WO2016015571A1 - 用于改善纸张白度的纤维氧化酶组合物及造纸方法和应用 - Google Patents

用于改善纸张白度的纤维氧化酶组合物及造纸方法和应用 Download PDF

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WO2016015571A1
WO2016015571A1 PCT/CN2015/084456 CN2015084456W WO2016015571A1 WO 2016015571 A1 WO2016015571 A1 WO 2016015571A1 CN 2015084456 W CN2015084456 W CN 2015084456W WO 2016015571 A1 WO2016015571 A1 WO 2016015571A1
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oxidase
fiber
composition
paper
whiteness
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PCT/CN2015/084456
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English (en)
French (fr)
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王祥槐
谢焱
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瑞辰星生物技术（广州）有限公司
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Priority claimed from CN201410366880.4A external-priority patent/CN104342425B/zh
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Publication of WO2016015571A1 publication Critical patent/WO2016015571A1/zh

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    • 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
    • C12N9/88—Lyases (4.)
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
    • D21H17/00—Non-fibrous material added to the pulp, characterised by its constitution; Paper-impregnating material characterised by its constitution
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
    • D21H21/00—Non-fibrous material added to the pulp, characterised by its function, form or properties; Paper-impregnating or coating material, characterised by its function, form or properties
    • D21H21/14—Non-fibrous material added to the pulp, characterised by its function, form or properties; Paper-impregnating or coating material, characterised by its function, form or properties characterised by function or properties in or on the paper
    • D21H21/30—Luminescent or fluorescent substances, e.g. for optical bleaching

Definitions

  • the present invention relates to the field of biological enzymes and the use of biological enzymes to improve the physical and chemical properties of fibers, and in particular to a fiber oxidase composition for improving paper whiteness and a papermaking method and application.
  • China's paper industry maintained a high growth rate for 10 consecutive years from 2000 to 2010. By 2011, China's paper and paperboard production and consumption exceeded 100 million tons, ranking first in the world.
  • the raw materials of paper or paperboard are mainly fiber. Because pulping and papermaking require a lot of raw materials and have certain influence on the environment, the paper industry in the world is compressing pulp production to ensure forest resources and domestic raw material forest. The construction of the base is sluggish and the supply of materials is limited. The development of non-wood pulp is affected by the lagging development of new technologies for clean production. The constraints on resources, energy and environment faced by the development of China's paper industry are increasingly prominent.
  • waste paper can not only protect forest resources to a certain extent, but also alleviate the huge demand pressure on the development of natural resources in the paper industry, reduce water and energy consumption, and reduce environmental pollution. .
  • China's recycling and recycling of waste paper resources is also increasing year by year.
  • the proportion of waste pulp in the national pulp consumption only accounted for 47%.
  • waste paper pulp consumed 56.6 million tons, accounting for the national pulp.
  • the proportion of consumption has risen to 62%. Therefore, research on waste paper deinking has received increasing attention as a key step in waste paper processing.
  • Lignin-degrading enzymes such as laccase
  • the biological enzyme deinking agent is preferably combined with a plurality of high-efficiency biological enzymes which are highly targeted, and is used together with an enzyme activation special auxiliary agent for deinking of newspapers, mixed office waste paper, books, magazines and the like.
  • biological enzymatic deinking has a series of outstanding advantages: 1 biological enzyme deinking conditions are mild, chemical use is small, fiber loss is small, maintaining good fiber characteristics; 2 greatly reducing sewage COD and BOD, reduce the pressure of sewage treatment; 3 improve pulp yield and improve paper strength; 4 clean production, save energy, reduce material consumption; 5 simple to use, and match most existing deinking process.
  • biological enzymatic deinking agents have been applied in the field of papermaking deinking; however, the effect of using the biological enzymes in the prior art is limited to increasing the dissociation of ink dots from the fiber surface.
  • one of the objects of the present invention is to provide a fiber oxidase composition for improving the whiteness of paper.
  • a fiber oxidase composition for improving whiteness of paper comprising the following components:
  • a biological enzyme component having an oxidizing function on the fiber the biological enzyme component being a fiber oxidase which changes the surface properties of the fiber by a redox reaction
  • auxiliary component having an increased catalytic activity on the fiber oxidase, the auxiliary component being a reaction substrate of the fiber oxidase;
  • the weight ratio of the biological enzyme component to the auxiliary component is from 1:1 to 100.
  • the cell oxidase is a family having a molecular weight of 20-50 KDa, A glycan-oxidizing enzyme of a copper-dependent small molecule metalloenzyme, the reaction substrate of which is an oxygen, air or oxygen releasing compound; the oxygen releasing compound is a peroxide or ozone.
  • the peroxide is at least one of hydrogen peroxide, sodium peroxide, calcium peroxide, or potassium peroxide.
  • the weight ratio of the biological enzyme component to the auxiliary component is from 1:1 to 75.
  • the weight ratio of the biological enzyme component to the auxiliary component is from 1:20 to 75.
  • the weight ratio of the biological enzyme component to the auxiliary component is from 1:30 to 70.
  • the cell oxidase composition further comprises a protein having a promoting effect on cell oxidase, wherein the protein having a promoting activity to the cell oxidase is a catalytically active biological enzyme or a non-catalytically active fiber. Protein binding protein.
  • the catalytically active biological enzyme is an oxidoreductase selected from the group consisting of cellobiose dehydrogenase, laccase, glucose oxidase, hydrogen peroxide dismutase, alcohol dehydrogenase or lignin.
