WO2016015570A1 - Fiber oxidase composition used for altering and improving whiteness of paper, papermaking method, and applications of the composition - Google Patents

Abstract

Provided are a fiber oxidase composition used for altering and improving the whiteness of a paper, a papermaking method, and applications of the composition. The fiber oxidase composition consists primarily of the following components: an enzyme component having an oxidation function for a fiber, where the enzyme component is a fiber oxidase that alters the surface properties of the fiber by means of a redox reaction; and, an auxiliary component having increased catalytic activities for the fiber oxidase, where the auxiliary component is a protein having catalytic effects for the fiber oxidase, and the weight ratio of the enzyme component to the auxiliary component is 1:0.4-50.

Description

用于改变和改善纤维性质的纤维氧化酶组合物及造纸方法和应用Fiber oxidase composition for changing and improving fiber properties, and papermaking method and application 技术领域Technical field

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，涉及一种用纤维氧化酶改变和改善纤维表面性质的组合物及造纸方法和应用。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 composition and papermaking method and application for modifying and improving the surface properties of fibers with fiber oxidase.

背景技术Background technique

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。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.

目前，中国造纸企业近80％的生产原料为非木纤维的原料，其中约70％为回收纤维和10％为草浆、芦苇浆和甘蔗渣浆等。这些原料的纤维本身很多性能远比木纤维差，而且其表面活性低，纤维之间的结合力差，由此带来很多生产和产品质量问题。At present, nearly 80% of the raw materials produced by Chinese papermaking enterprises are non-wood fiber raw materials, of which about 70% are recycled fiber and 10% are straw pulp, reed pulp and sugar cane pulp. The fibers of these raw materials have much lower performance than wood fibers, and their surface activity is low, and the bonding strength between fibers is poor, which causes many production and product quality problems.

例如，为使产品的物理指标满足要求，造纸企业目前采用的办法是：For example, in order to meet the requirements of the physical indicators of the product, the papermakers currently adopt the following methods:

(1)增加产品的定量，增加纸张密度；此方法的缺点是增加生产成本，浪费资源；(1) increase the quantification of products and increase the density of paper; the disadvantage of this method is to increase production costs and waste resources;

(2)增加磨浆，提高纤维表面分丝帚化，提高纸张强度；此方法的缺点是能耗高，降低制浆的游离度，降低纤维得率和增加烘干能耗；(2) Increasing refining, increasing the surface of the fiber and increasing the strength of the paper; the disadvantage of this method is high energy consumption, reducing the freeness of pulping, reducing the fiber yield and increasing the drying energy consumption;

(3)使用干强剂/湿强剂等化学品，例如使用阳离子淀粉、阴离子淀粉、羧甲基纤维素钠、聚丙烯酰胺、阴离子聚丙烯酰胺，以及低分子量阳离子聚合物等，来增加纸浆的脱水性能或强度，此方法的缺点是成本高，钝化纤维，对造纸水***循环不利，增加COD排放；和(3) using chemicals such as dry strength agents/wet strength agents, such as cationic starch, anionic starch, sodium carboxymethyl cellulose, polyacrylamide, anionic polyacrylamide, and low molecular weight cationic polymers to increase pulp The dewatering performance or strength, the disadvantage of this method is the high cost, passivated fiber, which is unfavorable to the papermaking water system cycle, increasing COD emissions;

(4)提高木纤维浆料比例，此方法缺点是成本高，增加进口原料的依赖性。可见，这些方法有多方面的问题，制浆造纸工业目前迫切需要一种高效的方法来解决这些难题。 (4) Increasing the proportion of wood fiber slurry, the disadvantage of this method is high cost and increasing dependence of imported raw materials. It can be seen that these methods have many problems, and the pulp and paper industry is currently in urgent need of an efficient method to solve these problems.

早在上世纪六十年代，人们开始研究使用生物酶来改变纸浆和改善造纸。1986年努等人报道了木聚糖酶对漂白化学浆的酶法打浆作用(Noe P.et al.，Action of xylanases on chemical pulp fibers，J.Wood Chern.Techno.，6：167，1986)。近一二十年来，人们对生物技术在制浆造纸工业的应用进行了大量的研究，研究范围几乎涉及了制浆造纸工业的各个方面(Bajpai，Biotechnology for Pulp and Paper Processing，2012，Springer，ISBN 978-1-4614-1408-7)。美国专利US4,923,565，US5,110,412和US5,308,449提出使用纤维素酶或半纤维素酶提高造纸纸浆的脱水性。US5,725,732提出使用纤维素酶和半纤维素酶解决纤维的掉毛缺陷，US6,066,233建议用纤维素酶和果胶酶结合使用可通过纸浆的滤水性。美国专利US5,582,681提出用纤维素酶、半纤维素酶和脂肪水解酶等混合酶制剂提高卫生纸的柔韧性。此外用纤维素酶、半纤维素酶处理纸浆，还可改善纤维压缩性，使纸页微孔性下降，密度提高，透明度提高。As early as the 1960s, people began to study the use of biological enzymes to change pulp and improve paper. In 1986, Nuo reported the enzymatic beating of xylanase on bleached chemical pulp (Noe P. et al., Action of xylanases on chemical pulp fibers, J. Wood Chern. Techno., 6: 167, 1986). . In the past decade or two, people have done a lot of research on the application of biotechnology in the pulp and paper industry. The research scope covers almost all aspects of the pulp and paper industry (Bajpai, Biotechnology for Pulp and Paper Processing, 2012, Springer, ISBN). 978-1-4614-1408-7). The use of cellulases or hemicellulases to increase the dewatering properties of papermaking pulp is taught in U.S. Patent Nos. 4,923,565, 5,110,412 and 5,308,449. U.S. Patent 5,725,732 teaches the use of cellulase and hemicellulase to address the hair loss of fibers. U.S. Patent No. 6,066,233, the use of cellulase and pectinase in combination with water filtration through pulp. U.S. Patent No. 5,582,681 teaches the use of a mixed enzyme preparation such as cellulase, hemicellulase and lipolytic enzyme to improve the flexibility of toilet paper. In addition, treatment of the pulp with cellulase or hemicellulase can also improve the compressibility of the fiber, reduce the microporosity of the paper, increase the density, and improve the transparency.

造纸行业目前使用的所谓“纤维改性酶”都是利用纤维素水解酶，包括内切纤维素酶和外切纤维素酶，对纤维的(-1，4苷糖键(b-1，4-linked D-glucose units)进行水解反应，逐步降解(depolymerization)成为较小分子量的多糖。应用的结果显示，纤维素水解酶对于降低磨浆能耗和提高网部脱水有一定的效果。但是，基于纤维水解酶的“打浆酶”技术有几个极大弊病：The so-called "fiber-modified enzymes" currently used in the paper industry are all utilizing cellulolytic enzymes, including endocellulases and exocellulases, on the fibers (-1,4 glycosidic bonds (b-1,4). -linked D-glucose units) undergo hydrolysis reaction and depolymerization into polysaccharides of smaller molecular weight. The results of application show that cellulose hydrolase has a certain effect on reducing the energy consumption of refining and improving the dehydration of the net. There are several major drawbacks to the "beating enzyme" technology based on fiber hydrolase:

(a)纤维素水解酶对纤维的作用在造纸过程中具有不可控性和不可逆性，当纤维素的浓度或作用时间过长时，纤维素酶会首先溃解纤维细胞，对纤维表面的纤丝象剃胡须一样的剪除，甚至对纤维整体进行切断，致使纤维损伤，其结果导致纸产品“发脆”和强度指标(特别是撕裂度和耐破度等)大幅度下降，甚至导致纸浆作废，给企业造成经济损失。(a) The effect of cellulose hydrolase on fiber is uncontrollable and irreversible in the papermaking process. When the concentration or duration of action of cellulose is too long, cellulase will first break down the fiber cells and fiber on the surface of the fiber. The silk is shaved like a shaving, and even the whole fiber is cut, resulting in fiber damage. As a result, the paper product is “brittle” and the strength index (especially tearing degree and bursting resistance) is greatly reduced, and even the pulp is caused. Obsolete, causing economic losses to the enterprise.

(b)纤维素水解酶对纤维的(-1，4苷糖键进行水解反应，降低纤维的聚合度(degree of polymerization)，最终产生葡聚糖和单糖，致使纤维溶解。当纤维素酶的用量过高或者停留时间过长时，纤维的降解率可高达1％，不仅造成纤维损失，降低纤维的综合利用率，而且大大增加白水中的COD含量，不利于节能减排。(b) Cellulolytic enzymes hydrolyze the (-1,4-glycosidic bond of the fiber to reduce the degree of polymerization of the fiber, eventually producing glucan and monosaccharide, causing the fiber to dissolve. When the cellulase When the dosage is too high or the residence time is too long, the degradation rate of the fiber can be as high as 1%, which not only causes fiber loss, reduces the comprehensive utilization rate of the fiber, but also greatly increases the COD content in the white water, which is not conducive to energy conservation and emission reduction.

(c)纤维素水解酶对底物“比较很挑剔”，只对较纯净的纤维素才有较显著作用，甚至只对某种纤维素有明显的作用。例如，现有纤维素酶大多数对漂白化学针叶浆有较显著的作用，而对漂白化学阔叶浆的作用较小。如果纤维含量木素，例如未漂针叶浆，受表面木素 的影响，纤维素分解酶的作用很小。对含木素高的机械浆，纤维素水解酶就失去其作用。(c) Cellulolytic enzymes are "very picky" on the substrate and only have a significant effect on purer cellulose, even on a certain cellulose. For example, most of the existing cellulases have a significant effect on bleaching chemical softwood pulp and less on bleaching chemical hardwood pulp. If the fiber content is lignin, such as unbleached needle pulp, surface lignin The effect of cellulolytic enzymes is small. For mechanical pulps containing high lignin, cellulose hydrolase loses its effect.

