉纖維之 FTIR-ATR 光譜係使用Nicolet IS10紅外分光光度計記錄，該分光光度計利用具有金剛石晶體的Golden Gate衰減全反射(ATR)附件(Thermo Nicolet，USA)。吸光度量測在400至3900 cm -1之波長範圍內以16 cm -1之解析度進行，且每一樣品記錄32次掃描。 FTIR-ATR spectra of freeze-dried oxidized cotton fibers were recorded using a Nicolet IS10 infrared spectrophotometer utilizing a Golden Gate attenuated total reflection (ATR) accessory with diamond crystals (Thermo Nicolet, USA). Absorbance measurements were performed in the wavelength range from 400 to 3900 cm -1 with a resolution of 16 cm -1 and 32 scans were recorded for each sample.

對未處理及經處理之棉纖維進行 元素分析以測定其碳、氫、氮含量。使用PerkinElmer 2400系列II分析儀，Toulouse在Laboratoire de Chimie de Coordination du CNRS可用之設施中進行元素分析。氮之偵測範圍在0.001 mg與6.0 mg之間。 Elemental analysis was performed on untreated and treated cotton fibers to determine their carbon, hydrogen, and nitrogen content. Using a PerkinElmer 2400 Series II analyzer, Toulouse performed elemental analysis at the facilities available at the Laboratoire de Chimie de Coordination du CNRS. The detection range of nitrogen is between 0.001 mg and 6.0 mg.

耐洗色牢度測試：織物有色樣品之色耐性測試由使用洗衣清潔劑之三個連續洗滌循環組成。各步驟在60℃下進行60分鐘。在最後一步後，將織物樣品在室溫下乾燥隔夜以評估顏色強度。 Color fastness to washing test : The color fastness test of colored fabric samples consists of three consecutive washing cycles using laundry detergent. Each step was performed at 60°C for 60 minutes. After the final step, the fabric samples were dried at room temperature overnight to evaluate color intensity.

測定氧化棉線中之羰基棉線中羰基之含量使用由Szabolcs (1961)公佈且由Strlič及Pihlar (1997)最佳化之分光光度法測定。「TTC試劑」係藉由將20 mL 0.5 M之氯化三苯基四唑鎓(TTC)與20 mL 0.3 M之KOH混合來製備。將溶液充分混合且在使用前在室溫下靜置10分鐘。 Determination of carbonyl groups in oxidized cotton threads The carbonyl content in cotton threads was determined using the spectrophotometric method published by Szabolcs (1961) and optimized by Strlič and Pihlar (1997). "TTC Reagent" was prepared by mixing 20 mL of 0.5 M triphenyltetrazolium chloride (TTC) and 20 mL of 0.3 M KOH. The solution was mixed thoroughly and allowed to stand at room temperature for 10 minutes before use.

將10 mg乾燥棉線***裝有2 mL水及2 mL新鮮製備之TTC試劑的玻璃管中，且隨後在80℃下在水浴中保溫6分鐘。將懸浮液(4 mL)與MeOH (41 mL)混合以溶解所產生之甲䐶三苯基四唑鎓(TTF)之淡紅色晶體。在482 nm下量測溶液之吸光度。由用D-葡萄糖確定之校準曲線計算羰基之量。自至少三個個別分析計算平均值。使用無水D-葡萄糖作為標準物建立校準曲線。製備5.5 mM之D-葡萄糖溶液，在室溫下靜置30分鐘，且隨後溶解於水中以獲得吸光度，該吸光度使用UV-Vis Cary 3500分光光度計(Agilent Technologies, Santa Clara, CA, United States)用CaryWinUV掃描軟體(版本5.0.0.999)在482 nm下進行量測。10 mg of dry cotton string was inserted into a glass tube containing 2 mL of water and 2 mL of freshly prepared TTC reagent, and then incubated in a water bath at 80 °C for 6 min. The suspension (4 mL) was mixed with MeOH (41 mL) to dissolve the resulting pale red crystals of triphenyltetrazolium (TTF). Measure the absorbance of the solution at 482 nm. The amount of carbonyl groups was calculated from the calibration curve determined with D-glucose. Averages were calculated from at least three individual analyses. A calibration curve was established using anhydrous D-glucose as standard. A 5.5 mM D-glucose solution was prepared, allowed to stand at room temperature for 30 minutes, and then dissolved in water to obtain absorbance using a UV-Vis Cary 3500 spectrophotometer (Agilent Technologies, Santa Clara, CA, United States) Measurements were performed at 482 nm using CaryWinUV scanning software (version 5.0.0.999).

實例 1.纖維素材料之染色方法 1) Glc5 纖維五糖 (Glc5) 之酶促氧化。氧化反應在27 mM Glc5、6 mM TEMPO及5.4 U.mL -1來自變色栓菌之蟲漆酶存在下，在5 mL乙酸鹽緩衝液(20 mM，pH 6.0)中在30℃下進行。將反應物在開口燒瓶中以500 rpm攪拌8小時且藉由對反應混合物取樣250 μL進行監測以用於後續分析。 Example 1. Dyeing method of cellulosic materials 1) Enzymatic oxidation of Glc5 cellopentose (Glc5) . The oxidation reaction was performed in the presence of 27 mM Glc5, 6 mM TEMPO, and 5.4 U.mL -1 laccase from Trametes versicolor in 5 mL acetate buffer (20 mM, pH 6.0) at 30°C. The reaction was stirred in an open flask at 500 rpm for 8 hours and monitored by sampling 250 μL of the reaction mixture for subsequent analysis.