  • One or more of peroxidases; or the catalytically active biological enzyme is a non-cellulolytic enzyme hydratase selected from the group consisting of an amylase, a xylanase, a pectinase or an esterase Or a variety.
  • the weight ratio of cell oxidase to protein that promotes fiber oxidase in the cell oxidase composition is 1: (0.5-3).
  • the catalytically inactive protein is a fibronectin and/or a fibroin.
  • Another object of the present invention is to provide a fiber oxidase composition or fiber oxidase for use as a bleaching, deinking or whitening agent for improving paper whiteness in a papermaking process.
  • Another object of the present invention is to provide a papermaking method comprising mainly the following steps:
  • step c) feeding the papermaking pulp obtained in step b) to the internet, thereby forming a paper sheet from the fibrous solid component by filtering out the water.
  • the amount of cell oxidase in the cell oxidase composition in step b) is 0.01-10 kg per ton of dry slurry, and the amount of auxiliary component is 0.05-10 kg per ton of dry slurry. .
  • the amount of cell oxidase in the cell oxidase composition in step b) is from 0.05 to 10 kg per ton of dry slurry, and the amount of auxiliary component is from 0.1 to 10 kg per ton of dry slurry. .
  • the amount of cell oxidase in the cell oxidase composition in step b) is from 0.1 to 1 kilogram per ton of dry slurry.
  • the amount of cell oxidase in the cell oxidase composition in step b) is from 0.1 to 0.5 kilograms per ton of dry slurry.
  • the cellulase composition and the papermaking stock have a reaction time of from 5 to 600 minutes, a pH of from 3 to 10, and a temperature of from 20 to 80 °C.
  • the cellulase composition and the papermaking stock have a reaction time of from 20 to 300 minutes, a reaction pH of from 5 to 10, and a reaction temperature of from 30 to 65 °C.
  • the cellulase composition and the papermaking stock have a reaction time of 60-120 minutes, a reaction pH of 8-10, and a reaction temperature of 50-65 °C.
  • the fiber oxidase and its composition can significantly improve the whiteness of the paper, and determine the optimum composition of the fiber oxidase composition and its ratio, in the treatment of waste paper pulp
  • the fiber oxidase composition or the fiber oxidase can be added to change the bonding bond on the surface of the fiber or near the ink ions, promote the swelling of the fiber, and weaken the connection between the fiber and the ink, and then through appropriate mechanical treatment. Further, the ink particles are further completely detached from the surface of the fiber, and then effectively removed by washing or flotation.
  • fiber oxidase or its composition can also catalyze the decomposition of lignin.
  • the chromophoric group in the lignin structure is the main source of pulping color, and the decomposition of lignin, fiber oxidase can destroy the chromophore.
  • the group which significantly increases the whiteness of the pulp, can also significantly reduce the amount of deinking agent used in the prior art and the amount of material added to increase the whiteness of the paper.
  • Cellulolytic enzyme refers to all biological enzymes which degrade cellulose by a hydrolysis reaction, and the terms such as cellulolytic enzyme and cellulase, fiber hydrolase and cellulase are often used. Alternate use. According to the different modes of action of cellulase degradation of substrates, they can be divided into three categories:
  • Endocellulase is also known as endoglucanase (Endoglucanase, EG; EC 3.2.1.4);
  • Exocellulase is also known as cellobiohydrolase (CBOH; EC 3.2.1.91);
  • endocellulase also known as endog-glucanase, endo-1,4- ⁇ -D-glucanase, EC 3.2.1.4
  • endocellulase also known as endog-glucanase, endo-1,4- ⁇ -D-glucanase, EC 3.2.1.4
  • endocellulase also known as endog-glucanase, endo-1,4- ⁇ -D-glucanase, EC 3.2.1.4
  • exocellulase also known as exog-glucanase, exo-1,4- ⁇ -D-glucanase, EC 3.2.1.91
  • exocellulase also known as exog-glucanase, exo-1,4- ⁇ -D-glucanase, EC 3.2.1.91
  • CBH cellobiohydrolase
  • Cellobiase also known as ⁇ -glucosidase, ⁇ -1,4-glucosidase, EC 3.2.1.21
  • BG Cellobiase
  • Such enzymes typically hydrolyze cellobiose or soluble cellodextrin to glucose molecules having a molecular weight of about 76 KD.
  • Cellulase Enzyme Activity The enzymatic activity of cellulase in the present invention refers to the glycan of the cellulase component in their ability to degrade cellulose into glucose, cellobiose and disaccharide. Enzyme activity. Cellulase activity is generally determined by a decrease in the viscosity of the solution of carboxymethylcellulose.
  • CBP Cellulose binding protein
  • cellulose-binding protein in the present invention means a cellulose having a special affinity for strongly adsorbing on the surface of cellulose, but itself is not catalyzed by cellulose. Hydrolyzed reactive protein.
  • CBD Cellulose Binding Domain
  • CBM Cellulose Binding Module
  • cellulose adsorption domain cellulose affinity domain.
