碱性木聚糖酶研究进展*

木聚糖是一种在自然界中含量仅次于纤维素的丰富的可再生资源,木聚糖酶是一类可以将木聚糖水解成单糖和寡糖的酶,利用木聚糖酶将木聚糖分解后的产物被广泛应用于食品、造纸以及纺织等行业。木聚糖酶按其对酸碱环境的耐受能力分为碱性木聚糖酶、中性木聚糖酶和酸性木聚糖酶,其中碱性木聚糖酶适合应用于造纸工业中,尤其在造纸的制浆、促进漂白及废纸脱墨等多种工艺中,可以显著提高纸张质量,有效降低氯气排放量,从而减少对环境的污染。随着生物技术的进步,利用基因工程技术可以对碱性木聚糖酶进行分子改造,以提高其耐碱、耐热能力,扩大其在工业应用中的条件范围。介绍碱性木聚糖酶在分子改造方面的研究进展以及其在造纸漂白和制浆、废纸脱墨中的应用。     

制浆造纸生物技术研究进展

制浆造纸工业是国民经济的主要支柱之一,但也是资源消耗和环境污染的大户。近年来,国外用于制浆造纸工业的生物技术研究异常活跃,除废水生物处理外,木聚糖酶助漂、脂肪酶控制树脂、木片真菌预处理和酶法废纸脱墨等工艺已经在生产中得到实际应用,生物制浆、漆酶漂白工艺也已进入中试阶段。结合以草浆为主的特点,我国的制浆造纸生物技术研究也已日趋活跃起来。     

嗜热和嗜碱木聚糖酶研究进展

木聚糖酶是降解半纤维素主要成分木聚糖的关键酶,广泛应用在食品、饲料、制浆造纸、生物脱胶等行业。特别是在造纸工业中,木聚糖酶显示出巨大的应用潜力,已成为国内外研究的热点。纸浆漂白工艺中需要酶在高温碱性条件下发挥作用。目前,主要通过筛选野生型木聚糖酶资源和对现有中性中温木聚糖酶分子改造的方法获得嗜热碱木聚糖酶。文中就嗜热嗜碱木聚糖酶的筛选、嗜热嗜碱机制研究及分子改造进展进行了综述,并对其前景进行了展望。     

常压室温等离子体快速诱变绿色糖单孢菌筛选 木聚糖酶高产菌株及其酶学性质研究

[目的]利用常压室温等离子体快速诱变绿色糖单孢菌,筛选耐热耐碱木聚糖酶高产菌株,并对其进行酶学性质分析,确保其适用于生物制浆漂白工艺.[方法]采用刚果红平板水解圈法结合摇瓶发酵胞外酶测定法进行菌株筛选,并通过DNS木聚糖酶活性测定等方法对来源于不同突变株的木聚糖酶进行酶学性质分析对比.[结果]筛选出遗传稳定性良好的两株木聚糖酶高产菌株AT24和AT22-2,以麦草浆为诱导底物的粗酶液中,突变株AT24及AT22-2所产的木聚糖酶活性分别为512.74、552.70U/mL,分别为原始菌株S.v的16和17倍的.来源于突变株AT22-2的木聚糖酶的最适反应pH为9.5,最适反应温度为90℃,在50℃-90℃温度范围内具有良好的热稳定性,在100℃条件下处理30 min剩余酶活仍为68％;突变株AT24所产木聚糖酶的最适反应温度为60℃,最适pH为10.0,在60℃-80℃的高温环境下,突变株AT24所产的木聚糖酶具有良好的热稳定性.[结论]突变株AT22-2所产具有耐碱耐高温性质的木聚糖酶,在应用领域尤其在纸浆造纸行业具有较大的潜在应用价值.     