为了避免常规纤维素酶对纤维过分剪切而降低纤维强度的缺陷，美国专利US6,294,366和US6,635,146公开了使用截短的纤维素酶(CBD-truncated cellulose)处理纸浆，截短的酶缺少纤维素结合结构域(CBD)，使用这样的纤维素酶处理纸浆，可以避免这样的纤维强度的损失。在相似的纺织工业应用中，US 5,916,799公开了含有纤维二糖水解酶和内切葡聚糖酶的纤维素酶组合物，这两种酶已经进行了有限的蛋白水解，从而将酶的核心和纤维素结合结构域分开，发现得到的酶组合物降低返染。WO96/2 3928公开了使用截短的纤维素酶处理含有纤维素的织物，发现能够减少染料的再沉积并增加磨损。U.S. Patent Nos. 6,294,366 and 6,635,146 disclose the use of truncated cellulase (CBD-truncated cellulose) to treat pulp, truncated enzymes are lacking in order to avoid the drawbacks of conventional cellulase to excessively shear the fibers to reduce fiber strength. The cellulose binding domain (CBD), which is treated with such cellulase, can avoid such fiber strength loss. In a similar textile industry application, US 5,916,799 discloses cellulase compositions containing cellobiohydrolases and endoglucanases, which have been subjected to limited proteolysis, thereby The cellulose binding domains were separated and the resulting enzyme composition was found to reduce back-staining. WO 96/2 3928 discloses the treatment of cellulose-containing fabrics using truncated cellulases, which have been found to reduce redeposition of dyes and increase wear.

Shoseyov等(Chapter 8：Modulation of Wood Fibers and Paper by Cellulose-Binding Domain，In Applications of Enzymes to Lign℃ellulosics；Edited by Mansfield et al..ACS Symposium Series；American Chemical S℃iety：Washington，DC，2003，page116-132)发现，单独使用纤维素亲合结构域(cellulose-binding domain，CBD)蛋白能提高纸张的强度，而且聚合纤维结合蛋白(即由两个或更多的单体纤维结合蛋白组成的聚合分子，又称纤维键联蛋白)比单体纤维结合蛋白对纸张强度的提高更有效。Levy等(Cellulose，2002，9：91-98)报道，经过纤维结合蛋白质处理后的纸张的强度和纸面防湿性都大大地提高。Pala等(Chapter 7：Cellulose-Binding Domain as a Tool for Paper Recycling，In Applications of Enzymes to Lign℃ellulosics；Edited by Mansfield et al..ACS Symposium Series；American Chemical S℃iety：Washington，DC，2003，pagel05-115)报道，用CBD处理后的回收废纸的强度有显著增加。Kitaoka等(J Wood Sci.，2001，47：322-324)描述了使用纤维素亲合结构域蛋白质和阴离子聚合物聚丙烯酰胺(CBD-APAM)合成新的聚合物，作为干强度或湿强度添加剂，比常规干强/湿度添加剂更有效。Shoseyov et al. (Chapter 8: Modulation of Wood Fibers and Paper by Cellulose-Binding Domain, In Applications of Enzymes to Lign ° Cellulosics; Edited by Mansfield et al.. ACS Symposium Series; American Chemical S° Ciety: Washington, DC, 2003, Page 116-132) found that the use of a cellulose-binding domain (CBD) protein alone can increase the strength of the paper, and that the polymeric fibronectin (ie consists of two or more monomeric fiber-binding proteins) Polymeric molecules, also known as fiber-bonded proteins, are more effective than monomer-fiber-binding proteins for increasing paper strength. Levy et al. (Cellulose, 2002, 9: 91-98) reported that the strength and paper moisture resistance of paper treated with fiber-bound protein were greatly improved. Pala et al. (Chapter 7: Cellulose-Binding Domain as a Tool for Paper Recycling, In Applications of Enzymes to Lign ° Cellulosics; Edited by Mansfield et al.. ACS Symposium Series; American Chemical S° Ciety: Washington, DC, 2003, pagel05 -115) reported a significant increase in the strength of recycled waste paper after treatment with CBD. Kitaoka et al. (J Wood Sci., 2001, 47: 322-324) describe the use of cellulose affinity domain proteins and anionic polymer polyacrylamide (CBD-APAM) to synthesize new polymers as dry strength or wet strength. Additives are more effective than conventional dry strength/humidity additives.

中国专利“一种用于改变和改善纤维表面性质的组合物和造纸方法”(专利号：ZL201010566132.2)提出联合使用纤维素酶和纤维结合蛋白(CBP)和纤维素酶处理纤维纸浆，通过纤维结合蛋白选择吸附在纤维的非晶型区域表面，庇护该区域不受纤维素酶的作用，从而避免纤维素酶对纤维非晶型纤维的过度剪切。但是在实际应用中发现，由于纤维结合蛋白(CBP)吸附的可逆性，特别是当聚能酶^TM产品在磨浆之前加入时，经过磨浆之后纸浆产生大量新生表面，这些新纤维表面将被暴露在纤维素酶的“攻击”之下，如果停 留时间较长，纤维被过度剪切，导致纸产品的强度下降。The Chinese patent "A composition for changing and improving the surface properties of fibers and a papermaking method" (Patent No.: ZL201010566132.2) proposes a combination of cellulase and fibronectin (CBP) and cellulase treatment of fiber pulp through The fibronectin is selectively adsorbed on the surface of the amorphous region of the fiber, protecting the region from the action of cellulase, thereby avoiding excessive shearing of the fibrous amorphous fiber by the cellulase. However, in practical applications, it has been found that due to the reversibility of the adsorption of fibronectin (CBP), especially when the ^polyenzymeTM product is added before refining, the pulp will produce a large number of new surfaces after refining, and these new fiber surfaces will be Exposure to the "attack" of cellulase, if the residence time is longer, the fibers are excessively sheared, resulting in a decrease in the strength of the paper product.

因此，制浆造纸工业迫切需要一种既能与纤维反应使纤维表面活化、又不会溃解纤维导致纤维强度下降的新型生物酶技术，而且这种生物酶能对不同纤维材料(包括木纤维、非木纤维和回收纤维)都具有显著活性的广谱性生物酶，这样的新型生物酶技术将对中国造纸工业的可持续性发展具有重要意义。Therefore, there is an urgent need in the pulp and paper industry for a novel bioenzyme technology that can react with fibers to activate the surface of the fibers without breaking the fibers, resulting in a decrease in fiber strength, and the bioenzyme can be used for different fiber materials (including wood fibers). Non-wood fiber and recycled fiber) have broad-spectrum biological enzymes with significant activity. Such new bio-enzyme technology will be of great significance to the sustainable development of China's paper industry.

发明内容Summary of the invention

本发明的目的之一在于提供一种用于改变和改善制浆造纸纤维材料性质的纤维氧化酶组合物，通过使用该组合物处理纸浆原料，能显著提高纤维之间的结合力，改善纸张的质量和纸浆的脱水效率。One of the objects of the present invention is to provide a fiber oxidase composition for modifying and improving the properties of a pulping and papermaking fiber material, which can significantly improve the bonding force between fibers and improve the paper by treating the pulp raw material with the composition. Quality and pulp dewatering efficiency.

实现上述目的的技术方案如下：The technical solution to achieve the above objectives is as follows:

一种用于改变和改善纤维性质的纤维氧化酶组合物，其含有：A fiber oxidase composition for modifying and improving fiber properties, comprising:

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i) 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;

ii)对所述纤维氧化酶具有增加催化活性的辅助组分，所述辅助组分为对纤维氧化酶具有促进作用的蛋白质；Ii) an auxiliary component having an increased catalytic activity for said cell oxidase, said auxiliary component being a protein having a promoting effect on cell oxidase;

所述生物酶组分和辅助组分的重量比例为1∶0.4-50。The weight ratio of the biological enzyme component to the auxiliary component is 1:0.4-50.

在其中一些实施例中，所述纤维氧化酶为一族分子量在20-50KDa、具有铜依赖性的小分子金属酶的聚糖加氧酶；或/及In some embodiments, the cell oxidase is a glycanoxygenase having a copper-dependent small molecular metalloenzyme having a molecular weight of 20-50 KDa; or/and

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。The protein having a promoting effect on the fiber oxidase is a catalytically active biological enzyme or a catalytically inactive cellulose binding protein.

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、非纤维素水解酶的水合酶或纤维素水解酶；所述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一种或多种，所述非纤维素水解酶的水合酶选自淀粉酶、木聚糖酶、果胶酶或酯酶中一种或多种。In some of the embodiments, the catalytically active biological enzyme is an oxidoreductase, a non-cellulolytic enzyme hydratase or a cellulolytic enzyme; the oxidoreductase is selected from the group consisting of cellobiose dehydrogenase, laccase One or more of glucose oxidase, hydrogen peroxide dismutase, alcohol dehydrogenase or lignin peroxidase, the non-cellulolytic enzyme hydratase is selected from the group consisting of amylase, xylanase and fruit One or more of a gelase or an esterase.

在其中一些实施例中，所述氧化还原酶为纤维二糖脱氢酶和漆酶，所述纤维二糖脱氢酶和漆酶的重量比为1∶1-5。 In some of these embodiments, the oxidoreductase is cellobiose dehydrogenase and laccase, and the cellobiose dehydrogenase and laccase are in a weight ratio of 1:1-5.

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。In some of these embodiments, the catalytically inactive protein is a fibronectin and/or a fibroin.