LC-HRMS分析顯示獲得醛及羧酸官能基： - t r= 2.45 min, [M-H] -: m/z 827.27, [(M-H+Na)-H] : m/z 863.24， - t r= 2.90 min, [M-H] -: m/z 843.26， - t r= 4.30 min, [M-H] -: m/z 841.24。 LC-HRMS analysis showed that aldehyde and carboxylic acid functional groups were obtained: - t r = 2.45 min, [MH] - : m/z 827.27, [(M-H+Na)-H] : m/z 863.24, - t r = 2.90 min, [MH] - : m/z 843.26, - t r = 4.30 min, [MH] - : m/z 841.24.

與染料 ( 對甲苯胺 (I-a)) 反應。在Glc5酶促氧化(如上文所描述) 8小時後，在室溫下將500 μL反應介質與2.4 mM對甲苯胺、10 mM 2-甲基吡啶-硼烷及10%乙酸混合3小時。 Reacts with dye ( p-toluidine (Ia)) . After 8 hours of enzymatic oxidation of Glc5 (as described above), 500 μL of reaction medium was mixed with 2.4 mM p-toluidine, 10 mM 2-methylpyridine-borane, and 10% acetic acid for 3 hours at room temperature.

LC-HRMS分析顯示對甲苯胺接枝於Glc5上： - t r= 4.76 min, [M-H] -: m/z 916.33， - t r= 4.89 min, [M-H] -: m/z 918.34， - t r= 41.69 min, [M-H] -: m/z 932.32， - t r= 44.19 min, [M-H] -: m/z 932.32。 LC-HRMS analysis showed that p-toluidine was grafted on Glc5: - t r = 4.76 min, [MH] - : m/z 916.33, - t r = 4.89 min, [MH] - : m/z 918.34, - t r = 41.69 min, [MH] - : m/z 932.32, - t r = 44.19 min, [MH] - : m/z 932.32.

與染料 ( 若丹明 123 (I-b)) 反應。使用若丹明123作為染料對Glc5應用類似程序。 Reacts with dye ( Rhodamine 123 (Ib)) . A similar procedure was applied for Glc5 using rhodamine 123 as dye.

LC-HRMS分析顯示若丹明123接枝於Glc5上： - t r= 2.63 min, [M-H] -: m/z 1154.37。 LC-HRMS analysis showed that rhodamine 123 was grafted onto Glc5: - t r = 2.63 min, [MH] - : m/z 1154.37.

2) 棉 棉之酶促氧化。將215 mg棉線(SAS Bruneel織物，Pérenchies，59，染色備用)用10 mL蒸餾水洗滌6次，且在空氣中乾燥隔夜。隨後將所得棉在含有10 mL含有以下之反應介質的開口燒瓶中浸漬8小時： -介質：變色栓菌蟲漆酶：5.4 U/mL (54U)，TEMPO：6 mM，磷酸鈉緩衝液(50 mM pH 6.0) -條件：500 rpm攪拌；開口燒瓶；30℃；8小時。 -分析。將所得棉用10 mL水洗滌6次，乾燥隔夜且藉由FTIR分析，其中相比於未處理之棉，在1605-1610 cm -1(諸如1608 cm -1)下出現新吸光帶，顯示新C=O鍵之出現(圖1)。固態NMR顯示，約2%之棉經氧化。 2) Enzymatic oxidation of cotton . 215 mg of cotton thread (SAS Bruneel fabric, Pérenchies, 59, ready for dyeing) was washed 6 times with 10 mL of distilled water and dried in air overnight. The resulting cotton was then soaked for 8 hours in an open flask containing 10 mL of reaction medium containing the following: - Medium: Trametes versicolor laccase: 5.4 U/mL (54U), TEMPO: 6 mM, sodium phosphate buffer (50 mM pH 6.0) - Conditions: 500 rpm stirring; open flask; 30°C; 8 hours. -analyze . The obtained cotton was washed 6 times with 10 mL water, dried overnight and analyzed by FTIR. Compared with the untreated cotton, a new absorption band appeared at 1605-1610 cm -1 (such as 1608 cm -1 ), indicating a new The emergence of the C=O bond (Figure 1). Solid-state NMR showed that approximately 2% of the cotton was oxidized.

羰基之初始量(亦即在用蟲漆酶/TEMPO氧化前)為59±8 µmol/g棉。在反應7小時後，量為178±9 µmol羰基/g棉線，對應於6173微莫耳當量葡萄糖。The initial amount of carbonyl groups (i.e. before oxidation with laccase/TEMPO) was 59±8 µmol/g cotton. After 7 hours of reaction, the amount was 178±9 µmol carbonyl/g cotton, corresponding to 6173 µmol equivalents of glucose.