产耐热木聚糖酶细菌的分离鉴定及酶易错PCR致突变条件优化

从福建省永泰县温泉采集样品中筛选到1株产耐热木聚糖酶嗜热菌株TC-W7,并获得该木聚糖酶基因。在此基础上,采用易错PCR技术在木聚糖酶基因中引入突变,研究Mg2＋浓度、Mn2＋浓度、dTTP/dCTP浓度等条件对突变率的影响。通过形态特征、生理生化试验及16S rRNA序列相似性比对分析,初步鉴定菌株TC-W7为土壤芽胞杆菌（Geobacillus）,菌株TC-W7在最适温度75℃和 pH 8.2条件下,其木聚糖酶活力为215.83 U/mL,Triton X-100和DDT能显著增强该酶的活性。在 Mg2＋浓度为20μmol/L,Mn2＋浓度为0.80μmol/L,dTTP/dCTP浓度为0.30 mmol/L的致突变条件下,碱基突变率为0.98%。 Geobacillus sp. TC-W7产木聚糖酶具有较好的耐热和耐碱等工业应用特性,对该酶易错PCR致突变条件优化结果,可用于后续木聚糖酶的耐热定向进化。     

耐碱性木聚糖酶

造纸工业是我国国民经济中具有可持续发展特点的重要产业,也是我国重要的工业污染源之一.近年来,世界范围内的造纸工业都受到资源、环境和效益等多方面的约束,都力求寻找能够达到节能减耗、保护生态环境、提高生产效率和经济效益及产品质量的先进生产技术.木聚糖酶及其潜在的工业应用前景引起人们关注,低(无)纤维素酶活性的木聚糖酶尤其有着诱人的应用前景,它可以应用于生物制浆、纸浆漂白、废纸二次纤维回收、废纸脱墨处理、纸浆纤维改性剂和纺织工业等,特别是其在纸浆漂白工艺中的巨大应用潜力,已成为各界同行的研究热点.应用于生物漂白过程中的木聚糖酶必须满足没有或者只有少量纤维素酶伴随产生,具有良好的耐碱性及耐高温性,在纸浆中具有稳定的酶活性等条件.自然界中微生物产生的木聚糖降解酶系比较复杂,许多会伴随纤维素酶的产生,这些酶的性质相近,不易分离纯化.此外,除了一些生长在极端环境中的微生物如嗜热菌、嗜碱菌等分泌的木聚糖酶能够满足生物漂白的要求之外,大多数来源于真菌、细菌或放线菌的木聚糖酶都需要在应用过程中调节反应条件.以上这些限制条件严重阻碍了木聚糖酶规模化生产及应用工艺的建立.     

细菌木聚糖酶高产菌的选育及产酶条件

木聚糖是一种在植物体内大量存在的半纤维素,是在自然界中含量仅次于纤维素的一种可再生植物纤维。木聚糖酶(ｘｙｌａｎａｓｅ,ＥＣ3.2.1.8)是一类能够特异降解木聚糖的酶类。近年来,人们将其广泛用于造纸工业的纸浆生物处理,与其他消化酶类一起用作饲料添加剂,以及应用于食品加工工业和纺织工业等。木聚糖酶可以由许多种微生物产生[1],我国多集中于霉菌木聚糖酶的研究。本文报告了一株细菌木聚糖酶产生菌的筛选及产酶条件的研究结果。1　材料和方法11　菌株本实验室分离、保存的木聚糖酶产生菌ＷＸＵＬＩ11及其突变株ＷＬＵＮ024。12　培养…     

耐热木聚糖酶研究进展

β1,4内切木聚糖酶(EC.3218)能够以内切方式作用于木聚糖主链产生不同长度的木寡糖和少量的木糖,因此是木聚糖降解酶系中最关键的酶。木聚糖酶具有很大的工业应用潜力和价值,由于许多工业应用木聚糖酶的单元操作都是在高温下进行的,寻求耐热木聚糖酶作为催化剂是非常重要的。重点介绍了耐热木聚糖酶的特性、分泌表达和结构区域的研究进展。     

绿色糖单孢菌木聚糖酶的分离纯化与酶学性质研究

木聚糖酶是一种重要的具有工业应用前景的木聚糖降解酶,在充分利用自然资源、保护生态环境等方面具有十分重要的意义.通过硫酸铵分级沉淀及Sephadex G-100凝胶柱层析方法从一株中度耐热耐碱放线菌—绿色糖单孢菌的胞外酶中纯化得到单一的木聚糖酶,相对分子质量为51 kD,酶纯度提高了13.01倍.纯酶最适反应温度为60℃,最适反应pH值为7.0;75℃以下该酶具有良好的热稳定性,在pH 7～10范围内具有较强的耐受力.金属离子Ca2+、Fe2+、Zn2+对该酶具有明显促进作用,Cu2+、Mn2+、Al3+和SDS具有抑制作用而K+,Na+,Mg2+没有明显的作用.该酶为大分子质量的糖基化蛋白,含糖量为21.96%.     