  • Fibroswellin Swollenin or Expansin: "Fibrous expanded protein” in the present invention means a protein/polypeptide compound which can loosen the fibrous structure of a natural substrate such as crystalline cellulose and hemicellulose. Cellulose does not have the catalytic function of degradation, but it can improve the hydrolysis ability of cellulase to microcrystalline cellulose. "Fibrous fiber expansion protein”, also known as fiber swelling factor, is called Swollenin and Expansin in English. It is a kind of plant cell wall expansion protein with non-hydrolysis activity of typical cellulase composition (including cellulose binding domain (CBD)). protein.
  • CBD cellulose binding domain
  • ⁇ -expansin has a molecular weight of about 25kDa, amino acid sequence is highly conserved, and homology is 70% to 90%; ⁇ -expansin has a molecular weight of about 29kDa, but the amino acid sequence big change.
  • Fibrous oxidase in the present invention means polysaccharide monooxygenase (PMO), and the "glycan oxygenase” refers to a family having a molecular weight of 20-50 KDa.
  • glycanoxygenase examples include polyglucose oxygenase or polysaccharide monooxygenase (PMO), lytic polysaccharide monooxygenase (LPMO), and glycosidation Enzyme 61 (glycosyl hydrolase 61, GH61).
  • cell oxidase and cellulolytic enzyme degrade cellulose by hydrolysis of ⁇ -1,4-D-14 glycosidic bonds; while cellulolytic enzymes oxidize cellulose.
  • the reduction reaction, the bond changed by the reaction is not limited to the ⁇ -1,4-D-14 glycosidic bond.
  • PMO was discovered as early as 1974, the hydrolysis reaction of PMO to ⁇ -1,4-D-14 glycosidic bonds was found to be very weak due to the analysis method using CMCase (the method for testing endocellulase). The role of this enzyme species and its reaction mechanism have not been further studied.
  • CAZy Carbohydrate Active Enzymes, CAZy, http://www.cazy.org/) divides them into Glycosyl Hydrolases Group 61 (GH61).
  • PMO is actually a copper-dependent monooxygenases (EC 1.14.17.x), which acts on the fibers through oxidation, rather than ordinary Hydrolysis reaction like cellulose hydrolase The fiber acts, so the PMO is an oxidase rather than a hydrolase.
  • Classical cellulolytic enzymes have a number of enzyme families.
  • PMO has only two families, namely carbohydrate binding module family 33 (CBM33, carbohydrate-binding domain family 33) and glycosyl hydrolase (GH61, glycoside hydrolase family 61).
  • CAZy recently reclassified PMOs into AA9 and AA10 biological enzymes.
  • the main source of the former is fungus (fungus), while the latter is mainly derived from bacteria, viruses and certain fungi.
  • PMO is present in many lignin-degrading microorganisms, which can be purified and expressed, or recombinantly expressed by modern protein engineering techniques.
  • U.S. Patent No. 7,273,738, U.S. Patent Application Serial No. U.S.A. U.S. Patent No. 8,298,795 and U.S. Patent Application Serial No. 2012/0083019 the disclosure of which is incorporated herein by reference to the entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire all Lignocellulosic materials increase biorefinery efficiency and increase alcohol production. So far, the application of PMO has been in biorefinery, and there is no report on the application of PMO in pulp and paper.
  • Fibrous oxidase composition in the present invention means that the composition contains the following two components, one component being a polysaccharide monooxygenase, and the other component being capable of Maintaining or increasing an adjuvant to the activity of the oxidative enzyme, wherein the auxiliary may be one or more compounds of the following compounds, (1) a reaction substrate of a cell oxidase, oxygen or oxygen precursors, (2) Reagents (radical scavengers), and (3) proteins that promote fiber oxidase.
  • reaction substrate of cellulase means an oxygen acceptor which is necessary for the reaction of a glycan oxidase in addition to a fiber; the reaction substrate can be provided by oxygen, air or
  • the oxygen releasing compound, the oxygen releasing compound in the present invention means a peroxide or ozone, wherein the peroxide includes hydrogen peroxide, sodium peroxide, calcium peroxide, potassium peroxide.
  • the "promoting protein for cell oxidase" of the present invention refers to a biological enzyme, a cellulose-binding protein, and a fibroin protein which can increase the activity of a glycan oxidase; among the organisms which promote the glycan oxidase
  • Enzymes refer to other hydrolase enzymes (including amylase, pectinase, lipase, esterase and xylanase) and oxidoreductases (including cellobiose dehydrogenase, glucose oxidation) in addition to cellulolytic enzymes. Enzyme, laccase and lignin peroxidase).
  • the cellulose oxidase composition of the present invention may be separately added to the papermaking process by a product of a single enzyme component thereof, or the two components may be separately added to different positions or mixed into one product formula to be added to the papermaking process. Position to achieve the best results.
  • the "kg/T" as used in the present invention refers to the number of kilograms of material added per ton of dry pulp.
  • Recycled old newspaper (ONP) and old magazine (OMG) waste paper are mixed at 80%: 20%. 200 grams of mixed waste paper and 1300 grams of white water (added CaCl 2 to adjust the hardness to 400 PPM; temperature 70 ° C), placed in a 10 liter hydraulic pulper, then add caustic soda, pulp 7.5 minutes; after the pulp is finished, then Add 2700 grams of white water with 5% pulp.
  • Fibrin oxidase is a GH61 supplied by the American company Dyadic, which is prepared from the strain Myceliophthora thermophila, and its amino acid sequence is described in detail in U.S. Patent No. 8,298,795 and U.S. Patent Application Serial No. 2012/0083019.
  • Glucose Oxidase (GOX) is a commercial product of Genencor, a subsidiary of the DuPont Group.