與染料對甲苯胺 (I-a) 反應。隨後將213 mg氧化棉置放於40℃之開口容器中，該容器中含有10 mL 10%乙酸水性溶液，其組分如下： - 介質：對甲苯胺：0.1 M；2-甲基吡啶硼烷(Pic-BH 3)：48 mM (羰基、胺基及Pic-BH 3之莫耳比為1:5:1.2)。 - 條件：T℃=40℃；時間=3小時；500 rpm Reacts with the dye p-toluidine (Ia) . Then 213 mg of oxidized cotton was placed in an open container at 40°C. The container contained 10 mL of 10% acetic acid aqueous solution with the following components: - Medium: p-toluidine: 0.1 M; 2-methylpyridineborane (Pic-BH 3 ): 48 mM (the molar ratio of carbonyl group, amine group and Pic-BH 3 is 1:5:1.2). - Conditions: T℃=40℃; time=3 hours; 500 rpm

用8×10 mL 50:50 H 2O:EtOH及2×10 mL H 2O洗滌所得棉。在各次洗滌之間，將樣品攪拌30秒且最後在室溫下乾燥兩天。 The cotton obtained was washed with 8×10 mL 50:50 H 2 O:EtOH and 2×10 mL H 2 O. Between washes, the samples were stirred for 30 seconds and finally dried at room temperature for two days.

光譜分析顯示氧化後之未接枝棉與接枝棉之間的醛鍵量減少10倍。接枝棉之酸水解(H 2SO 472% (13.5 M)，體積約2 mL，T℃=30℃，時間=1小時)顯示出現在286 nm下吸光之兩個新峰(圖2)。此顯示對甲苯胺在C1及C6處之接枝。 Spectral analysis shows that the amount of aldehyde bonds between ungrafted cotton and grafted cotton after oxidation is reduced by 10 times. Acid hydrolysis of grafted cotton (H 2 SO 4 72% (13.5 M), volume about 2 mL, T°C = 30°C, time = 1 hour) showed the appearance of two new peaks absorbing light at 286 nm (Figure 2) . This shows grafting of p-toluidine at C1 and C6.

使用來自里氏木黴( Trichoderma reesei)之纖維素酶混合物對染色棉進行酶促水解且藉由LC-MS/UV分析水解產物：在洗滌後，將50 mg染色棉與500 µL纖維素酶在乙酸鈉緩衝液(50 mM，pH 4.5)中在40℃下保溫24小時。未處理之棉纖維亦在相同條件下水解以充當對照物，且其比染色線更快降解。24小時後停止反應。離心後，反應上清液中回收之水解產物之HPLC-UV 286nm分析顯示存在對應於產物 P1(t r=9.34 min)及 P2(t r=9.62 min)之兩個主峰，兩者均在286 nm下吸光且不存在於對照實驗中。正模式LC-UV/MS分析進一步展現 P1(m/z 432 [M+H] +)對應於在分子之C-6位置中之一者處接枝有對甲苯胺之纖維二糖分子， P2(m/z 270 [M+H] +)係在C-6位置處接枝有對甲苯胺之葡萄糖分子，且 P3(t r= 24.7 min) (在UV中未偵測到，m/z 521 [M+H] +)係在其各C-6位置處接枝有兩個對甲苯胺分子之纖維二糖(圖3A及圖3B)。 Enzymatic hydrolysis of dyed cotton using a cellulase mixture from Trichoderma reesei and analysis of the hydrolyzate by LC-MS/UV: After washing, 50 mg of dyed cotton was mixed with 500 µL of cellulase in Incubate in sodium acetate buffer (50 mM, pH 4.5) at 40°C for 24 hours. Untreated cotton fiber was also hydrolyzed under the same conditions to serve as a control and degraded faster than dyed thread. The reaction stopped after 24 hours. After centrifugation, HPLC-UV 286nm analysis of the hydrolyzate recovered in the reaction supernatant showed the presence of two main peaks corresponding to products P1 (t r =9.34 min) and P2 (t r =9.62 min), both at 286 nm and was not present in control experiments. Positive mode LC-UV/MS analysis further revealed that P1 (m/z 432 [M+H] + ) corresponds to a cellobiose molecule grafted with p-toluidine at one of the C-6 positions of the molecule, P2 (m/z 270 [M+H] + ) is a glucose molecule grafted with p-toluidine at the C-6 position, and P3 (t r = 24.7 min) ( not detected in UV, m/z 521 [M+H] + ) is cellobiose grafted with two p-toluidine molecules at each C-6 position (Figure 3A and Figure 3B).

與染料若丹明 123 (I-b) 反應。使用若丹明123 (0.1 M)作為染料對氧化棉應用類似程序。 Reacts with dye rhodamine 123 (Ib) . A similar procedure was applied to oxidized cotton using Rhodamine 123 (0.1 M) as dye.

用若丹明123染色之棉線之纖維素酶水解在如上文所描述之相同條件下進行。HPLC-UV 500nm/MS分析顯示存在產物 P4(t r=22.29 min)，m/z 507 [M+H] +，對應於在C-6位置處共價接枝有若丹明123之葡萄糖。另一實驗係用1 g線進行。使過量纖維素酶經72小時的氧化線水解產物離心，且藉由LC-MS/UV分析上清液。除已鑑別之產物 P4外，另一產物 P5(m/z 669 [M+H] +)經鑑別，其對應於在兩個可能的C6位置中之一者上接枝有若丹明123的纖維二糖(圖4A及圖4B)。 Cellulase hydrolysis of cotton threads dyed with Rhodamine 123 was carried out under the same conditions as described above. HPLC-UV 500nm /MS analysis showed the presence of product P4 (t r =22.29 min), m/z 507 [M+H] + , corresponding to glucose covalently grafted with rhodamine 123 at the C-6 position. Another experiment was performed using a 1 g line. The excess cellulase oxidation line hydrolyzate was centrifuged for 72 hours and the supernatant was analyzed by LC-MS/UV. In addition to the identified product P4 , another product P5 (m/z 669 [M+H] + ) was identified, which corresponds to rhodamine 123 grafted at one of the two possible C6 positions. Cellobiose (Figure 4A and Figure 4B).