嗜碱细菌Cellulomonas bogoriensis 69B4T木聚糖酶的表达纯化及性质研究

【目的】对嗜碱细菌Cellulomonas bogoriensis 69B4~T产碱性木聚糖酶进行研究,克隆来源于该菌株的木聚糖酶基因,并对其进行异源表达、纯化及酶学性质的表征,为后续研究碱性木聚糖酶的耐碱机制及应用奠定基础。【方法】采用单因素分析法对菌株产碱性木聚糖酶情况进行研究;通过基因组分析,锚定5个内切木聚糖酶基因,利用同源扩增的方法进行克隆,并在大肠杆菌中重组表达,利用亲和层析对重组酶进行纯化,以木聚糖为底物表征木聚糖酶的酶学性质。【结果】来源于C. bogoriensis 69B4~T的5种木聚糖酶Xyn370、Xyn393、Xyn425、Xyn466和Xyn486均在大肠杆菌内实现了异源表达,并经亲和层析获得纯酶组分,其最适反应温度分别为60、50、40、40、60°C,在50°C范围内保温2h,残余酶活均在90%以上;最适反应p H分别为7.0、8.0、8.0、8.0、9.0,在p H5.0–9.0时具有较好的稳定性;5种重组木聚糖酶对部分金属离子和高浓度盐表现出较好的耐受性,对榉木木聚糖的酶活性最高,均为内切型木聚糖酶。【结论】本研究表达纯化的5种重组木聚糖酶具有耐盐碱的优良特性,且对温度、某些金属离子和化学试剂耐受,为研究木聚糖酶的耐碱机制及工业应用提供了酶源。     

草酸脱羧酶及其应用

草酸脱羧酶是一种含锰的酶,在白腐菌中广泛存在,少数低等真菌和细菌中也能产生。目前,至少10多种草酸脱羧酶得到了分离和纯化。该酶是一种氨基酸残基在379个左右的单体酶,一般都为酸性糖蛋白,含有2个锰离子,形成2个活性区域;表面一些氨基酸被不同程度地糖基化。晶体结构和其它一些波谱学研究解释了其空间结构和可能的电子传递机制。运用PCR技术和cDNA文库技术,越来越多的草酸脱羧酶基因被克隆。已研究的该酶基因中都含有17个左右的内含子,这些内含子在活性域位置上有比较高的保守性。一些特殊氨基酸序列的存在决定了该酶的表达形式为诱导型,菌株的基因调控序列中含有一段受草酸化合物作用的序列。该酶在一些酵母和植物等异源表达系统中有成功表达的报道。该酶的应用主要集中在以下几方面：造纸废水中的草酸盐降解;食品中的草酸降解;草酸生物检测（如,临床诊断）等。     

Application of enzymes in the pulp and paper industry

The pulp and paper industry processes huge quantities of lignocellulosic biomass every year. The technology for pulp manufacture is highly diverse, and numerous opportunities exist for the application of microbial enzymes. Historically, enzymes have found some uses in the paper industry, but these have been mainly confined to areas such as modifications of raw starch. However, a wide range of applications in the pulp and paper industry have now been identified. The use of enzymes in the pulp and paper industry has grown rapidly since the mid 1980s. While many applications of enzymes in the pulp and paper industry are still in the research and development stage, several applications have found their way into the mills in an unprecedented short period of time. Currently the most important application of enzymes is in the prebleaching of kraft pulp. Xylanase enzymes have been found to be most effective for that purpose. Xylanase prebleaching technology is now in use at several mills worldwide. This technology has been successfully transferred to full industrial scale in just a few years. The enzymatic pitch control method using lipase was put into practice in a large-scale paper-making process as a routine operation in the early 1990s and was the first case in the world in which an enzyme was successfully applied in the actual paper-making process. Improvement of pulp drainage with enzymes is practiced routinely at mill scale. Enzymatic deinking has also been successfully applied during mill trials and can be expected to expand in application as increasing amounts of newsprint must be deinked and recycled. The University of Georgia has recently opened a pilot plant for deinking of recycled paper. Pulp bleaching with a laccase mediator system has reached pilot plant stage and is expected to be commercialized soon. Enzymatic debarking, enzymatic beating, and reduction of vessel picking with enzymes are still in the R&D stage but hold great promise for reducing energy. Other enzymatic applications, i.e., removal of shives and slime, retting of flax fibers, and selective removal of xylan, are also expected to have a profound impact on the future technology of the pulp and paper-making process.     