  • A biological enzyme treatment conditions: taking 600 grams of the above prepared slurry, placed in a 1000ml mixing stirrer, using a thermostat controller to control the temperature of the slurry at 60 ° C;
  • the control group is: adding 1-10 kg/T of caustic soda, 15-30 kg/T of water glass, 10-20 kg/T of hydrogen peroxide, and 0.5-3 kg/T of deinking agent; (Note: all dosages are For the dry pulp, the same reason as below).
  • the experimental components are three groups, wherein
  • Experimental group 1 was: adding fiber oxidase (PMO) 150-300g/T (absolute dry pulp) to the slurry, deinking agent 0.5kg/T, adding caustic soda 2kg/T, water glass 15kg/T, hydrogen peroxide 10kg/T;
  • PMO fiber oxidase
  • Experimental group 2 is: adding glucose-oxidase 250-500g/T, deinking agent 1kg/T, adding caustic soda 2kg/T, water glass 15kg/T, hydrogen peroxide 10kg/T;
  • the experimental group 3 is: adding a combination of cell oxidase and glucose oxidase in the slurry, PMO 150-300g/T, GOX 250-500g/T, deinking agent 0.5kg/T, adding caustic soda 1-2kg/T , water glass 15kg / T, hydrogen peroxide 10kg / T;
  • reaction conditions were as follows: a temperature of 60 ° C, a reaction time of 90 minutes, a reaction pH of 8-10, and constant stirring.
  • Handsheet preparation and strength measurement Dilute the prepared deinked pulp, add tap water to 1.0%, mix, measure temperature and slurry concentration, and accurately prepare 10-12 sheets of 6.5 g according to TAPPI method. Heavy hand paper. After drying, the handsheets were placed in a constant temperature and constant humidity control box for 24 hours, and then measured for whiteness by the TAPPI measurement method.

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Abstract

提供了一种用于改善纸张白度的纤维氧化酶组合物及造纸方法和应用，所述纤维氧化酶组合物主要由以下组分组成：对纤维具有氧化功能的生物酶组分，所述生物酶组分为通过氧化还原反应来改变纤维表面性质的纤维氧化酶；和对纤维氧化酶具有增加催化活性的辅助组分，所述辅助组分为纤维氧化酶的反应底物；还提供了上述组合物用于造纸的方法。

Description

用于改善纸张白度的纤维氧化酶组合物及造纸方法和应用 技术领域
本发明涉及生物酶和使用生物酶改善纤维物理化学性质的技术领域，具体地，涉及一种用于改善纸张白度的纤维氧化酶组合物及造纸方法和应用。
背景技术
中国造纸工业在2000-2010年间连续10年保持高增长率，到2011年我国纸及纸板的生产量和消费量均超过1亿吨，均居世界第一位。纸或纸板的组成原料主要是纤维，由于制浆、造纸需要大量的原料，而且对环境有一定的影响，目前世界各国造纸行业都在压缩制浆生产，以保证森林资源，加上国内原料林基地建设迟缓，供材有限，而非木浆发展受到清洁生产新技术开发滞后的影响，中国造纸工业发展面临的资源、能源和环境的约束日益突显。