與染料酸性紅 33 (I-f) 反應。使用酸性紅33 (0.1 M)作為染料對氧化棉應用類似程序。 Reacts with dye Acid Red 33 (If) . Apply a similar procedure to oxidized cotton using Acid Red 33 (0.1 M) as dye.

用酸性紅33染色之棉線之纖維素酶水解在如上文所描述之相同條件下進行。Cellulase hydrolysis of cotton thread dyed with Acid Red 33 was performed under the same conditions as described above.

HPLC-UV 532nm/MS分析突出顯示兩種產物 P6(t r=18.69 min)及 P ' 6(t r=19.54 min)，其具有相同m/z 745 [M+H] +值，對應於在兩個可能的C-6位置(在還原或非還原單元上)中之一者處接枝有酸性紅33的纖維二糖，且在不同滯留時間下溶離(圖4C及圖4D)。 HPLC-UV 532nm /MS analysis highlighted two products, P6 (t r =18.69 min) and P ' 6 (t r =19.54 min), which had the same m/z 745 [M+H] + value, corresponding to Cellobiose of Acid Red 33 was grafted at one of the two possible C-6 positions (on reducing or non-reducing units) and eluted at different residence times (Figure 4C and Figure 4D).

比較測試 ：圖5A和圖5B顯示在反應及洗滌步驟後的根據本發明之分析(A1、B1)及對照組(A2、A3、B2、B3)的分別用若丹明123及酸性紅33染料染色之棉線(表1)。 表1    染料    若丹明123 酸性紅33 本發明 樣品A1：+ 樣品B1：+ 未氧化之棉+染料+Pic-BH 3 樣品A2：̶ 樣品B2：̶ 經氧化之棉+染料( 無 Pic-BH 3 ) 樣品A3：̶ 樣品B3：̶ 「+」意謂在染色及洗滌後獲得材料之強著色。 「-」意謂在染色及洗滌後獲得材料之低著色。 Comparative test : Figure 5A and Figure 5B show the analysis according to the invention (A1, B1) and the control group (A2, A3, B2, B3) after the reaction and washing steps using Rhodamine 123 and Acid Red 33 dyes respectively. Dyed cotton thread (Table 1). Table 1 dye Rhodamine 123 Acid red 33 invention Sample A1: + Sample B1: + Unoxidized cotton+dye+Pic-BH 3 Sample A2: ̶ Sample B2: ̶ Oxidized cotton + dye ( no Pic-BH 3 ) Sample A3: ̶ Sample B3: ̶ "+" means strong coloration of the material obtained after dyeing and washing. "-" means low coloration of the material obtained after dyeing and washing.

值得注意地，相比於對照物，與若丹明123 (樣品A1)或酸性紅33 (樣品B1)及pic-BH 3一起保溫之氧化棉線在洗滌後保持強著色，指示提高之濕牢度。在類似條件下處理之未氧化纖維(樣品A2及B2)及在洗滌前僅與胺化染料一起保溫的氧化棉(樣品A3及B3)獲得低得多的著色。此證實氧化及還原胺化步驟對獲得高著色及濕牢度而言均至關重要。更明確而言，在不存在還原劑之情況下獲得之結果顯示，藉由胺鍵(A1、B1)獲得之著色及濕牢度相對於藉由亞胺鍵(A3、B3)獲得者有所改良。 Notably, oxidized cotton threads insulated with Rhodamine 123 (Sample A1) or Acid Red 33 (Sample B1) and pic-BH 3 retained strong coloration after washing, indicating improved wet fastness compared to the control . Much lower coloration was achieved with unoxidized fibers treated under similar conditions (samples A2 and B2) and with oxidized cotton insulated with only amined dyes before washing (samples A3 and B3). This confirms that both the oxidative and reductive amination steps are critical to achieving high color and wet fastness. More specifically, the results obtained in the absence of reducing agents show that the coloration and wet fastness obtained through amine bonds (A1, B1) are better than those obtained through imine bonds (A3, B3). Improvement.