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例为1∶0.4-30。In some of these embodiments, the weight ratio of the bio-enzyme component to the auxiliary component is 1:0.4-30.

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例为1∶0.4-10。In some of these embodiments, the weight ratio of the bio-enzyme component to the auxiliary component is 1:0.4-10.

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例为1∶0.4-5。In some of these embodiments, the weight ratio of the biological enzyme component to the auxiliary component is 1:0.4-5.

在其中一些实施例中，所述纤维氧化酶组合物还包括纤维氧化酶反应产物的清除剂；所述纤维氧化酶和清除剂的重量比为1∶1-5。In some of these embodiments, the cell oxidase composition further comprises a scavenger of a cell oxidase reaction product; the fiber oxidase and scavenger are present in a weight ratio of 1:1-5.

在其中一些实施例中，所述清除剂选自抗坏血酸、没食子酸、木质素、锰(II)盐、铜(II)盐或铁(II)盐中的一种或多种。In some of these embodiments, the scavenger is selected from one or more of the group consisting of ascorbic acid, gallic acid, lignin, manganese (II) salt, copper (II) salt, or iron (II) salt.

在其中一些实施例中，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。In some of these embodiments, the reaction substrate of the cell oxidase is an oxygen, air or oxygen releasing compound; the oxygen releasing compound is a peroxide or ozone.

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。In some of these embodiments, the peroxide is at least one of hydrogen peroxide, sodium peroxide, calcium peroxide, or potassium peroxide.

本发明的另一目的提供新的造纸方法，该方法比现有技术更有效地利用纤维原料、提高纸产品的性能和品质，改善生产效率和降低能耗。Another object of the present invention is to provide a new papermaking process which utilizes fiber raw materials more efficiently than the prior art, improves the performance and quality of paper products, improves production efficiency and reduces energy consumption.

实现上述目的的技术方案如下：The technical solution to achieve the above objectives is as follows:

一种造纸方法，主要包括以下步骤：A papermaking method mainly comprises the following steps:

a)形成含水的纤维素造纸浆料，a) forming an aqueous cellulose papermaking slurry,

b)将上述一种用于改变和改善纤维性能的纤维氧化酶组合物加入到纸浆料中充分反应；b) adding a fiber oxidase composition as described above for modifying and improving fiber properties to the paper slurry for complete reaction;

c)将纸浆送上网，由此通过滤出水而由纤维等固体组分形成纸页，c) sending the pulp to the Internet, thereby forming a sheet of paper from solid components such as fibers by filtering out the water.

d)将纸页经过压榨段和干燥段，最终生产出纸产品。d) The paper sheet is passed through the pressing section and the drying section to finally produce a paper product.

在其中一些实施例中，所述纤维氧化酶用量为每吨干浆料0.01-10千克，所述辅助组分的用量为每吨干浆料0.05-10千克。In some of these embodiments, the amount of cell oxidase is from 0.01 to 10 kilograms per ton of dry slurry, and the amount of the auxiliary component is from 0.05 to 10 kilograms per ton of dry slurry.

在其中一些实施例中，所述纤维氧化酶用量为每吨干浆料0.05-10千克，所述辅助组分的用量为每吨干浆料0.1-5千克。In some of these embodiments, the amount of cell oxidase is from 0.05 to 10 kilograms per ton of dry slurry, and the amount of the auxiliary component is from 0.1 to 5 kilograms per ton of dry slurry.

在其中一些实施例中，所述纤维氧化酶用量为每吨干浆料0.05-2千克，所述辅助组分的用量为每吨干浆料0.1-5千克。 In some of these embodiments, the amount of cell oxidase is from 0.05 to 2 kilograms per ton of dry slurry, and the amount of the auxiliary component is from 0.1 to 5 kilograms per ton of dry slurry.

在其中一些实施例中，所述反应的时间为5-600分钟，pH为3-10，温度为20-80℃。In some of these embodiments, the reaction is carried out for a period of from 5 to 600 minutes, at a pH of from 3 to 10, and at a temperature of from 20 to 80 °C.

在其中一些实施例中，所述反应的时间为20-300分钟，pH为5-10，温度为30-65℃。In some of these embodiments, the reaction is carried out for a period of from 20 to 300 minutes, a pH of from 5 to 10, and a temperature of from 30 to 65 °C.

在其中一些实施例中，所述反应的时间为20-200分钟，pH为5-7，温度为40-65℃。In some of these embodiments, the reaction is carried out for a period of from 20 to 200 minutes, a pH of from 5 to 7, and a temperature of from 40 to 65 °C.

本发明的另一目的在于提供纤维氧化酶组合物或纤维氧化酶在造纸工艺中作为改变和改善纤维性质的增强剂或助留助滤剂的应用。Another object of the present invention is to provide a fiber oxidase composition or fiber oxidase for use as a reinforcing agent or retention and drainage aid in a papermaking process for modifying and improving fiber properties.

现有造纸技术中纸浆制浆存在的问题是主要是纤维的活性低，纤维之间的反应度低，为了达到纸产品的质量要求，通常的办法是增加打浆度，但是增加磨浆会导致纤维被切断，产生大量的细小纤维，不仅降低制浆的脱水性能，增加造纸的烘干能耗，而且可能导致细小纤维流失，原料利用率下降。现有的“打浆酶”是利用纤维素水解酶对纤维进行水解反应，虽然目前该技术还在初级应用阶段，但已经暴露出严重问题，即纤维素水解酶对纸浆纤维的反应是不可控性的和不可逆性的，稍有不当，将造成纸浆强度明显下降，甚至产生次品或废品。The problem of pulp pulping in the existing papermaking technology is that the fiber has low activity and low reactivity between fibers. In order to meet the quality requirements of paper products, the usual method is to increase the degree of beating, but increasing the refining leads to fiber. It is cut and produces a large amount of fine fibers, which not only reduces the dewatering performance of pulping, but also increases the drying energy consumption of papermaking, and may cause the loss of fine fibers and the utilization of raw materials. The existing "beating enzyme" is a hydrolysis reaction of cellulose by using cellulolytic enzyme. Although the technology is still in the primary application stage, serious problems have been exposed, that is, the reaction of cellulose hydrolase to pulp fiber is uncontrollable. And irreversible, a little improper, will cause a significant drop in pulp strength, and even produce defective or scrap.

与现有技术相比，本发明具有如下优点和有益效果：Compared with the prior art, the present invention has the following advantages and benefits:

本发明经发明人的实验和研究，得出纤维氧化酶及其组合物能改变和改善纤维表面性质，并确定了纤维氧化酶组合物的最佳组分及其配比，所述纤维氧化酶组合物或纤维氧化酶对不同纤维材料(包括木纤维、非木纤维和回收纤维)都具有显著反应活性，采用所述纤维氧化酶组合物或纤维氧化酶处理纸浆原料，如：木浆/化学浆，主要是漂白和未漂白的木浆/化学浆的处理，包括二次回收纤维；还有木浆/机械浆，及非木浆(秸秆、稻草、芦苇和烟叶等)，处理过程中，纤维氧化酶集中在晶型纤维表面的反应，激活该部分纤维的表面活性，增加纤维反应活性，提高纤维间的结合力，而不会溃解纤维导致纤维强度下降；采用该种纤维氧化酶及其组合物处理上述纸浆，不仅能改善制得的纸张的各种物理强度，如：叩解度、抗张指数、撕裂度、层间结合力、耐破度和白度(特别是废纸的白度)，还能改善纸浆的滤水性能(尤其是非木浆的滤水性能)，同时降低制浆的有游离度，还能够改变纸浆(尤其是木片浆)的打浆性能，大大地降低打浆能耗，且环保，这对造纸生产产业具有重要意义。According to the experiments and studies of the present inventors, it has been found that the fiber oxidase and its composition can change and improve the surface properties of the fiber, and determine the optimum components of the fiber oxidase composition and the ratio thereof, the fiber oxidase. The composition or cell oxidase has significant reactivity with different fiber materials, including wood fibers, non-wood fibers and recycled fibers, and the pulp material is treated with the fiber oxidase composition or cell oxidase, such as: wood pulp/chemistry Pulp, mainly bleached and unbleached wood pulp / chemical pulp treatment, including secondary recycled fiber; wood pulp / mechanical pulp, and non-wood pulp (straw, straw, reed and tobacco), during processing, The reaction of fiber oxidase on the surface of the crystal fiber, activates the surface activity of the fiber, increases the fiber reactivity, and improves the bonding force between the fibers without breaking the fiber to cause the fiber strength to decrease; using the fiber oxidase and The composition of the above-mentioned pulp can not only improve various physical strengths of the produced paper, such as: degree of decomposition, tensile index, tearing degree, interlaminar bonding force, bursting resistance and Whiteness (especially the whiteness of waste paper) can also improve the water filtration performance of pulp (especially the water filtration performance of non-wood pulp), while reducing the freeness of pulping, and also changing the pulp (especially wood pulp) The beating performance greatly reduces the energy consumption of beating and is environmentally friendly, which is of great significance to the paper production industry.

附图说明DRAWINGS

图1为实施例3中生物酶处理木片的流程示意图；1 is a schematic flow chart of biological enzyme treatment of wood chips in Example 3;

图2为实施例3中不同处理条件对TMP浆的游离度与磨浆能耗的关系图； 2 is a graph showing the relationship between the freeness of the TMP pulp and the refining energy consumption under different processing conditions in Example 3;

图3为实施例4中复合生物酶处理TMP浆对磨浆能耗的影响结果图；3 is a graph showing the effect of the composite biological enzyme-treated TMP slurry on the energy consumption of refining in Example 4;

图4为实施例6中纤维氧化酶处理非木浆对纸张抗张强度的影响结果图。Fig. 4 is a graph showing the effect of the fiber oxidase-treated non-wood pulp on the tensile strength of the paper in Example 6.