Modification of paper properties by the pretreatment of wastepaper pulp with Graphiumputredinis, Trichodermaharzianum and fusant xylanases

Graphiumputredinis, Trichodermaharzianum and fusant were used in the present study to produce extracellular xylanases, an important industrial enzyme used in pulp and paper industry produced in a minimal medium supplemented with oat spelt xylan (1%, w/v) pH 7.0 at 27+/-2 degrees C. The enzyme was purified to homogeneity by DEAE-Cellulose and Superdex 75 FPLC column, respectively. The enzyme was found to be a monomer as determined by SDS gel electrophoresis. The optimum pH and temperature for purified G. putredinis, T. harzianum and fusant xylanases were 5.0-6.0 and 50-70 degrees C, respectively. Pretreatment of paper pulp with G. putredinis, T. harzianum and fusant xylanases decreased pulp kappa number. Xylanases particularly that of fusant at 5 IU/g pulp concentration and 1.5% pulp consistency at 60 degrees C for 18 h followed by EDED process yielded good quality paper from waste paper pulp. A significant increase in pulp brightness and improvement in various pulp properties, viz. burst capacity, thickness and bulkness of the treated pulp were observed in comparison to the conventional chemical bleaching. Easy purification and high stability of these enzymes makes it amicable for industrial applications.     

Delignification of wood pulp by a thermostable xylanase from Bacillus stearothermophilus strain T-6

During the bleaching of wood pulp for the paper industry, large amounts of chlorinated aromatic compounds are produced and released into the environment. These compounds are extremely toxic and are a major source of pollution. The paper and pulp industry is seeking for alternative methods for bleaching pulp. One such method involves the use of hemicellulases to release the colored lignohemicellulose. We have isolated and characterized several thermophilic bacteria which produce xylanases. One such strain, T-6, produced high levels of extracellular xylanase, free of cellulase and proteinase activities. Strain T-6 was classified as a strain of Bacillus stearothermophilus and was able to grow on defined medium containing xylose, methionine and asparagine at 65 °C. Xylanase activity was induced by either xylose or xylan; no activity was detected with other carbon sources, such as glycerol, acetate, lactose, glucose, maltose, fructose, mannose, galactose or sucrose. Xylanase constitutive mutants were obtained following mutagenesis and detection on p-nitrophenol -d-xylopyranoside containing agar plates. Xylanase T-6 was produced on large scale, and was purified and concentrated by a single adsorption-desorption step from a cation exchanger. The overall purification yield of a 1000 liter fermentation was 45%, resulting in a 98% pure enzyme. Xylanase T-6 was shown to partially remove lignin from unbleached pulp at 65 °C and pH 9.0, without loss in pulp viscosity. The enzyme-treated pulp was used to make handsheets that had higher brightness than untreated pulp.     

A novel thermostable xylanase of Paenibacillus macerans IIPSP3 isolated from the termite gut

Xylanase is an enzyme in high demand for various industrial applications, such as those in the biofuel and pulp and paper fields. In this study, xylanase-producing microbes were isolated from the gut of the wood-feeding termite at 50°C. The isolated microbe produced thermostable xylanase that was active over a broad range of temperatures (40-90°C) and pH (3.5-9.5), with optimum activity (4,170 ± 23.5 U mg?1) at 60°C and pH 4.5. The enzyme was purified using a strong cation exchanger and gel filtration chromatography, revealing that the protein has a molecular mass of 205 kDa and calculated pI of 5.38. The half-life of xylanase was 6 h at 60°C and 2 h at 90°C. The isolated thermostable xylanase differed from other xylanases reported to date in terms of size, structure, and mode of action. The novelty of this enzyme lies in its high specific activity and stability at broad ranges of temperature and pH. These properties suggest that this enzyme could be utilized in bioethanol production as well as in the paper and pulp industry.     