表2顯示各系列實驗(A系列及B系列)獲得之要素組成。經若丹明123及酸性紅33、以及pic-BH 3處理之氧化線中發現之氮的量分別為0.30%及0.08%。在對照物中未偵測到氮的跡象，顯示在洗滌樣品A2/B2及A3/B3後保持之存留顏色係歸因於極低量之殘餘染料。 表2： 系列 A N (%) C (%) H (%) 未氧化之棉 0.00±0.00 42.29±0.01 6.79±0.05 使用TvL/TEMPO氧化之棉 0.00±0.00 41.49±0.06 6.55±0.01 經氧化之棉+若丹明123 + pic-BH 3(A1) 0.30±0.00 42.82±0.12 6.32±0.01 未氧化之棉+若丹明123 + pic-BH 3(A2) 0.00±0.00 42.10±0.06 6.48±0.04 經氧化之棉+若丹明123 (A3) 0.00±0.00 41.93±0.13 6.31±0.01 系列 B N (%) C (%) H (%) 經氧化之棉+酸性紅33 +用pic-BH 3(B1) 0.08±0.01 41.93±0.04 6.32±0.06 未氧化之棉+酸性紅33 + pic-BH 3(B2) 0.00±0.00 42.47±0.08 6.49±0.10 經氧化之棉+酸性紅33 (B3) 0.00±0.00 42.04±0.13 6.31±0.04 兩個量測獲得的平均值 Table 2 shows the element composition obtained from each series of experiments (Series A and B). The amount of nitrogen found in the oxidized lines treated with Rhodamine 123, Acid Red 33, and Pic-BH 3 was 0.30% and 0.08% respectively. No evidence of nitrogen was detected in the controls, indicating that the residual color maintained after washing samples A2/B2 and A3/B3 was due to very low amounts of residual dye. Table 2: Series A N(%) C(%) H(%) unoxidized cotton 0.00±0.00 42.29±0.01 6.79±0.05 Use TvL/TEMPO oxidized cotton 0.00±0.00 41.49±0.06 6.55±0.01 Oxidized cotton + Rhodamine 123 + pic-BH 3 (A1) 0.30±0.00 42.82±0.12 6.32±0.01 Unoxidized cotton + Rhodamine 123 + pic-BH 3 (A2) 0.00±0.00 42.10±0.06 6.48±0.04 Oxidized Cotton + Rhodamine 123 (A3) 0.00±0.00 41.93±0.13 6.31±0.01 Series B N(%) C(%) H(%) Oxidized cotton + Acid Red 33 + Use pic-BH 3 (B1) 0.08±0.01 41.93±0.04 6.32±0.06 Unoxidized cotton + acid red 33 + pic-BH 3 (B2) 0.00±0.00 42.47±0.08 6.49±0.10 Oxidized cotton + acid red 33 (B3) 0.00±0.00 42.04±0.13 6.31±0.04 The average value obtained from two measurements

3) 棉織物a)將一塊5.8 cm×4 cm之棉織物浸沒於含有5.4 U/mL之蟲漆酶纖維及0.6 mM TEMPO之溶液中。將溶液在30℃下以140 rpm攪拌7小時。在氧化後，織物用125 mL水沖洗，且隨後用100 mL EtOH/H 2O 70/30洗滌，最後用125 mL蒸餾水沖洗。經洗滌之織物在室溫下乾燥隔夜。 3) Cotton fabric a) Immerse a piece of 5.8 cm×4 cm cotton fabric in a solution containing 5.4 U/mL laccase fiber and 0.6 mM TEMPO. The solution was stirred at 30°C and 140 rpm for 7 hours. After oxidation, the fabric was rinsed with 125 mL of water and subsequently washed with 100 mL of EtOH/H 2 O 70/30 and finally with 125 mL of distilled water. Laundered fabrics were dried at room temperature overnight.

還原胺化在40℃下於含有10%乙酸之溶液中進行，使用1:1.5:1.2之羰基、胺基及pic-BH 3莫耳比。由於pic-BH 3具有低水溶性，其預先在80% MeOH (v/v)中製備。還原胺化後，染色棉材料用150 mL水沖洗三次，隨後用50 mL 50 mM NaOH溶液洗滌兩次，最後各用150 mL蒸餾水連續沖洗三次。經洗滌之纖維在室溫下乾燥隔夜。 Reductive amination was performed at 40°C in a solution containing 10% acetic acid, using a 3 molar ratio of carbonyl, amine and pic-BH of 1:1.5:1.2. Since pic-BH 3 has low water solubility, it was prepared in advance in 80% MeOH (v/v). After reductive amination, the dyed cotton material was washed three times with 150 mL of water, followed by two washes with 50 mL of 50 mM NaOH solution, and finally three consecutive washes with 150 mL of distilled water each. The washed fibers were dried at room temperature overnight.

圖6顯示根據本發明方法用AR33染色之棉織物在洗滌後相較於對照物(樣品2：無氧化，樣品3：無還原劑)保持強著色，表明提高之濕牢度。 Figure 6 shows that cotton fabrics dyed with AR33 according to the method of the invention retain strong coloration after washing compared to the controls (Sample 2: no oxidation, sample 3: no reducing agent), indicating improved wet fastness.

b)在工業規模下：在30℃下將1 kg編織棉織物浸漬於50 kg含有54 mg變色栓菌蟲漆酶(270千單位(kUnit)，50單位/mg)、47 g TEMPO (6 mM，0.93 g/L)及6 kg磷酸鹽緩衝液(50 mM，120 g/L)之水浴中保持8小時。隨後將所得織物在40℃下在50 kg水中及在含有45 kg水、5 kg乙酸(10%)、0.1 mM染料、250 g甲基吡啶硼烷(48 mM，5 g/L)之另一槽中沖洗3小時。所得棉用水洗滌且在空氣中乾燥。 b) At an industrial scale: 1 kg of woven cotton fabric is immersed in 50 kg of 54 mg of Trametes versicolor laccase (270 kilounits (kUnit), 50 units/mg), 47 g of TEMPO (6 mM) at 30°C , 0.93 g/L) and 6 kg of phosphate buffer (50 mM, 120 g/L) in a water bath for 8 hours. The obtained fabric was then incubated at 40°C in 50 kg water and another layer containing 45 kg water, 5 kg acetic acid (10%), 0.1 mM dye, 250 g picolidine borane (48 mM, 5 g/L). Rinse in tank for 3 hours. The cotton obtained was washed with water and dried in air.