具体实施方式detailed description

对于本发明所述技术术语的说明如下：The description of the technical terms of the present invention is as follows:

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：Cellulolytic enzyme: The "cellulolytic enzyme" in the present invention 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:

(1)内切纤维素酶又称之为内切葡聚糖酶(Endoglucanase，EG；EC 3.2.1.4)；(1) Endocellulase is also known as endoglucanase (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)；(2) Exocellulase is also known as cellobiohydrolase (CBOH; EC 3.2.1.91);

(3)β-葡萄糖苷酶(β-glucosidase，BGL；EC 3.2.1.21)。(3) 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；(a) endocellulase (also known as endog-glucanase, endo-1,4-β-D-glucanase, EC 3.2.1.4), which acts mainly on the amorphous region inside the cellulose. Randomly hydrolyzed β-1,4-glycosidic bond, truncating long-chain cellulose molecules, producing a large number of non-reducing terminal small-molecule cellulose, the molecular weight of which is about 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。(b) exocellulase (also known as exog-glucanase, exo-1,4-β-D-glucanase, EC 3.2.1.91), which acts on the ends of cellulose linear molecules and hydrolyzes β - 1,4-D-14 glycosidic bond, which in turn cuts a cellobiose molecule, so it is also called cellobiohydrolase (CBH), and has a molecular weight of about 38-118 KD.

(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***解成葡萄糖分子，其分子量约为76KD。(c) Cellobiase (also known as β-glucosidase, β-1,4-glucosidase, EC 3.2.1.21), abbreviated as BG. Such enzymes typically hydrolyze cellobiose or soluble cellodextrin to glucose molecules having a molecular weight of about 76 KD.

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。Cellulase Enzyme Activity (CMCase): 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.

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。Cellulose binding protein (CBP): "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. In the literature, it is also known as Cellulose Binding Domain (CBD), Cellulose Binding Module (CBM), cellulose adsorption domain, Cellulose affinity domain.

纤维膨胀蛋白(Swollenin或者Expansin)：本发明中的“纤维膨胀蛋白”系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。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. There are two main types of plant-derived fibronectin, named α-expansin and β-expansin. The two types of fibronectin have high homology in structure and function: α-expansin has a molecular weight of about 25kDa, amino acid sequence is highly conserved, and homology is 70% to 90%; β-expansin has a molecular weight of about 29kDa, but the amino acid sequence big change.

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)。Cellulose Oxidative Enzymes: "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. A metalloenzyme containing copper (II), which is an enzyme that can oxidize or dehydrogenate the surface of a fiber to change the chemical properties of the surface of the fiber. Other names for glycanoxygenase include polyglucose oxygenase or polysaccharide monooxygenase (PMO), lytic polysaccharide monooxygenase (LPMO), and glycosidation Enzyme 61 (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。The difference between cell oxidase and cellulolytic enzyme is that cellulolytic enzymes degrade cellulose by hydrolysis of β-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 β-1,4-D-14 glycosidic bond. Although PMO was discovered as early as 1974, the hydrolysis reaction of PMO to β-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). . However, the latest research surface, PMO is actually a copper-dependent monooxygenases (EC 1.14.17.x), which acts on the fibers through oxidation, rather than ordinary The cellulose hydrolyzing enzyme acts on the fiber by a hydrolysis reaction, and thus the PMO is an oxidase rather than a hydrolase. Classical cellulolytic enzymes have many enzyme families. In contrast, PMO has only two families, namely carbohydrate binding module family 33 (CBM33, carbon-water 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US Pat.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。PMO is present in many lignin-degrading microorganisms, which can be purified and expressed, or recombinantly expressed by modern protein engineering techniques. For example, U.S. Pat. No. 7,273,738, U.S. Patent Application Serial No 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 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.

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/JGlBlast的搜索结果显示，目前共有761套GH61的基因***。随着研究深入，可以预见PMO的数据会继续迅速增加。As of February 2013, CAZy's database has collected 249 PMO (ie GH61) protein amino acid sequence data, most of which belong to ascomycetous and basidiomycetous. The search results in NCBI/JGlBlast show that there are currently 761 sets of GH61 gene systems. As the research progresses, it is foreseeable that the PMO data will continue to increase rapidly.

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。Fibrous oxidase composition: The "fibrin 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.

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。The "reaction substrate of cellulase" of the present invention 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: releasing oxygen, air or oxygen The 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.

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、酯酶或木聚糖酶)和氧化还原酶(包括纤维二糖脱氢酶、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)。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 Enzyme refers to other hydrolase enzymes (including amylase, pectinase, lipase, esterase or xylanase) and oxidoreductases (including cellobiose dehydrogenase, laccase, etc.) other than cellulolytic enzymes. Glucose oxidase, hydrogen peroxide dismutase, alcohol dehydrogenase Or lignin peroxidase).

本发明的“纤维氧化酶的反应产物清除剂”是指能降低聚糖氧化酶的反应产物过氧化氢浓度的化合物，包括抗坏血酸(ascorbate)、没食子酸(gallate)、木质素(lignin)、铜(II)盐即二价铜盐、铁(II)盐即二价铁盐、锰(II)盐即二价锰盐和锌(II)盐即二价锌盐。The "reaction product scavenger of cellulase" of the present invention means a compound capable of reducing the concentration of hydrogen peroxide of a reaction product of glycan oxidase, including ascorbate, gallate, lignin, copper. (II) The salt is a divalent copper salt, an iron (II) salt, that is, a divalent iron salt, a manganese (II) salt, that is, a divalent manganese salt, and a zinc (II) salt, that is, a divalent zinc salt.

本发明涉及使用纤维氧化酶来处理制浆造纸的纤维原料，以改变纤维表面的化学性质，提高纤维的反应活性，增加纤维之间的结合力，从而提高纸张的强度指标，增加脱水烘干效率，降低能耗，为造纸企业节约生产成本，提高经济利润。The invention relates to the use of fiber oxidase to treat fiber raw materials for pulping and papermaking, to change the chemical properties of the fiber surface, to improve the reactivity of the fiber, increase the bonding force between the fibers, thereby improving the strength index of the paper and increasing the dewatering and drying efficiency. Reduce energy consumption, save production costs for paper companies, and increase economic profits.

本发明所涉及的纤维素氧化酶可以是以其单个酶组分的产品分别加入造纸过程中，以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。The cellulose oxidase involved in the present invention may be separately added to the papermaking process as a product of its individual enzyme components, so that the two components can be separately added to different positions or mixed into one product formula to be added to the agreed position in the papermaking process. To achieve the best results.

本发明所涉及的使用纤维氧化酶的目的和效果是通过氧化酶对纤维表面的晶型区域反应，产生表面反应活性很高的键位，使纤维之间的结合力增加。而且纤维氧化酶对纤维的反应可以通过控制在纤维表面，不会对纤维有过度的反应，从而达到选择性的纤维表面改性和修复。因此，与传统的纤维素水解酶相比，从而避免了传统纤维素水解酶的降解纤维的问题。The object and effect of using the fiber oxidase according to the present invention is to cause a bond having a high surface reactivity by an oxidase to react with a crystal region on the surface of the fiber to increase the binding force between the fibers. Moreover, the reaction of fiber oxidase on the fiber can be controlled on the surface of the fiber without excessive reaction to the fiber, thereby achieving selective fiber surface modification and repair. Therefore, the problem of degrading fibers of the conventional cellulolytic enzyme is avoided as compared with the conventional cellulolytic enzyme.

本发明所述单位“kg/T”是指每吨绝干浆中添加物料的千克数。The unit "kg/T" in the present invention refers to the number of kilograms of material added per ton of dry pulp.

以下将结合具体实施例对本发明做进一步说明。The invention will be further described below in conjunction with specific embodiments.

实施例1纤维氧化酶和氧化酶组合处理机械阔叶浆对其纸张强度的影响Example 1 Combination of Cellulose Oxidase and Oxidase to Treat the Effect of Mechanical Hardwood Pulp on Paper Strength

一、实验目的First, the purpose of the experiment

通过对比分析评价纤维氧化酶组合物处理机械阔叶浆对其纸张强度的影响。The effect of the fiber oxidase composition on the mechanical strength of the mechanical broadleaf pulp was evaluated by comparative analysis.

二、实验方法Second, the experimental method

(1)试验材料准备(1) Preparation of test materials

本实验所述机械阔叶浆由山东万国纸业26号机的纸浆厂提供。The mechanical broadleaf pulp described in this experiment was provided by the pulp mill of Shandong Wanguo Paper No. 26 machine.

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。Fibrin oxidase (PMO) 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.

漆酶和纤维二糖脱氢酶为DuPont集团Genencor公司的商业产品。 Laccase and cellobiose dehydrogenase are commercial products of the DuPont Group Genencor.

(2)造纸方法(2) Paper making method

A、制浆：将200克浆板和1300克去离子水放置在10升的水力碎浆机中，碎浆10分钟，然后再加2500克水，配得得到5％的浆。A. Pulping: 200 g of pulp plate and 1300 g of deionized water were placed in a 10 liter hydropulper, slurried for 10 minutes, and then 2500 g of water was added to obtain a 5% slurry.

B：反应：B: Reaction:

(1)取上述配好的浆400克，放入1000ml混合搅拌器中，用恒温控制器控制浆的温度在50℃。(1) Take 400 g of the above prepared slurry, put it into a 1000 ml mixing stirrer, and control the temperature of the slurry at 50 ° C with a thermostatic controller.