而废纸作为一种可再利用的二次纤维资源，回收造纸既可以保护森林资源在一定程度上缓解造纸行业发展对自然资源的巨大需求压力，又能降低水和能源的消耗，减少环境污染。我国对废纸资源的回收再利用力度也在逐年加大，据中国造纸协会统计，2002年全国纸浆消耗中，废纸浆比例仅占47％，2011年废纸浆消耗了5660万吨，占全国纸浆消耗的比例上升至62％，因此，废纸脱墨的研究作为废纸处理的关键步骤受到越来越多的关注。
传统的化学脱墨是加入氢氧化钠、硅酸钠、过氧化氢、螯合剂和表面活性剂等化学试剂，在碱性条件下各种化学因子促使油墨被皂化而从纤维上脱落，然后利用浮选或洗涤的方法将油墨除去。脱墨剂在废纸回用中主要用于破坏油墨对纤维的粘附力，使油墨从纤维上剥离并分散于水中。化学脱墨法由于采用较多化学品，不仅会引起污染环境，而且不适用于激光打印和复印的办公废纸。以激光打印和静电复印为代表的混合办公废纸，因为表面施胶度高，油墨熔化在纤维上，仅通过常规浮选和洗涤依靠机械搅拌和相互摩擦很难将油墨剥离。生物酶的引入能适当地打断油墨和纤维之间的联接并使之脱离纤维表面，提高 废纸浆的白度，同时改善纸张的物理性能。
在过去的几十年中，很多酶(例如纤维素酶/半纤维素、脂肪酶、漆酶)在废纸脱墨回收工业中对化学法的可替代性已经得到研究。各种酶的脱墨机理不同且尚待进一步研究，目前较为认同的是：纤维素酶/半纤维素酶水解纤维表面的非结晶区部分，使油墨与纤维间的连接变弱，从而有利于分离；脂肪酶针对性的将油墨中的油基连接料降解，使油墨中的炭黑及颜料从纸面散出、脱离。木质素降解酶，如漆酶，可以选择性地移除纸张表面的木质素，从而促进油墨的脱除。生物酶脱墨剂是优选针对性强的多种高效生物酶复配而成，与酶激活专用助剂配合使用，用于报纸、混合办公废纸、书籍、杂志等的脱墨。
与传统化学脱墨相比，生物酶脱墨具有一系列突出的优点：①生物酶脱墨条件温和，化学品用量少，纤维损失小，保持良好的纤维特性；②大幅度降低污水COD和BOD，减轻污水处理压力；③提高纸浆得率及提高纸张强度；④清洁生产，节省能源，降低物耗；⑤使用简单，与现有大多数脱墨工艺匹配。目前，生物酶脱墨剂已经应用于造纸脱墨领域；但采用现有技术中的生物酶的作用效果只限于提高油墨点从纤维表面的解离。
发明内容
基于此，本发明的目的之一为提供一种用于改善纸张白度的纤维氧化酶组合物。
解决上述技术问题的具体技术方案如下：
一种用于改善纸张白度的纤维氧化酶组合物，包括以下组成：
i)对纤维具有氧化功能的生物酶组分，所述生物酶组分为通过氧化还原反应来改变纤维表面性质的纤维氧化酶；和
ii)对纤维氧化酶具有增加催化活性的辅助组分，所述辅助组分为纤维氧化酶的反应底物；
所述生物酶组分和辅助组分的重量比为1:1-100。
在其中一些实施例中，所述纤维氧化酶是一族分子量在20-50KDa、具有 铜依赖性的小分子金属酶的聚糖加氧酶，所述纤维氧化酶的反应底物为氧气、空气或者氧气释放化合物；所述氧气释放化合物为过氧化物或者臭氧。
在其中一些实施例中，所述过氧化物为过氧化氢、过氧化钠、过氧化钙或过氧化钾中的至少一种。
在其中一些实施例中，所述生物酶组分和辅助组分的重量比为1:1-75。
在其中一些实施例中，所述生物酶组分和辅助组分的重量比为1:20-75。
在其中一些实施例中，所述生物酶组分和辅助组分的重量比为1:30-70。
在其中一些实施例中，所述纤维氧化酶组合物还包括对纤维氧化酶具有促进作用的蛋白质，所述对纤维氧化酶具有促进作用的蛋白质为有催化活性的生物酶或无催化活性的纤维素结合蛋白。
在其中一些实施例中，所述有催化活性的生物酶为氧化还原酶，选自纤维二糖脱氢酶、漆酶、葡糖氧化酶、过氧化氢歧化酶、酒精脱氢酶或木质素过氧化酶中的一种或多种；或所述有催化活性的生物酶为非纤维素水解酶的水合酶，选自淀粉酶、木聚糖酶、果胶酶或酯酶中的一种或多种。
在其中一些实施例中，所述纤维氧化酶组合物中纤维氧化酶和对纤维氧化酶具有促进作用的蛋白质的重量比为1:(0.5-3)。
在其中一些实施例中，所述无催化活性的蛋白质为纤维结合蛋白和/或纤维膨胀蛋白。
本发明的另一目的为提供一种纤维氧化酶组合物或纤维氧化酶在造纸工艺中作为改善纸张白度的漂白剂、脱墨剂或增白剂的应用。
本发明的另一目的为提供一种造纸方法，主要包括以下步骤：
a)形成含水的纤维素造纸浆料，
b)将上述用于改善纸张白度的纤维氧化酶组合物加入到造纸浆料中进行反应，
c)将步骤b)中得到的造纸浆料送上网，由此通过滤出水而由纤维固体组分形成纸页，
d)将纸页经过压榨段和干燥段，最终生产出纸产品。
在其中一些实施例中，步骤b)中所述纤维氧化酶组合物中的纤维氧化酶用量为每吨干浆料0.01-10千克，辅助组分的用量为每吨干浆料0.05-10千克。
在其中一些实施例中，步骤b)中所述纤维氧化酶组合物中的纤维氧化酶用量为每吨干浆料0.05-10千克，辅助组分的用量为每吨干浆料0.1-10千克。
在其中一些实施例中，步骤b)中所述纤维氧化酶组合物中的纤维氧化酶用量为每吨干浆料0.1-1千克。
在其中一些实施例中，步骤b)中所述纤维氧化酶组合物中的纤维氧化酶用量为每吨干浆料0.1-0.5千克。
在其中一些实施例中，所述纤维氧化酶组合物和造纸浆料的反应时间为5-600分钟，pH为3-10，温度为20-80℃。