經 接枝棉織物之色牢度研究 ：用AR33染色之棉織物用於染料耐洗色牢度之研究。針對該目的，將一塊23.2 cm 2之AR33有色棉織物在60℃下用洗衣清潔劑進行三個洗滌循環(在洗衣機中)，各持續一小時。 Study on the color fastness of grafted cotton fabrics : Cotton fabrics dyed with AR33 were used to study the color fastness of the dye to washing. For this purpose, a 23.2 cm 2 piece of AR33 colored cotton fabric was subjected to three washing cycles (in a washing machine) with laundry detergent at 60°C for one hour each.

在60℃下進行三個洗滌循環後，織物之染色保持接近起始材料之染色(圖7)。此結果表明染色材料之強度及良好耐洗色牢度。After three washing cycles at 60°C, the dyeing of the fabric remained close to that of the starting material (Fig. 7). This result demonstrates the strength of the dyed material and its good color fastness to washing.

圖 1.根據本發明方法之步驟(a)氧化之棉及未處理之棉的FTIR (傅立葉(Fourier)轉換紅外光譜)光譜。 Figure 1. FTIR (Fourier Transform Infrared Spectroscopy) spectra of cotton oxidized and untreated cotton according to step (a) of the method of the present invention.

圖 2.根據本發明方法染色之棉之水解產物及未處理之棉之水解產物的LCUV (液體層析-紫外線)層析圖。 Figure 2. LCUV (liquid chromatography-ultraviolet) chromatograms of the hydrolyzate of cotton dyed according to the method of the present invention and the hydrolyzate of untreated cotton.

圖 3.經用對甲苯胺染色之棉線之纖維素酶酶水解後上清液的LC-MS/UV分析(A：UV 286nm層析圖- B：對於各產物，偵測 P1、 P2及 P3之 m/z，且對應於正模式中之H +加成物)。 Figure 3. LC-MS/UV analysis of the supernatant after cellulase enzymatic hydrolysis of cotton thread dyed with p-toluidine (A: UV 286nm chromatogram - B: For each product, detection of P1 , P2 and P3 m/z , and corresponds to H + additive in positive mode).

圖 4.經用RHO123或AR33染色之棉線之纖維素酶酶水解後上清液的LC-MS/UV分析(圖4A、4C：自分別用RHO123或AR33接枝之棉線獲得的UV層析圖；圖4B、圖4D：偵測各產品之m/z且對應於正模式中之H +加成物)。 Figure 4. LC-MS/UV analysis of the supernatant after cellulase enzymatic hydrolysis of cotton threads dyed with RHO123 or AR33 (Figure 4A, 4C: UV chromatograms obtained from cotton threads grafted with RHO123 or AR33, respectively ; Figure 4B, Figure 4D: The m/z of each product is detected and corresponds to the H + adduct in the positive mode).

圖 5.根據本發明方法用RHO123 (A1)或AR33 (B1)染色之棉線的圖像，及沒有氧化(A2、B2)或沒有還原劑(A3、B3)之棉線染料的比較樣品。 Figure 5. Images of cotton threads dyed with RHO123 (A1) or AR33 (B1) according to the method of the invention, and comparative samples of cotton thread dyes without oxidation (A2, B2) or without reducing agents (A3, B3).

圖 6.根據本發明方法用AR33染色(樣品1)、沒有氧化(樣品2)及沒有還原劑(樣品3)之棉織物。 Figure 6. Cotton fabric dyed with AR33 according to the method of the invention (sample 1) without oxidation (sample 2) and without reducing agent (sample 3).

圖 7.根據本發明方法用AR33染色之棉織物在洗滌前及在三個洗滌循環後的圖像。 Figure 7. Images of cotton fabric dyed with AR33 according to the method of the invention before washing and after three washing cycles.

TW202336316A_111148843_SEQL.xmlTW202336316A_111148843_SEQL.xml

Claims (22)