(2)本实验分为实验组和对照组；(2) This experiment is divided into experimental group and control group;

对照组1为空白样品：不加任何药剂；Control group 1 was a blank sample: no drug was added;

对照组1为：100g/T、400g/T漆酶处理；Control group 1 was: 100g/T, 400g/T laccase treatment;

对照组2为：100g/T、250g/T纤维二糖脱氢酶处理；Control group 2 was: 100 g/T, 250 g/T cellobiose dehydrogenase treatment;

实验组为经酶处理的样品，其中，The experimental group is an enzyme-treated sample, wherein

实验组1为：250g/T(绝干浆)纤维氧化酶(PMO)处理；Experimental group 1 was: 250 g/T (absolute dry pulp) fiber oxidase (PMO) treatment;

实验组2为纤维氧化酶+漆酶组合物处理：100g/T，250g/T纤维氧化酶(PMO)，加上的漆酶一起处理，漆酶用量为400g/T；Experimental group 2 is treated with a fiber oxidase + laccase composition: 100 g / T, 250 g / T fiber oxidase (PMO), plus laccase treatment together, the amount of laccase is 400 g / T;

实验组3为纤维氧化酶+纤维二糖脱氢酶组合物处理：250g/T纤维氧化酶(PMO)，加上100g/T纤维二糖脱氢酶一起处理；Experimental group 3 was treated with a fiber oxidase + cellobiose dehydrogenase composition: 250 g/T cell oxidase (PMO), plus 100 g/T cellobiose dehydrogenase;

实验组4为纤维氧化酶+漆酶+纤维二糖脱氢酶组合物处理：100g/T纤维氧化酶+200g/T漆酶+100g/T纤维二糖脱氢酶一起处理；Experimental group 4 was treated with a fiber oxidase + laccase + cellobiose dehydrogenase composition: 100 g / T fiber oxidase + 200 g / T laccase + 100 g / T cellobiose dehydrogenase;

上述各组的反应条件均为：反应温度为50℃，反应时间为150分钟，反应pH为5.7-6.0，恒速搅拌。The reaction conditions of the above respective groups were as follows: a reaction temperature of 50 ° C, a reaction time of 150 minutes, a reaction pH of 5.7-6.0, and constant stirring.

C、手抄纸准备和强度测量：将上述制备好的浆，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测量方法测量其厚度(密度)、抗张强度、撕裂强度和结合强度。C. Handsheet preparation and strength measurement: Dilute the prepared slurry, 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. Hand paper. After drying, the handsheets were placed in a constant temperature and constant humidity control box for 24 hours, and then their thickness (density), tensile strength, tear strength, and bond strength were measured according to the TAPPI measurement method.

三、实验结果Third, the experimental results

实验结果参见表1，从表1中可知：纤维氧化酶单独处理纸浆和与其他氧化酶(漆酶和/或纤维二糖脱氢酶)联合使用时对纸张的各种强度性质的影响结果，如：叩解度、抗张指 数、撕裂度和层间结合力。与空白条件相比，加入单独加入漆酶或者纤维二糖脱氢酶处理机械阔叶浆，对纸张的各种强度性质的影响不显著。但是，当漆酶或者纤维二糖脱氢酶与纤维氧化酶联合使用时，纸张各种强度都显著增加；特别是当纤维氧化酶、漆酶和纤维二糖脱氢酶三者一起使用时，纸张的抗张强度和撕裂度提高了40％和60％。这些结果表明，纤维氧化酶和漆酶及纤维二糖脱氢酶，纤维氧化酶集中在晶型纤维表面的反应，激活该部分纤维的表面活性，增加纤维反应活性，并且提高纤维间的结合力，进而显著提高了机械阔叶浆制得的纸张的各种物理强度。The experimental results are shown in Table 1. From Table 1, it can be seen that the effect of the fiber oxidase alone on the pulp and the various strength properties of the paper when combined with other oxidases (laccase and/or cellobiose dehydrogenase), Such as: the degree of resolution, anti-tension Number, tear and bond between layers. Compared with the blank conditions, the addition of laccase or cellobiose dehydrogenase alone to treat mechanical broadleaf pulp had no significant effect on the various strength properties of the paper. However, when laccase or cellobiose dehydrogenase is used in combination with cell oxidase, the various strengths of the paper are significantly increased; especially when fiber oxidase, laccase and cellobiose dehydrogenase are used together, The tensile strength and tear of the paper increased by 40% and 60%. These results indicate that fiber oxidase and laccase and cellobiose dehydrogenase, fiber oxidase concentrate on the surface of the crystal fiber, activate the surface activity of the fiber, increase fiber reactivity, and improve the adhesion between fibers. , in turn, significantly increases the physical strength of paper made from mechanical broadleaf pulp.

表1实验组和对照组处理机械阔叶浆对纸张物理指标的影响结果表Table 1 Effect of mechanical broadleaf pulp on paper physical indicators in experimental group and control group

PMO用量PMO dosage	漆酶用量Laccase dosage	纤维二糖脱氢酶Cellobiose dehydrogenase	叩解度Degree of resolution	抗张指数Tensile index	撕裂度Tear degree	层间结合力Interlayer bonding
							g/Tg/T	g/Tg/T	g/Tg/T	°SR°SR	Nm/gNm/g	mN*m/gmN*m/g	KpaKpa
空白blank	00	00	42.542.5	18.918.9	6.556.55	210.8210.8
							00	00	100100	41.041.0	19.219.2	6.376.37	220.1220.1
250250	00	00	43.043.0	21.421.4	7.707.70	249.5249.5
							00	100100	00	42.042.0	19.719.7	6.706.70	223.7223.7
00	00	250250	42.542.5	20.420.4	6.706.70	227.5227.5
							00	400400	00	42.542.5	21.121.1	7.007.00	247.6247.6
100100	400400	00	43.843.8	21.921.9	8.128.12	280.6280.6
							250250	400400	00	45.045.0	22.722.7	8.458.45	270.7270.7
250250	00	100100	43.543.5	24.124.1	8.648.64	290.0290.0
							100100	200200	100100	46.046.0	26.626.6	10.7610.76	358.6358.6

实施例2纤维氧化酶和漆酶组合处理回收OCC浆对其纸张强度的影响Example 2 The effect of fiber oxidase and laccase treatment on the strength of paper by OCC pulp

一、实验目的First, the purpose of the experiment

通过对比分析评价纤维氧化酶组合物处理OCC浆对其纸张强度的影响。The effect of the fiber oxidase composition on the strength of the paper by the ODC pulp was evaluated by comparative analysis.

二、实验方法Second, the experimental method

(1)试验材料准备(1) Preparation of test materials

回收OCC废纸浆从广东明天纸业多盘浓缩机良浆池取得，浆浓为4.8-5％；The recovered OCC waste paper pulp was obtained from the Guangdong Tobacco Paper Multi-plate Concentrate Pulp Pool, and the pulp concentration was 4.8-5%;

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；Fibrin oxidase (PMO) is supplied by Dyadic, USA, GH61 from the strain Myceliophthora Prepared by thermophila, the amino acid sequence of which is described in detail in U.S. Patent No. 8,298,795 and U.S. Patent Application Serial No. 2012/0083019;

漆酶为DuPont集团下属子公司Genencor公司的商业产品。Laccase is a commercial product of Genencor, a subsidiary of the DuPont Group.

(2)造纸方法(2) Paper making method

A、反应：A, reaction:

(1)取上述配好的浆400克，放入1000ml混合搅拌器中，用恒温控制器控制浆的温度在50℃。(1) Take 400 g of the above prepared slurry, put it into a 1000 ml mixing stirrer, and control the temperature of the slurry at 50 ° C with a thermostatic controller.

本实验分为实验组和对照组；This experiment is divided into experimental group and control group;

所述对照组为空白样品：不加任何药剂；The control group is a blank sample: no drug is added;

实验组为经酶或酶和清除剂处理的样品，其中，The experimental group is a sample treated with an enzyme or an enzyme and a scavenger, wherein

实验组1为：纤维氧化酶(PMO)250g/T+硫酸铁用量为500g/T；Experimental group 1 is: fiber oxidase (PMO) 250g / T + iron sulfate dosage is 500g / T;

实验组2为：漆酶250g/T+硫酸铁用量为500g/T；Experimental group 2 is: laccase 250g / T + iron sulfate dosage is 500g / T;

实验组3为：纤维氧化酶250g/T+漆酶250g/T+硫酸铁用量为500g/T；Experimental group 3 is: fiber oxidase 250g / T + laccase 250g / T + iron sulfate dosage is 500g / T;

上述各组反应的条件均为：温度为50℃，时间为90分钟，pH为5.7-6.0，恒速搅拌。The conditions of the above respective groups of reactions were as follows: temperature was 50 ° C, time was 90 minutes, pH was 5.7-6.0, and stirring was continued at a constant rate.

B、手抄纸准备和强度测量：B. Handsheet preparation and strength measurement:

将上述制备好的浆，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测量方法测量其厚度(密度)、抗张强度、撕裂强度和结合强度。The prepared slurry was diluted with tap water to 1.0% strength, mixed, and the temperature and slurry concentration were measured. According to the TAPPI method, 10-12 sheets of 6.5 g weight handsheets were accurately prepared. After drying, the handsheets were placed in a constant temperature and constant humidity control box for 24 hours, and then their thickness (density), tensile strength, tear strength, and bond strength were measured according to the TAPPI measurement method.