在其中一些实施例中，所述纤维氧化酶组合物和造纸浆料的反应时间为20-300分钟，反应pH为5-10，反应温度为30-65℃。
在其中一些实施例中，所述纤维氧化酶组合物和造纸浆料的反应时间为60-120分钟，反应pH为8-10，反应温度为50-65℃。
本发明所述一种用于改善纸张白度的纤维氧化酶组合物及造纸方法和应用具有以下优点和有益效果：
本发明经发明人大量的实验和研究，得出纤维氧化酶及其组合物可显著改善纸张白度，并确定出纤维氧化酶组合物的最佳组分及其配比，在处理废纸纸浆的过程中，加入该纤维氧化酶组合物或纤维氧化酶，能够改变纤维表面或油墨离子附近的连接键，促进纤维的润胀，致使纤维和油墨之间的连接减弱，再通过适当的机械处理，进一步使油墨粒子比较完整地从纤维表面脱离，再经洗涤或浮选即可有效脱除。同时，纤维氧化酶或其组合物还能催化木质素的分解，由于木质素结构中的发色基团，是制浆颜色的主要来源，而木质素的分解，纤维氧化酶可以破坏发色基团，从而显著提高了纸浆的白度，还可显著减少现有技术中所用的脱墨剂及为增加纸张白度所添加的物料的用量。
具体实施方式
纤维素水解酶:本发明中的“纤维素水解酶”是指所有的通过水解反应来降解纤维素的生物酶，目前常常把纤维素水解酶和纤维素酶、纤维水解酶和纤维酶等术语交替使用。根据纤维素酶降解底物时不同的作用方式可将其分成3类：
(1)内切纤维素酶又称之为内切葡聚糖酶(Endoglucanase，EG；EC 3.2.1.4)；
(2)外切纤维素酶又称之为纤维二糖水解酶(Cellobiohydrolase，CBH；EC 3.2.1.91)；
(3)β-葡萄糖苷酶(β-glucosidase，BGL；EC 3.2.1.21)。
(a)内切纤维素酶(又称内切葡聚糖酶，endo-1,4-β-D-glucanase,EC 3.2.1.4)，该类酶主要作用于纤维素内部的非结晶区，随机水解β-1,4-糖苷键，将长链纤维素分子截短，产生大量非还原性末端的小分子纤维素，其分子量大小约为23-146KD；
(b)外切纤维素酶(又称外切葡聚糖酶，exo-1,4-β-D-glucanase,EC 3.2.1.91)，该类酶作用于纤维素线状分子末端，水解β-1,4-D-14糖苷键，依次切下一个纤维二糖分子，故又称为纤维二糖水解酶(cellobiohydrolase,CBH)，分子量约38-118KD。
(c)纤维二糖酶(又称β-葡萄糖苷酶，β-1,4-glucosidase，EC 3.2.1.21),简称BG。这类酶一般将纤维二糖或可溶性的纤维糊精水解成葡萄糖分子，其分子量约为76KD。
纤维素酶的酶活性(CMCase)：本发明中纤维素水解酶的酶活性是指纤维素酶组分在它们将纤维素降解成葡萄糖、纤维二糖和二糖的能力方面的萄聚糖的酶活性。纤维素酶的活性一般用羧甲基纤维素的溶液粘度的降低来确定。
纤维素结合蛋白质(cellulose binding protein，CBP)：本发明中的“纤维素结合蛋白质”是指对纤维素表面具有特别亲和力、能强烈地吸附在纤维素表面的、但其本身对纤维素没有催化水解反应活性的蛋白质。在文献中，又称为纤维素结合结构域(Cellulose Binding Domain,CBD)，纤维素结合模块(Cellulose Binding Module,CBM)，纤维素吸附结构域，纤维素亲合结构域。
纤维膨胀蛋白(Swollenin或者Expansin)：本发明中的“纤维膨胀蛋白”是指一种能使结晶纤维素和半纤维素等天然底物的纤维结构膨胀疏松的蛋白/多肽化合物，这类蛋白对纤维素没有降解的催化功能，但能提高纤维素酶对微晶纤维素的水解能力。“纤维膨胀蛋白”也称为纤维膨胀因子，英文称Swollenin和Expansin,是一类植物细胞壁扩展蛋白,其具有纤维素酶典型结构域组成(含纤维素结合结构域(CBD))的非水解活性蛋白。源于植物的纤维扩张蛋白主要有两类，分别命名为α-expansin和β-expansin。两类纤维扩张蛋白在结构和功能上有较高的同源性：α-expansin分子量约25kDa，氨基酸序列高度保守,同源性达70％～90％；β-expansin分子量约29kDa，但氨基酸序列变化较大。
纤维氧化酶(Cellulose Oxidative Enzymes)：本发明中的“纤维氧化酶”是指聚糖氧合酶(polysaccharide monooxygenase，PMO)，所述“聚糖氧合酶”是指是一族分子量在20-50KDa、含有铜(II)的小分子金属酶(metalloenzyme)，能与纤维表面进行加氧或者去氢的氧化反应，从而改变纤维表面化学性质的生物酶。聚糖氧合酶的其它名称包括聚葡糖加氧酶或者称聚葡糖单加氧酶(polysaccharide monooxygenase,PMO)，裂解性聚糖氧合酶(lytic polysaccharide monooxygenase,LPMO)，以及糖苷水化酶61族(glycosyl hydrolase 61,GH61)。