一種纖維素材料之染色方法，該方法包含以下步驟： (a)使纖維素材料與氧化還原酶及氧化還原介質接觸，以獲得氧化纖維素材料； (b)在還原劑存在下，使步驟(a)之該氧化纖維素材料與包含至少一個NH 2基團之染料接觸，以獲得染色纖維素材料；及 (c)回收染色纖維素材料。 A dyeing method for cellulose materials, which method includes the following steps: (a) contacting cellulose materials with oxidoreductase and redox media to obtain oxidized cellulose materials; (b) in the presence of a reducing agent, making the step ( a) contacting the oxidized cellulosic material with a dye containing at least one NH 2 group to obtain a dyed cellulosic material; and (c) recovering the dyed cellulosic material. 如請求項1之方法，其中步驟(b)中之該染料為式(I)化合物： [H 2N-(X-Y) n] m-Z   (I)， 其中： n為0或1， m為1至4之整數， X為伸芳基或伸雜芳基，該基團視情況經至少一個選自由以下組成之群的取代基取代：(C 1-C 12)烷基、(C 2-C 12)烯基、(C 2-C 12)炔基、(C 3-C 12)環烷基、(C 2-C 12)雜環烷基、芳基、雜芳基、鹵素、-CN、-NO 2、-C(O)R 1、-C(O)OR 2、-C(O)NR 3R 4、-S(O) 2R 5、-S(O) 2OR 6、-NHS(O) 2R 7、-NHS(O) 2OR 8、-OR 9、-SR 10及-NR 11R 12， R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R 11及R 12各自獨立地選自氫、(C 1-C 12)烷基、(C 2-C 12)烯基、(C 2-C 12)炔基、(C 3-C 12)環烷基、(C 2-C 12)雜環烷基、芳基及雜芳基， Y為選自以下基團之間隔基：(C 1-C 12)伸烷基、(C 2-C 12)伸烯基、(C 2-C 12)伸炔基、(C 3-C 12)伸環烷基、(C 2-C 12)伸雜環烷基、伸芳基及伸雜芳基，該基團視情況間雜有至少一個選自以下之基團：-O-、-S-、-NH-、-C(O)-及-S(O) 2，及 Z為發色團部分， 或其鹽。 The method of claim 1, wherein the dye in step (b) is a compound of formula (I): [H 2 N-(XY) n ] m -Z (I), where: n is 0 or 1, m is An integer from 1 to 4, X is an aryl or heteroaryl group, and the group is optionally substituted by at least one substituent selected from the group consisting of: (C 1 -C 12 ) alkyl, (C 2 - C 12 )alkenyl, (C 2 -C 12 )alkynyl, (C 3 -C 12 )cycloalkyl, (C 2 -C 12 )heterocycloalkyl, aryl, heteroaryl, halogen, -CN , -NO 2 , -C(O)R 1 , -C(O)OR 2 , -C(O)NR 3 R 4 , -S(O) 2 R 5 , -S(O) 2 OR 6 , - NHS(O) 2 R 7 , -NHS(O) 2 OR 8 , -OR 9 , -SR 10 and -NR 11 R 12 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7. R 8 , R 9 , R 10 , R 11 and R 12 are each independently selected from hydrogen, (C 1 -C 12 ) alkyl, (C 2 -C 12 ) alkenyl, (C 2 -C 12 ) Alkynyl, (C 3 -C 12 ) cycloalkyl, (C 2 -C 12 ) heterocycloalkyl, aryl and heteroaryl, Y is a spacer selected from the following groups: (C 1 -C 12 )Alkylene group, (C 2 -C 12 ) alkenyl group, (C 2 -C 12 ) alkynylene group, (C 3 -C 12 ) cycloalkylene group, (C 2 -C 12 ) heterocycloalkane group, aryl group and heteroaryl group, the group is optionally mixed with at least one group selected from the following: -O-, -S-, -NH-, -C(O)- and -S(O ) 2 , and Z is the chromophore part, or its salt. 如請求項2之方法，其中n為0。Such as the method of request item 2, where n is 0. 如請求項2或3之方法，其中m為1或2，較佳m為1。For example, the method of claim 2 or 3, where m is 1 or 2, preferably m is 1. 如請求項2至4中任一項之方法，其中該式(I)化合物使得Z為選自由以下組成之群的發色團部分：苯基、若丹明(rhodamine)、蒽醌、三芳基甲烷、酞菁、單偶氮染料、雙偶氮染料、三偶氮染料、聚偶氮染料、雙亞吲哚基二酮、茚二酮、喹啉茚二酮、吖啶、噻𠯤、噻唑、㗁𠯤、啡㗁𠯤、𠮿、氯、二酮吡咯并吡咯、喹吖啶酮、花青素、類黃酮及其衍生物。The method of any one of claims 2 to 4, wherein the compound of formula (I) is such that Z is a chromophore moiety selected from the group consisting of: phenyl, rhodamine, anthraquinone, triaryl Methane, phthalocyanine, monoazo dyes, disazo dyes, triazo dyes, polyazo dyes, bisindolylidenedione, indenedione, quinoline indenedione, acridine, thiazole, thiazole , 㗁𠯤, Brown 㗁𠯤, 𠮿 , chlorine, diketopyrrolopyrrole, quinacridone, anthocyanins, flavonoids and their derivatives. 如請求項1至5中任一項之方法，其中該染料由下式中之任一者表示： 。 The method of any one of claims 1 to 5, wherein the dye is represented by any one of the following formulas: . 如請求項1至6中任一項之方法，其中該染料由下式中之任一者表示： 。 The method of any one of claims 1 to 6, wherein the dye is represented by any one of the following formulas: . 如請求項1至7中任一項之方法，其中該染料由下式中之任一者表示： 。 The method of any one of claims 1 to 7, wherein the dye is represented by any one of the following formulas: . 如請求項1至8中任一項之方法，其中該氧化還原介質為TEMPO。The method of any one of claims 1 to 8, wherein the redox medium is TEMPO. 如請求項1至9中任一項之方法，其中該氧化還原酶為蟲漆酶(laccase)。The method of any one of claims 1 to 9, wherein the oxidoreductase is laccase. 