三、实验结果Third, the experimental results

结果参见表2，从表2可知：纤维氧化酶、漆酶和硫酸铁单独或者联合使用处理OCC废纸纸浆对纸张的各种强度性质的影响结果。与空白条件相比，单独加入纤维氧化酶处理后，短纤浆或长纤浆所制得的纸张的耐破度显著增加；单独加入漆酶处理后，短纤浆或长纤浆所制得的纸张的耐破度略有增加，但不如纤维氧化酶显著；单独加入硫酸铁处理后，短纤浆或长纤浆所制得的纸张的耐破度基本没发生变化；仅当纤维氧化酶、漆酶和硫酸铁联合使用时，效果最显著，无论长纤维浆或是短纤维浆，其纸张的耐破度增加了25-30％。The results are shown in Table 2. From Table 2, it is known that fiber oxidase, laccase and ferric sulfate are used alone or in combination to treat the effects of OCC waste paper pulp on various strength properties of paper. Compared with the blank conditions, after the fiber oxidase treatment alone, the burst strength of the paper made from the fluff pulp or the fluff pulp is significantly increased; after the laccase treatment alone, the fluff pulp or the fluff pulp is prepared. The paper has a slightly increased bursting resistance, but not as significant as the fiber oxidase; after the addition of ferric sulfate alone, the bursting resistance of the paper made from the fluff pulp or the fluff pulp is almost unchanged; only when the fiber oxidase When combined with laccase and ferric sulfate, the effect is most pronounced. Regardless of long fiber pulp or short fiber pulp, the paper's bursting strength is increased by 25-30%.

表2实验组和对照组处理回收OCC废纸浆对纸张物理指标的影响结果表 Table 2 Effect of the recovery of OCC waste paper pulp on the physical indicators of paper in the experimental group and the control group

浆料Slurry	PMOPMO	漆酶Laccase	硫酸铁Ferric sulfate	温度temperature	反应时间Reaction time	游离度Freeness	耐破度Burst resistance
								用量Dosage	g/Tg/T	g/Tg/T	g/Tg/T	℃°C	MinMin	CSF(ml)CSF (ml)	kPa.m2/gkPa.m2/g
	短纤浆Fleece pulp	00	00	00	4040	9090	265265	1.451.45
短纤浆 Fleece pulp									00	00	500500	4040	9090	290290	1.431.43
	短纤浆Fleece pulp	250250	00	500500	4040	9090	280280	1.761.76
短纤浆 Fleece pulp									00	250250	500500	4040	9090	270270	1.541.54
	短纤浆Fleece pulp	250250	250250	500500	4040	9090	255255	1.861.86
长纤浆 Fibrous pulp									00	00	00	6060	120120	330330	1.621.62
	长纤浆 Fibrous pulp	00	00	500500	6060	120120	360360	1.491.49
长纤浆Fibrous pulp									250250	00	500500	6060	120120	320320	1.881.88
	长纤浆 Fibrous pulp	00	250250	500500	6060	120120	350350	1.751.75
长纤浆Fibrous pulp									250250	250250	500500	6060	120120	345345	2.042.04

实施例3纤维氧化酶及其不同组合物处理木片对磨浆能耗的影响Example 3 Effect of fiber oxidase and its different compositions on the energy consumption of refining wood chips

一、实验目的First, the purpose of the experiment

通过对比分析评价纤维氧化酶及其不同组合物处理木片对磨浆能耗的影响。The effects of fiber oxidase and its different compositions on the energy consumption of refining were evaluated by comparative analysis.

二、实验方法Second, the experimental method

(1)试验材料(1) Test materials

纤维氧化酶(PMO)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。Cellulose oxidase (PMO) is prepared from the strain Myceliophthora thermophila, the amino acid sequence of which is described in detail in U.S. Patent No. 8,298,795 and U.S. Patent Application Serial No. 2012/0083019.

纤维素水解酶为Dyadic公司的商业产品FiberZyme G200。The cellulolytic enzyme is the commercial product FiberZyme G200 from Dyadic.

(2)制浆木片制备：某纸厂使用南方松木制造TMP，木头在去树皮会，经过切片机切成木片，在进入木片储仓或堆放场。(2) Preparation of pulp wood chips: A paper mill uses southern pine wood to make TMP. When the wood is removed from the bark, it is cut into wood chips by a slicer and enters the wood chip storage bin or stacking yard.

(3)生物酶处理：在木片进入储仓之前的皮带卸料口装置生物酶喷淋到木片上，然后放在2-3天(流程图参见图1)；所述生物酶为纤维水解酶(酶A)、纤维氧化酶(酶B)或其组合(酶A+酶B＝1∶2)，用量为200-400g/T(绝干浆)。(3) Biological enzyme treatment: the biological enzyme is sprayed onto the wood chip before the wood chip enters the storage bin, and then placed on the wood chip, and then placed for 2-3 days (see Figure 1 for the flow chart); the biological enzyme is a fiber hydrolase (Enzyme A), cell oxidase (Enzyme B) or a combination thereof (Enzyme A + Enzyme B = 1:2) in an amount of 200-400 g/T (absolute dry pulp).

(4)制浆流程/工艺：以上准备好的木片，经过木片清洗除砂之后，进入木片蒸汽螺旋压榨机，随即进入一级盘磨，之后进入一级磨后疏解池，经粗筛和双网压滤压榨之后再进入二次磨浆机，磨后浆经过精筛之后进入多盘浓缩机，最后进入TMP储浆塔。(4) Pulping process/process: The wood chips prepared above are cleaned and removed by wood chips, then enter the wood chip steam screw press, and then enter the first-stage disc grinding, and then enter the first-stage grinding and decontamination pool, after coarse screening and double After the net press filter presses, it enters the secondary refiner. After the grind is passed through the fine screen, it enters the multi-plate concentrator and finally enters the TMP storage tower.

在制浆过程中，对总磨浆能耗、TMP浆的游离度、TMP浆的强度指标进行抄片测试 跟踪。In the pulping process, the total refining energy consumption, the freeness of TMP pulp, and the strength index of TMP pulp are tested. track.

三、实验结果Third, the experimental results

结果参见图2，从图2可知：木片经过纤维水解酶(酶A)处理后，其磨浆性能变化不大；经过纤维氧化酶(酶B)处理之后，磨浆性能有明显改善，磨浆能耗降低；而最佳的效果是使用纤维氧化酶和纤维素水解酶同时使用，与空白相比，达到同样的游离度的所需能耗降低100KWH/T以上。The results are shown in Fig. 2. It can be seen from Fig. 2 that the refining performance of the wood chips after the treatment with the fiber hydrolase (enzyme A) has little change; after the treatment with the fiber oxidase (enzyme B), the refining performance is obviously improved, and the refining is improved. The energy consumption is reduced; and the best effect is to use both fiber oxidase and cellulolytic enzyme, and the energy consumption required to achieve the same freeness is reduced by more than 100 KWH/T compared with the blank.

实施例4纤维氧化酶及其不同组合物处理木片对磨浆能耗的影响Example 4 Effect of fiber oxidase and its different compositions on the energy consumption of refining wood chips

一、实验目的First, the purpose of the experiment

通过对比分析评价纤维氧化酶及其不同组合物处理木片对磨浆能耗的影响。The effects of fiber oxidase and its different compositions on the energy consumption of refining were evaluated by comparative analysis.

二、实验方法Second, the experimental method

(1)试验材料(1) Test materials

纤维氧化酶(PMO)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。Cellulose oxidase (PMO) is prepared from the strain Myceliophthora thermophila, the amino acid sequence of which is described in detail in U.S. Patent No. 8,298,795 and U.S. Patent Application Serial No. 2012/0083019.

纤维素水解酶为Dyadic公司的商业产品FiberZyme G200。The cellulolytic enzyme is the commercial product FiberZyme G200 from Dyadic.

(2)制浆流程/工艺(2) Pulping process / process

木片经过清洗除砂之后，进入木片蒸汽螺旋压榨机，随即进入一级盘磨，磨后进入一级磨后疏解池，疏解浆经粗筛和双网压滤压榨之后再进入二次磨浆机，磨后浆经过精筛之后进入多盘浓缩机，最后进入TMP储浆塔。After the wood chips are cleaned and sanded, they enter the wood chip steam screw press, and then enter the first-stage disc grinding. After grinding, they enter the first-stage grinding and decontamination pool. After the coarse slurry and the double mesh filter press are squeezed, they enter the secondary refiner. After grinding, the slurry enters the multi-plate concentrator after fine screening, and finally enters the TMP storage tower.

(3)生物酶处理：生物酶加入到一级磨浆的疏解池，其浆浓大约3.5％，温度75-80℃，停留时间45-60分钟；纤维氧化酶组合物(纤维氧化酶和纤维素酶1∶3)用量为0.30-0.6kg/T。(3) Biological enzyme treatment: the biological enzyme is added to the first-stage refining decontamination tank, the pulp concentration is about 3.5%, the temperature is 75-80 ° C, the residence time is 45-60 minutes; the fiber oxidase composition (fibrous oxidase and fiber) The amount of the enzyme 1:3) is 0.30-0.6 kg/T.

三、实验结果Third, the experimental results

实验结果参见图3，从图3可知：加入生物酶时总磨浆能耗的变化情况；具体为：与加入之前和停用之后相比，纤维氧化酶和纤维素酶联合使用，总磨浆能耗下降了200-300KWH/T。The experimental results are shown in Fig. 3. From Fig. 3, the change of the total refining energy consumption when the biological enzyme is added; specifically, the fiber oxidase and the cellulase are used in combination with the total refining before and after the deactivation. Energy consumption has dropped by 200-300KWH/T.