纤维氧化酶与纤维素水解酶的区别在于：纤维素水解酶是通过对β-1,4-D-14糖苷键进行水解反应，使纤维素降解；而纤维氧化酶是通过对纤维素进行氧化还原反应，反应所改变的键位不限于β-1,4-D-14糖苷键。虽然PMO早在1974年就已经被发现，由于用CMCase(即测试内切纤维素酶的活性方法)分析方法，发现PMO对β-1,4-D-14糖苷键的水解反应非常微弱，于是人们对该酶种的作用和其反应机理没有进行更深入的研究认识，CAZy(Carbohydrate Active Enzymes，简称CAZy，http://www.cazy.org/)把它们划分为Glycosyl Hydrolases 61族(GH61)。然而，最新研究表面，PMO实际是一种铜-依赖性单氧加合酶(Copper-dependent monooxygenases，EC 1.14.17.x)，它是通过氧化反应而对纤维进行作用的，而不像普通的纤维素水解酶那样通过水解反应 对纤维进行作用，因此PMO是氧化酶而非水解酶。经典的纤维素水解酶具有许多酶族，相反，PMO只有两个家族，即carbohydrate binding module family 33(CBM33，碳水结合域家族33)和glycosyl hydrolase(GH61,糖苷水解酶家族61)。CAZy最近将PMO重新列分为AA9类和AA10类生物酶。前者主要来源是真菌(fungus)，而后者主要来自于细菌、病毒和某些真菌。
PMO存在于很多木素纤维降解的微生物中，可以依此提纯和表达，或者通过现代蛋白工程技术对酶蛋白进行重组表达。例如，美国专利USPat.7,273,738,美国专利申请USA2009/099079，美国专利申请USA2013/0052698公开了一系列新的GH61酶种的列序。美国专利US8,298,795和美国专利申请USA2012/0083019公开了从真菌宿主菌株Myceliophthora thermophila(又称Chrysosporium lucknowense)获得重组GH61蛋白(recombinant GH61proteins)以及联合使用该GH61和和纤维素酶(cellulase)组合物处理木质纤维类物质增加生物精炼效率、提高酒精产量的技术。迄今，有关PMO的应用都在生物精炼(biorefinery)，还没有PMO在制浆造纸中应用的报道。
截止2013年2月，CAZy的数据库已经收集了249种PMO(即GH61)蛋白氨基酸列序数据，大部分属于子囊属菌株(ascomycetous)和担子菌(basidiomycetous)。而在NCBI/JGIBlast的搜索结果显示，目前共有761套GH61的基因系统。随着研究深入，可以预见PMO的数据会继续迅速增加。
纤维氧化酶组合物：本发明中的“纤维氧化酶组合物”是指该组合物含有如下两种组分，一个组分是聚糖氧合酶(polysaccharide monooxygenase)，和另外一个组分是能保持或者增加对维氧化酶活性的辅助物，其中该辅助物可以是以下化合物的一个或多个化合物，(1)纤维氧化酶的反应底物氧气或者氧气释放化合物(oxygen precursors)、(2)反应产物的清除剂(radical scavengers)、和(3)对纤维氧化酶具有促进作用的蛋白质。
本发明的“纤维氧化酶的反应底物”是指除了纤维以外，聚糖氧化酶发生反应所必需的电子受体--氧气；该反应底物可以通过如下方式提供：氧气、空气或 者氧气释放化合物，本发明中的氧气释放化合物是指过氧化物或者臭氧，其中过氧化物包括过氧化氢、过氧化钠、过氧化钙、过氧化钾。
本发明的“对纤维氧化酶的有促进作用的蛋白质”是指能增加聚糖氧化酶反应活性的生物酶、纤维素结合蛋白、和纤维膨胀蛋白；其中对聚糖氧化酶有促进作用的生物酶是指除了纤维素水解酶以外的其它类水解酶(包括淀粉酶、果胶酶、脂肪酶、酯酶和木聚糖酶)和氧化还原酶(包括纤维二糖脱氢酶、葡糖氧化酶、漆酶和木质素过氧化酶)。
本发明所涉及的纤维素氧化酶组合物可以是以其单个酶组分的产品分别加入造纸过程中，也可以将两组分分别加入不同的位置或者是混合成一个产品配方加入造纸过程中同意位置，以达到最佳的效果。
本发明所述“kg/T”是指每吨绝干浆中添加物料的千克数。
下述实施例所述原料均为常规市售产品。
以下将结合具体实施例对本发明做进一步说明。
实施例1纤维氧化酶及其组合物处理ONP纸浆
一、实验目的
通过对比分析评价纤维氧化酶及其组合物对纸张白度的影响。
二、实验方法
(1)试验材料准备
回收旧报纸(ONP)和旧杂志(OMG)废纸按照80％:20％的比例进行混合。将200克混合废纸和1300克白水(加入CaCl2调节硬度到400PPM；温度70℃)，放置在10升的水力碎浆机中，再加入烧碱，碎浆7.5分钟；碎浆完毕后，再加2700克白水，配得5％的浆。
纤维氧化酶(PMO)由美国Dyadic公司提供的GH61，该酶种从菌株Myceliophthora thermophila制取，其氨基酸列序在美国专利US8,298,795和美国专利申请USA2012/0083019有详细表述。
葡糖氧化酶(Glucose Oxidase，GOX)为DuPont集团下属子公司Genencor公司的商业产品。
(2)造纸
A、生物酶处理条件：取上述配好的浆600克，放入1000ml混合搅拌器中，用恒温控制器控制浆的温度在60℃；
B、设置实验组和对照组，
所述对照组为：在浆料中加入烧碱1-10kg/T，水玻璃15-30kg/T，过氧化氢10-20kg/T，脱墨剂0.5-3kg/T；(注：所有用量均为绝干浆，下述同理)。
所述实验组分为3组，其中，