如請求項10之方法，其中該蟲漆酶為來自以下之蟲漆酶：貝萊斯芽孢桿菌( Bacillus velezensis)、平流層芽孢桿菌( Bacillus stratosphericus)、無氧芽孢桿菌( Anoxybacillus)、曲黴菌屬( Aspergillus)或變色栓菌( Trametes versicolor)，較佳為變色栓菌。 The method of claim 10, wherein the laccase is a laccase from the following: Bacillus velezensis, Bacillus stratosphericus , Anoxybacillus , Aspergillus ( Aspergillus ) or Trametes versicolor, preferably Trametes versicolor. 如請求項10或11之方法，其中該蟲漆酶包含選自由SEQ ID NO: 1、SEQ ID NO: 2、SEQ ID NO: 3、SEQ ID NO: 4、SEQ ID NO: 5及其變異體組成之群的胺基酸序列，其中該等變異體表現出蟲漆酶活性且具有至少60%之胺基酸序列一致性。 The method of claim 10 or 11, wherein the laccase enzyme comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and variants thereof A group of amino acid sequences, wherein the variants exhibit laccase activity and have at least 60% amino acid sequence identity. 如請求項1至12中任一項之方法，其中步驟(a)在包含於10℃與60℃之間的溫度下進行，較佳20℃與50℃之間，且更佳25℃與35℃之間。The method of any one of claims 1 to 12, wherein step (a) is performed at a temperature comprised between 10°C and 60°C, preferably between 20°C and 50°C, and more preferably between 25°C and 35°C between ℃. 如請求項1至13中任一項之方法，其中步驟(a)在包含於2.5與12之間的pH下進行，較佳在4與10之間，更佳在4與8之間，甚至更佳在5與8之間。The method of any one of claims 1 to 13, wherein step (a) is carried out at a pH comprised between 2.5 and 12, preferably between 4 and 10, more preferably between 4 and 8, or even Better between 5 and 8. 如請求項1至14中任一項之方法，其中步驟(b)在包含於10℃與60℃之間的溫度下進行，較佳在30℃與50℃之間，更佳在35℃與45℃之間。The method of any one of claims 1 to 14, wherein step (b) is carried out at a temperature comprised between 10°C and 60°C, preferably between 30°C and 50°C, more preferably between 35°C and 50°C. between 45℃. 如請求項1至15中任一項之方法，其中步驟(b)在酸存在下進行，諸如乙酸。A method as claimed in any one of claims 1 to 15, wherein step (b) is carried out in the presence of an acid, such as acetic acid. 如請求項1至16中任一項之方法，其中步驟(a)及(b)在水中進行。The method of any one of claims 1 to 16, wherein steps (a) and (b) are performed in water. 如請求項1至17中任一項之方法，其中該還原劑為硼烷化合物。The method of any one of claims 1 to 17, wherein the reducing agent is a borane compound. 如請求項1至18中任一項之方法，其中該還原劑為硼烷化合物，該硼烷化合物選自由以下組成之群：硼氫化鈉或硼氫化鉀、硼氫化鋅、氰基硼氫化鈉、硼烷(BH 3)、二硼烷(B 2H 6)、吡啶硼烷、甲基吡啶硼烷、5-乙基-2-甲基吡啶硼烷、𠰌啉硼烷、4-甲基𠰌啉硼烷、三乙胺硼烷、9-硼雙環[3.3.1]壬烷(9-BBN)、單異松蒎硼烷、二環己基硼烷、二基硼烷、二異戊基硼烷(disiamylborane)、兒茶酚硼烷、頻哪醇硼烷、三仲丁基硼氫化鋰(L-selectride)及其混合物，較佳甲基吡啶硼烷。 The method of any one of claims 1 to 18, wherein the reducing agent is a borane compound, and the borane compound is selected from the group consisting of: sodium borohydride or potassium borohydride, zinc borohydride, sodium cyanoborohydride , borane (BH 3 ), diborane (B 2 H 6 ), pyridine borane, methylpyridine borane, 5-ethyl-2-methylpyridine borane, 𠰌line borane, 4-methyl 𠰌lineborane, triethylamineborane, 9-boranebicyclo[3.3.1]nonane (9-BBN), monoisopinepineborane, dicyclohexylborane, dicyclohexylborane borane, diisoamylborane (disiamylborane), catecholborane, pinacolborane, tri-sec-butyl lithium borohydride (L-selectride) and mixtures thereof, preferably methylpyridine borane. 如請求項1至19中任一項之方法，其中該纖維素材料係選自棉、亞麻、***、黃麻、人絲(viscose)、萊賽爾(lyocell)、螺縈(rayon)及莫代爾(modal)，較佳為棉。The method of any one of claims 1 to 19, wherein the cellulosic material is selected from the group consisting of cotton, linen, hemp, jute, viscose, lyocell, rayon and modal (modal), preferably cotton. 如請求項1至20中任一項之方法，其包含以下連續步驟： (a)使纖維素材料與蟲漆酶及TEMPO或其衍生物接觸，以獲得氧化纖維素材料； (b)在硼烷化合物存在下，使步驟(a)之該氧化纖維素材料與如請求項2至6中任一項所定義之式(I)染料接觸，以獲得染色纖維素材料；及 (c)回收染色纖維素材料。 For example, the method of any one of claims 1 to 20 includes the following consecutive steps: (a) contacting cellulose materials with laccase and TEMPO or derivatives thereof to obtain oxidized cellulose materials; (b) contacting the oxidized cellulose material of step (a) with a dye of formula (I) as defined in any one of claims 2 to 6 in the presence of a borane compound to obtain a dyed cellulose material; and (c) Recycling of dyed cellulosic materials. 一種染色纖維素材料，其藉由如請求項1至21中任一項之方法獲得。A dyed cellulose material obtained by a method according to any one of claims 1 to 21.
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