实施例5纤维氧化酶组合物处理ONP纸浆Example 5 Treatment of ONP Pulp by Cellulose Oxidase Composition

一、实验目的 First, the purpose of the experiment

通过对比分析评价纤维氧化酶组合物对纸张白度的影响。The effect of the fiber oxidase composition on the whiteness of the paper was evaluated by comparative analysis.

二、实验方法Second, the experimental method

(1)试验材料准备(1) Preparation of test materials

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。Recycled old newspaper (ONP) and old magazine (OMG) waste paper are mixed at a ratio of 80% to 20%. 200 grams of mixed waste paper and 1300 grams of white water (added CaCl _{2 to} adjust the hardness to 400PPM; 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.

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。Fibrin oxidase (PMO) 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.

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。Glucose Oxidase (GOX) is a commercial product of Genencor, a subsidiary of the DuPont Group.

(2)造纸(2) Papermaking

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℃；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;

B、设置实验组和对照组，B. Set the experimental group and the control group.

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；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;

实验组为采用纤维氧化酶组合物处理，其中，The experimental group is treated with a fiber oxidase composition, wherein

实验组1为：在浆料中加入纤维氧化酶(PMO)150-300g/T，脱墨剂0.5kg/T，加入烧碱2kg/T，水玻璃15kg/T，过氧化氢10kg/T；Experimental group 1 is: adding fiber oxidase (PMO) 150-300g / T, deinking agent 0.5kg / T, adding caustic soda 2kg / T, water glass 15kg / T, hydrogen peroxide 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；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;

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；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;

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。The above 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.

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。C. Deinking test: Add the above slurry to a 3 liter Denver flotation machine and add white water (50 ° C, 400 PPM). Hardness) dilute the slurry to about 1%, stir and mix for 3 minutes, then open the compressed air valve and adjust the flow rate to form a uniform fine bubble and a stable foam layer, scrape the foam, and float for 4 minutes to obtain deinked pulp. .

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。D. 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.

三、实验结果Third, the experimental results

结果参见表3，从表3中可看出：常规脱墨化学品(双.氧水，水玻璃和烧碱)、PMO和GOX单独或者联合使用处理废纸纸浆对脱墨的影响。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。The results are shown in Table 3. As can be seen from Table 3, the effects of conventional deinking chemicals (double oxygen water, water glass and caustic soda), PMO and GOX alone or in combination on the treatment of waste paper pulp on deinking. Under the condition of 80% reduction of caustic soda consumption, 50% reduction of water glass and 50% reduction of hydrogen peroxide, after treatment with fiber oxidase, the whiteness of paper increased significantly before flotation, and the whiteness of the contrast condition increased by 2- 4% ISO; after flotation, the whiteness increase is more significant, which is 3-4% higher than the contrast condition. When added by GOX alone, the whiteness of the paper increased slightly compared to the contrast conditions, but it was not significant. When fiber oxidase and glucose oxidase are used in combination, the effect is most remarkable, and the whiteness of the paper reaches 54% ISO or more.

表3实验组和对照组处理回收废纸浆对纸张物理指标的影响Table 3 Effect of treatment of recycled pulp on the physical parameters of paper in experimental group and control group

实施例6纤维氧化酶对非木浆(烟梗)磨浆能耗和强度的影响 Example 6 Effect of cell oxidase on energy consumption and strength of non-wood pulp (tobacco stem) refining

一、实验目的First, the purpose of the experiment

研究纤维氧化酶对非木浆(烟梗)磨浆能耗和强度的影响。The effect of fiber oxidase on the energy consumption and strength of non-wood pulp (tobacco) refining was studied.

二、实验方法Second, the experimental method

试验材料：纤维氧化酶(PMO)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。Test material: Fibrous oxidase (PMO) was 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.

浆料：取自某再生烟叶生产公司的2号机叩前池的烟叶/烟梗混合浆料，并根据其烟梗浆料生产工艺和流程条件进行模拟实验。即在2号机的烟梗制浆的一次萃取和挤压之后，将纤维氧化酶组合物入到二次萃取的反应釜，萃取20分钟，然后在经过挤压，去除萃取液，之后再对浆料进行磨浆，然后测试浆料的各种物理性能。Slurry: The tobacco/tobacco mixed slurry from the front tank of No. 2 machine of a regenerated tobacco leaf production company, and the simulation experiment was carried out according to the production process and process conditions of the tobacco stem slurry. That is, after one extraction and extrusion of the tobacco stem pulping of the No. 2 machine, the fiber oxidase composition is introduced into the secondary extraction reactor, extracted for 20 minutes, and then squeezed to remove the extract, and then The slurry is refined and then tested for various physical properties of the slurry.

具体步骤和条件如下：The specific steps and conditions are as follows:

许多生产采用“浸泡式”施胶或涂布技术，在纸张/薄片烘干之后再将涂布液(或者表胶液)涂上纸张的表面。纸张的湿强对纸机的稳定运行，特别是提高纸机车速，非常重要。因此，生物酶处理对薄片的湿强的影响进行了测试。测试方法如下：Many production uses "soaking" sizing or coating techniques to apply a coating solution (or surface glue) to the surface of the paper after the paper/sheet has been dried. The wet strength of the paper is very important for the stable operation of the paper machine, especially for increasing the speed of the paper machine. Therefore, the effect of the biological enzyme treatment on the wet strength of the sheet was tested. The test method is as follows:

(1)样品制取与干强(即抗张强度)的测试样品相同；(1) The sample preparation is the same as the test sample of dry strength (ie tensile strength);

(2)将样品放置在干净的吸水纸表面，然后用毛细液滴管取去离子水，将一滴水轻轻滴在样品纸条上，计时3秒，迅速用吸水纸将样品的水滴吸干； (2) Place the sample on the surface of a clean absorbent paper, then take the deionized water with a capillary tube, and gently drop a drop of water on the sample strip for 3 seconds. Quickly blot the sample with water. ;

(3)测试拉力。(3) Test the tension.

三、实验结果Third, the experimental results

(1)纤维氧化酶处理非木浆对磨浆和纸张物理指标的影响结果(1) Effect of fiber oxidase treatment on non-wood pulp on refining and paper physical indexes

结果参见表4，从表4可知：在空白(即未添加生物酶)条件下，随着磨浆转数(即能耗)的增加，浆料的叩解度迅速增加，薄片的抗张强度也增加，但是浆料的湿重明显下降，表明磨浆对纤维有破坏，细小纤维增加，将导致细小纤维的流失。因此增加磨浆对浆料在抄纸的保留率不利。The results are shown in Table 4. It can be seen from Table 4 that under the condition of blank (ie no biological enzyme added), the degree of decomposing of the slurry increases rapidly with the increase of the number of refining revolutions (ie, energy consumption), and the tensile strength of the flakes is also increased. Increased, but the wet weight of the slurry decreased significantly, indicating that the refining has damage to the fibers, and the increase in fine fibers will result in the loss of fine fibers. Therefore, increasing the refining is unfavorable for the retention rate of the slurry in the papermaking.

与空白条件相比，添加纤维氧化酶之后，磨浆之后浆料的叩解度和抗张强度随酶产品的用量的增加而增加，例如，在磨浆转数为250转时，生物酶的用量2kg/T时，薄片的抗张指数比空白在250转时增加了将近100％，与空白磨浆1500转的抗张指数相近；但是，生物酶处理后浆料的湿重没有明显变化，而空白条件时在磨浆转数达到1500转时，湿重下降到30％，这说明新型生物酶对纤维没有任何破坏。以上结果表明，使用纤维氧化酶可以通过优化用量和处理时间来增加薄片的强度，同时保持浆料较高的滤水性能。Compared with the blank condition, after adding the fiber oxidase, the degree of decomposability and tensile strength of the slurry after refining increase with the amount of the enzyme product, for example, the amount of the enzyme used when the refining number is 250 rpm. At 2kg/T, the tensile index of the flakes increased by nearly 100% compared with the blank at 250 rpm, which is similar to the tensile index of 1500 rpm of the blank refining; however, the wet weight of the slurry after bio-enzyme treatment did not change significantly, In the blank condition, when the refining revolution reaches 1500 rpm, the wet weight drops to 30%, which indicates that the new biological enzyme does not cause any damage to the fiber. The above results indicate that the use of fiber oxidase can increase the strength of the flakes by optimizing the amount and treatment time while maintaining the higher drainage performance of the slurry.

表4纤维氧化酶处理非木浆对磨浆和纸张物理指标的影响Table 4 Effect of fiber oxidase treatment on non-wood pulp on refining and paper physical indexes

(2)纤维氧化酶处理非木浆对纸张湿强的影响(2) Effect of fiber oxidase treatment on non-wood pulp on paper wet strength

试验结果如表5和图4所示。从表5可见，经纤维氧化酶处理之后，纸张的湿强度显 著增加。从图4可见，在再生烟叶薄片抄纸过程中添加本发明的纤维氧化酶之后纸张的抗张指数的变化趋势。可见，纤维氧化酶体显著提高薄片的强度，有利于纸机生产。The test results are shown in Table 5 and Figure 4. As can be seen from Table 5, the wet strength of the paper after treatment with cell oxidase Increase. As can be seen from Fig. 4, the tendency of the tensile index of the paper after the addition of the cell oxidase of the present invention in the process of papermaking of the reconstituted tobacco leaf sheet was observed. It can be seen that the fiber oxidase body significantly increases the strength of the sheet, which is beneficial to the production of paper machines.

表5纤维氧化酶处理非木浆对纸张湿强的影响Table 5 Effect of fiber oxidase treatment on non-wood pulp on paper wet strength

实施例7纤维氧化酶对非木浆(秸秆)制浆的影响Example 7 Effect of Cellulose Oxidase on Non-wood Pulp (Straw) Pulping

一、实验目的First, the purpose of the experiment

研究纤维氧化酶对非木浆(秸秆)制浆的影响。The effect of fiber oxidase on non-wood pulp (stalk) pulping was studied.