实验组1为：在浆料中加入纤维氧化酶(PMO)150-300g/T(绝干浆)，脱墨剂0.5kg/T，加入烧碱2kg/T，水玻璃15kg/T，过氧化氢10kg/T；
实验组2为：在浆料中加入葡糖氧化酶250-500g/T，脱墨剂1kg/T，加入烧碱2kg/T，水玻璃15kg/T，过氧化氢10kg/T；
实验组3为：在浆料中加入纤维氧化酶和葡糖氧化酶组合处理，PMO 150-300g/T，GOX 250-500g/T，脱墨剂0.5kg/T，加入烧碱1-2kg/T，水玻璃15kg/T，过氧化氢10kg/T；
上述反应条件均为：温度为60℃，反应时间为90分钟，反应pH为8-10，恒速搅拌。
C、脱墨试验：取将以上浆料加入到3升的丹佛式浮选机，加入白水(50℃，400PPM硬度)稀释浆料到1％左右，搅拌混合3分钟，然后打开压缩空气阀门和调节流量到形成均匀细小气泡和稳定的泡沫层，将泡沫刮出，浮选时间为4分钟，得到脱墨浆。
D、手抄纸准备和强度测量：将上述制备好的脱墨浆，加自来水稀释到1.0％的浆浓，混匀，测量温度和浆浓度，按TAPPI方法，精确制备10-12张6.5克重的手抄纸。烘干后，手抄纸放在恒温和恒湿度的控制箱中24小时，然后按TAPPI测量方法测量其白度。
三、实验结果
结果参见表1，从表1中可看出：常规脱墨原料、PMO和GOX单独或者联合使用处理废纸纸浆对脱墨效果的影响。在烧碱用量减少80％，水玻璃减少 50％和双氧水减少50％的条件下，纤维氧化酶及其组合物处理后，纸张的白度在浮选之前就有显著增加，比对比条件的白度提高了2-4％ISO；经过浮选之后，白度增加更显著，比对比条件提高了3-4％ISO。单独加入GOX处理，纸张的白度增加比对比条件略有提高，但不显著。当纤维氧化酶和葡糖氧化酶联合使用时，效果最显著，其纸张的白度达到54％ISO以上。
表1纤维氧化酶和葡糖氧化酶联合使用处理回收废纸浆对纸张物理指标的影响
Figure PCTCN2015084456-appb-000001
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种用于改善纸张白度的纤维氧化酶组合物，其特征在于，包括以下组成：
    i)对纤维具有氧化功能的生物酶组分，所述生物酶组分为通过氧化还原反应来改变纤维表面性质的纤维氧化酶；和
    ii)对纤维氧化酶具有增加催化活性的辅助组分，所述辅助组分为纤维氧化酶的反应底物；
    所述生物酶组分和辅助组分的重量比为1:1-100。
  2. 根据权利要求1所述的用于改善纸张白度的纤维氧化酶组合物，其特征在于，所述纤维氧化酶是一族分子量在20-50KDa、具有铜依赖性的小分子金属酶的聚糖加氧酶，所述纤维氧化酶的反应底物为氧气、空气或者氧气释放化合物；所述氧气释放化合物为过氧化物或者臭氧。
  3. 根据权利要求2所述的用于改善纸张白度的纤维氧化酶组合物，其特征在于，所述过氧化物为过氧化氢、过氧化钠、过氧化钙或过氧化钾中的至少一种。
  4. 根据权利要求1所述的用于改善纸张白度的纤维氧化酶组合物，其特征在于，所述生物酶组分和辅助组分的重量比为1:30-70。
  5. 根据权利要求1-4任一项所述的用于改善纸张白度的纤维氧化酶组合物，其特征在于，所述纤维氧化酶组合物还包括对纤维氧化酶具有促进作用的蛋白质，所述对纤维氧化酶具有促进作用的蛋白质为有催化活性的生物酶或无催化活性的纤维素结合蛋白，所述纤维氧化酶组合物中纤维氧化酶和对纤维氧化酶具有促进作用的蛋白质的重量比为1:(0.5-3)。
  6. 根据权利要求5所述的用于改善纸张白度的纤维氧化酶组合物，其特征在于，所述有催化活性的生物酶为氧化还原酶，选自纤维二糖脱氢酶、漆酶、葡糖氧化酶、过氧化氢歧化酶、酒精脱氢酶或木质素过氧化酶中的一种或多种；或所述有催化活性的生物酶为非纤维素水解酶的水合酶，选自淀粉酶、木聚糖酶、果胶酶或酯酶中的一种或多种。
  7. 根据权利要求5所述的用于改善纸张白度的纤维氧化酶组合物，其特征在于，所述无催化活性的蛋白质为纤维结合蛋白和/或纤维膨胀蛋白。
  8. 纤维氧化酶或如权利要求1-7任一项所述的纤维氧化酶组合物在造纸工艺中作为改善纸张白度的漂白剂、脱墨剂或增白剂的应用。
  9. 一种造纸方法，其特征在于，主要包括以下步骤：
    a)形成含水的纤维素造纸浆料，
    b)将权利要求1-7任一项所述的用于改善纸张白度的纤维氧化酶组合物加入到造纸浆料中进行反应，
    c)将步骤b)中得到的造纸浆料送上网，由此通过滤出水而由纤维固体组分形成纸页，
    d)将纸页经过压榨段和干燥段，最终生产出纸产品。
  10. 根据权利要求9所述的造纸方法，其特征在于，步骤b)中所述纤维氧化酶组合物中的纤维氧化酶用量为每吨干浆料0.01-10千克，辅助组分的用量为每吨干浆料0.05-10千克；或/及
    所述纤维氧化酶组合物和造纸浆料反应的时间为5-600分钟，pH为3-10，温度为20-80℃。
PCT/CN2015/084456 2014-07-29 2015-07-20 用于改善纸张白度的纤维氧化酶组合物及造纸方法和应用 WO2016015571A1 (zh)

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