Engineering Thermostable Microbial Xylanases Toward its Industrial Applications

Xylanases are one of the important hydrolytic enzymes which hydrolyze the β-1, 4 xylosidic linkage of the backbone of the xylan polymeric chain which consists of xylose subunits. Xylanases are mainly found in plant cell walls and are produced by several kinds of microorganisms such as fungi, bacteria, yeast, and some protozoans. The fungi are considered as most potent xylanase producers than that of yeast and bacteria. There is a broad series of industrial applications for the thermostable xylanase as an industrial enzyme. Thermostable xylanases have been used in a number of industries such as paper and pulp industry, biofuel industry, food and feed industry, textile industry, etc. The present review explores xylanase–substrate interactions using gene-editing tools toward the comprehension in improvement in industrial stability of xylanases. The various protein-engineering and metabolic-engineering methods have also been explored to improve operational stability of xylanase. Thermostable xylanases have also been used for improvement in animal feed nutritional value. Furthermore, they have been used directly in bakery and breweries, including a major use in paper and pulp industry as a biobleaching agent. This present review envisages some of such applications of thermostable xylanases for their bioengineering.     

纤维素酶脱墨机理的研究进展

废纸是制浆造纸纤维原料的重要来源,利用生物酶法对废纸浆进行生物脱墨是一种新的造纸生物技术,酶法脱墨所使用的酶种主要是纤维素酶。本文主要就近十年来国内外纤维素酶酶法脱墨的研究特别是影响纤维素酶酶法脱墨的因素及脱墨机理作了综述报道。     

Critical factors affecting laccase-mediated biobleaching of pulp in paper industry

Next to xylanases, laccases from fungi and alkali-tolerant bacteria are the most important biocatalysts that can be employed for eco-friendly biobleaching of hard and soft wood pulps in the paper industry. Laccases offer a potential alternative to conventional, environmental-polluting chlorine and chlorine-based bleaching and has no reductive effect on the final yield of pulp as compared to hemicellulases (xylanases and mannanases). In the last decade, reports on biobleaching with laccases are based on laboratory observations only. There are several critical challenges before this enzyme can be implemented for pulp bleaching at the industrial scale. This review discusses significant factors like redox potential, laccase mediator system (LMS)—synthetic or natural, pH, temperature, stability of enzyme, unwanted grafting reactions of laccase, and cost-intensive production at large scale which constitute a great hitch for the successful implementation of laccases at industrial level.     

Biotechnology of microbial xylanases: enzymology, molecular biology, and application

Xylanases are hydrolases depolymerizing the plant cell wall component xylan, the second most abundant polysaccharide. The molecular structure and hydrolytic pattern of xylanases have been reported extensively and the mechanism of hydrolysis has also been proposed. There are several models for the gene regulation of which this article could add to the wealth of knowledge. Future work on the application of these enzymes in the paper and pulp, food industry, in environmental science, that is, bio-fueling, effluent treatment, and agro-waste treatment, etc. require a complete understanding of the functional and genetic significance of the xylanases. However, the thrust area has been identified as the paper and pulp industry. The major problem in the field of paper bleaching is the removal of lignin and its derivatives, which are linked to cellulose and xylan. Xylanases are more suitable in the paper and pulp industry than lignin-degrading systems.     

Anaerobic fluidised bed for the purification of effluents from chemical and mechanical pulping

Anaerobic treatment has seldom been used for wastewaters from the pulp and paper industry and other branches of the chemical industry. Escape of volatile pollutants to the atmosphere, which always occurs during aerobic treatment, is avoided, and much less sludge is being produced than in an aerobic process. The greatest obstacle for using anaerobic treatment in the pulp and paper industry is the large wastewater volume, which necessitates short hydraulic detention times, because the treatment is to occur in an enclosed space. We used solid carrier particles to prevent wash-out of biomass from the reactor at high hydraulic loading, and an up-flow system in order to be able to use very small carrier particles, maximizing the surface area for biomass attachment. In this paper we describe and discuss the results obtained with this type of anaerobic reactor (fluidised bed) at bench and semitechnical scale for wastewaters from pressurized ground wood pulping and paper manufacture, sulphite pulp evaporator condensate and bleach waste. Earlier work with Kraft pulp bleaching effluent and thermomechanical pulping wastewater and evaporator condensates using anaerobic reactors is also discussed. The results obtained thus far show that there are several wastewater streams from the pulping industry, where 60 to 90% of the dissolved organic pollutants (measured as COD_Cr or TOC) was biodegraded within 4 to 24 h. The high strength waste streams (COD_Cr 2000 mg O₂ 1⁻¹) allowed organic space load of 4 to 10 kg COD_Cr m⁻³ reactor volume d⁻¹. With low strength wastes the hydraulic loading was the limiting factor.