二、实验方法Second, the experimental method

试验材料：纤维氧化酶(PMO)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。Test material: Fibrous oxidase (PMO) was 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.

浆料：取自秸秆制浆生产厂的浆料，具体步骤和条件如下：称取20个绝干浆的浆料，放置1000ml的烧杯中，然后将烧杯放置在温度设置为60℃的恒温水浴中，使之达到平衡。之后，根据所需的生物酶处理条件，加入一定量的纤维氧化酶组合物，反应60分钟，并不断搅拌，使反应均衡。之后，取样测试浆料的叩解度。Slurry: taken from the slurry of the straw pulping production plant. The specific steps and conditions are as follows: Weigh 20 pieces of dry slurry, place it in a 1000ml beaker, and then place the beaker in a constant temperature water bath set at 60 °C. In, to make it balanced. Thereafter, a certain amount of the fiber oxidase composition was added according to the desired biological enzyme treatment conditions, reacted for 60 minutes, and continuously stirred to equilibrate the reaction. After that, the degree of resolution of the test slurry was sampled.

三、实验结果Third, the experimental results

结果参见表6，从表6可知：生物酶处理后对叩解度和制浆得率的变化趋势。与未处理的结果相比，在生物酶用量达到较高(3kg/T)时，浆料的叩解度显著上升，增加了8度，或相当增加了50％(从16增加到24)。同样，湿重下降了20％。这说明，在高用量情况下，酶会降低浆料的滤水性能。从浆料的得率来看，在经过较低用量处理之后，其得率变化不大。但是，在高用量的情况下，浆料得率会下降。The results are shown in Table 6. From Table 6, it can be seen that the tendency of the degree of decomposing and the yield of pulping after the treatment with biological enzymes. Compared to the untreated results, when the amount of biological enzymes reached a higher level (3 kg/T), the degree of decompression of the slurry increased significantly, by 8 degrees, or by a considerable increase of 50% (from 16 to 24). Similarly, the wet weight has dropped by 20%. This shows that at high dosages, the enzyme will reduce the drainage performance of the slurry. From the viewpoint of the yield of the slurry, the yield did not change much after the treatment with a lower amount. However, in the case of high usage, the slurry yield will decrease.

表6纤维氧化酶处理秸秆浆料对制浆和得率的影响 Table 6 Effect of fiber oxidase treatment of straw slurry on pulping and yield

编号Numbering	叩解度Degree of resolution	湿重Ww	挤干浆重量Squeeze dry weight	挤干浆浓度Extruded dry pulp concentration	纤维得率Fiber yield
							(oSR)(oSR)	(g)(g)	(g)(g)	(％)(%)	(％)(%)
空白blank	1616	4.634.63	83.3483.34	20.820.8	86.786.7
						0.5kg/T0.5kg/T	16.516.5	3.983.98	78.978.9	22.522.5	88.888.8
1.0kg/T1.0kg/T	1717	3.643.64	76.976.9	22.622.6	86.986.9
						3.0kg/T3.0kg/T	24twenty four	3.623.62	75.8475.84	20.820.8	78.978.9

表7是生物酶产品处理浆料后对抄片强度的影响。很明显，随着酶用量的增加，纸张的强度随之增加。在用量为0.5kg/T时，抗张指数增加了16％；用量为1.0kg/T时，抗张指数增加了36％；而当用量增加到3.0kg/T时，抗张指数增加了近80％。Table 7 shows the effect of the bioenzyme product on the sheet strength after treating the slurry. Obviously, as the amount of enzyme increases, the strength of the paper increases. When the dosage is 0.5kg/T, the tensile index increases by 16%; when the dosage is 1.0kg/T, the tensile index increases by 36%; and when the dosage increases to 3.0kg/T, the tensile index increases nearly 80%.

表7纤维氧化酶处理秸秆浆料对纸张强度指标的影响Table 7 Effect of fiber oxidase treatment of straw slurry on paper strength index

试验编号Test number	总吸附能Total adsorption energy	抗张强度tensile strength	伸长率Elongation	抗张指数Tensile index	裂断长Long break	弹性模数Elastic modulus
							(聚能酶用量)(concentration of concentrating enzyme)	TEATEA	N/mN/m	％%	mN.m/gmN.m/g	kmKm	N/mm2N/mm2
空白blank	2.312.31	422.6422.6	0.790.79	5.715.71	0.580.58	280.9280.9
								2.532.53	566.6566.6	0.720.72	7.867.86	0.780.78	440.0440.0
	2.002.00	446.2446.2	0.620.62	6.036.03	0.610.61	341.7341.7
							平均average	2.282.28	478.5478.5	0.710.71	6.536.53	0.660.66	354.2354.2
0.5kg/T0.5kg/T	2.272.27	551.7551.7	0.650.65	7.467.46	0.760.76	515.39+515.39+
								2.162.16	568.1568.1	0.610.61	7.687.68	0.780.78	528.4528.4
平均average	2.222.22	559.9559.9	0.630.63	7.577.57	0.770.77	528.4528.4
							1.0kg/T1.0kg/T	2.692.69	654.4654.4	0.650.65	8.848.84	0.900.90	55.955.9
	2.552.55	680.6680.6	0.590.59	8.968.96	0.910.91	599.9599.9
							平均average	2.622.62	667.5667.5	0.620.62	8.908.90	0.910.91	327.9327.9
3.0kg/T3.0kg/T	3.573.57	923.2923.2	0.640.64	12.4812.48	1.271.27	783.3783.3
								3.013.01	811.5811.5	0.640.64	10.9710.97	1.121.12	772.1772.1
平均average	3.293.29	867.3867.3	0.640.64	11.7311.73	1.201.20	777.7777.7

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。 The above-mentioned embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims (7)

1. 一种用于改变和改善纤维性质的纤维氧化酶组合物，其特征是，包括以下组成：A fiber oxidase composition for modifying and improving fiber properties, characterized by comprising the following composition:

   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i) 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;

   ii)对纤维氧化酶具有增加催化活性的辅助组分，所述辅助组分为对纤维氧化酶具有促进作用的蛋白质；Ii) an auxiliary component having an increased catalytic activity on the fiber oxidase, the auxiliary component being a protein having a promoting effect on the fiber oxidase;

   所述生物酶组分和辅助组分的重量比例为1∶0.4-50。The weight ratio of the biological enzyme component to the auxiliary component is 1:0.4-50.
2. 根据权利要求1所述的用于改变和改善纤维性质的纤维氧化酶组合物，其特征是，所述纤维氧化酶为一族分子量在20-50KDa、具有铜依赖性的小分子金属酶的聚糖加氧酶；或/及The cellulase composition for modifying and improving fiber properties according to claim 1, wherein the cell oxidase is a family of glycans having a copper-dependent small molecular metalloenzyme having a molecular weight of 20 to 50 KDa. Oxygenase; or/and

   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。The protein having a promoting effect on the fiber oxidase is a catalytically active biological enzyme or a catalytically inactive cellulose binding protein.
3. 根据权利要求2所述的用于改变和改善纤维性质的纤维氧化酶组合物，其特征是，所述有催化活性的生物酶为氧化还原酶、非纤维素水解酶的水合酶或纤维素水解酶；所述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一种或多种，所述非纤维素水解酶的水合酶选自淀粉酶、木聚糖酶、果胶酶或酯酶中一种或多种。The fiber oxidase composition for modifying and improving fiber properties according to claim 2, wherein the catalytically active biological enzyme is an oxidoreductase, a non-cellulolytic enzyme hydratase or cellulose hydrolysis. An enzyme; the oxidoreductase is selected from one or more of cellobiose dehydrogenase, laccase, glucose oxidase, hydrogen peroxide dismutase, alcohol dehydrogenase or lignin peroxidase, the non- The hydratase of the cellulolytic enzyme is selected from one or more of an amylase, a xylanase, a pectinase or an esterase.
4. 根据权利要求2所述的用于改变和改善纤维性质的纤维氧化酶组合物，其特征是，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。The cellulase composition for modifying and improving fiber properties according to claim 2, wherein the catalytically inactive protein is fibronectin and/or fibroin.
5. 根据权利要求1所述的用于改变和改善纤维性质的纤维氧化酶组合物，其特征是，所述生物酶组分和辅助组分的重量比例为1∶0.4-10。The cellulase composition for modifying and improving fiber properties according to claim 1, wherein the weight ratio of the biological enzyme component to the auxiliary component is 1:0.4-10.
6. 根据权利要求1-5任一项所述的用于改变和改善纤维性质的纤维氧化酶组合物，其特征是，所述纤维氧化酶组合物还包括纤维氧化酶的反应产物的清除剂；所述纤维氧化酶和清除剂的重量比为1∶1-5。A cellulase composition for modifying and improving fiber properties according to any one of claims 1 to 5, wherein the cell oxidase composition further comprises a scavenger of a reaction product of cell oxidase; The weight ratio of the fiber oxidase to the scavenger is 1:1 to 5.
7. 根据权利要求6所述的用于改变和改善纤维性质的纤维氧化酶组合物，其特征是，所述清除剂选自抗坏血酸、没食子酸、木质素、锰(II)盐、铜(II)盐或铁(II)盐中的一种或多种。 The cellulase composition for modifying and improving fiber properties according to claim 6, wherein the scavenger is selected from the group consisting of ascorbic acid, gallic acid, lignin, manganese (II) salt, copper (II) salt. Or one or more of iron (II) salts.

Images