青霉生产木质纤维素降解酶系的研究进展

木质纤维素降解酶系的高效生产是实现植物生物质大规模生物炼制的重要支撑。就地生产木质纤维素降解酶,有助于降低其使用成本,提高技术经济效益。青霉是自然界常见的木质纤维素降解真菌,可以合成分泌种类多样、组分齐全的木质纤维素降解酶系,已被应用于纤维素酶制剂的工业生产。文中从就地生产降解酶,为木质纤维素生物炼制构建“糖平台”的角度,综述了青霉木质纤维素降解酶系的性质、菌株遗传改造及发酵工艺的研究进展。     

纤维素酶的糖基化

木质纤维素价格低廉,供应充足,且未得到充分开发利用。把纤维素降解成葡萄糖,进而生产纤维素乙醇的技术已经进入商业应用阶段。提高纤维素酶的活性,有利于充分利用自然界中大量存在的木质纤维素,开发生物质资源,以缓解能源危机。糖基化修饰对纤维素酶的活性、稳定性以及其他性质有着重要的影响。因此,对纤维素酶糖基化的了解,以及合理地改善糖基化修饰,可以极大地提高木质纤维素降解速率,有利于工业上液体燃料的生产。     

产纤维素酶黏细菌在生物转化的作用

目的:预处理对木质纤维素降解的影响.方法:从土壤中分离筛选到高纤维素酶活的黏细菌菌株So ce sh1008.该菌具有CMC酶活(CMCase)及微晶纤维素酶活性.研究NaOH联合黏细菌降解盐蒿、稻草、棉花秸秆和甘蔗渣四种木质纤维素的情况.结果:碱(2％ NaOH) -黏细菌处理的方法优于黏细菌-碱的方法,其中降解棉花秸秆降解效果最明显,以5.0g木质纤维素为原料,其最终干重损失达2.1g,溶液中总糖含量和还原糖含量均值分别为12.8 mg/mL和0.93 mg/mL.酵母菌发酵产乙醇的研究结果表明,最佳发酵时间为47h,碱-黏细菌甘蔗渣降解液发酵效果最好,乙醇产出达6.0％.结论:黏细菌联合2％ NaOH能有效降解甘蔗渣,提高乙醇产量.     

里氏木霉产纤维素酶研究进展

木质纤维素类生物质被认为是重要且可持续的可再生能源,其主要组成部分是纤维素.纤维素酶是一种能将纤维素分解为葡萄糖的复合酶,能有效地降解木质纤维素生物质.真菌、细菌、放线菌、酵母等多种微生物均可以产生纤维素酶,其中里氏木霉具有完整的纤维素酶系结构,常作为生物技术领域中一个重要菌株,广泛应用于纤维素酶的商业生产.介绍了纤维...     

粗糙脉孢菌木质纤维素降解利用研究进展简

粗糙脉孢菌作为木质纤维素降解真菌,不仅具有完整的木质纤维素降解酶系,而且还拥有全基因组基因敲除突变体库,是研究丝状真菌纤维素酶表达分泌和木质纤维素降解机制的优秀体系。近年来,国内外利用粗糙脉孢菌系统,在木质纤维素降解机制方面取得了显著进展,包括纤维素酶信号传导、调控以及生物质降解后糖的转运利用等。笔者就相关方面的进展进行综述,并对利用粗糙脉孢菌研究木质纤维素降解利用进行展望,总结和分析木质纤维素降解机制研究的国际前沿动态,有助于加深本领域研究人员对真菌体系纤维素降解机制的理解。     

粗糙脉孢菌木质纤维素降解利用研究进展

粗糙脉孢菌作为木质纤维素降解真菌,不仅具有完整的木质纤维素降解酶系,而且还拥有全基因组基因敲除突变体库,是研究丝状真菌纤维素酶表达分泌和木质纤维素降解机制的优秀体系。近年来,国内外利用粗糙脉孢菌系统,在木质纤维素降解机制方面取得了显著进展,包括纤维素酶信号传导、调控以及生物质降解后糖的转运利用等。笔者就相关方面的进展进行综述,并对利用粗糙脉孢菌研究木质纤维素降解利用进行展望,总结和分析木质纤维素降解机制研究的国际前沿动态,有助于加深本领域研究人员对真菌体系纤维素降解机制的理解。     

丝状真菌纤维素酶合成诱导及转录调控

利用廉价可再生木质纤维素资源水解产生的可发酵糖生产生物能源和生物基化学品是近年来国内外研究的热点。纤维素酶酶解是木质纤维素原料生物降解的重要手段,但目前纤维素酶生产成本过高,限制了纤维素生物转化和生物炼制的工业化应用。对丝状真菌纤维素酶基因表达和调控进行研究,有利于进一步选育纤维素酶高产菌株,降低纤维素酶生产成本。随着高通量测序及丝状真菌遗传操作等技术的进步,对丝状真菌纤维素酶诱导和基因表达调控机理有了更深入的认识。本文综述了近年来丝状真菌纤维素酶诱导和纤维素酶基因表达调控的最新进展,重点论述糖转运蛋白、转录因子和染色质重塑对纤维素酶表达调控的影响,并对利用人工锌指蛋白进行丝状真菌纤维素酶诱导调控研究进行了展望。     

酿酒酵母纤维素乙醇统合加工(CBP)的策略及研究进展

木质纤维素乙醇的统合生物加工过程(Consolidated bioprocessing,CBP)是将纤维素酶和半纤维素酶生产、纤维素水解和乙醇发酵过程组合或部分组合,通过一种微生物完成。统合生物加工过程有利于降低生物转化过程的成本,越来越受到研究者的普遍关注。酿酒酵母Saccharomyces cerevisiae是传统的乙醇发酵菌株。介绍了影响外源基因在酿酒酵母中表达水平的因素,纤维素酶和半纤维素酶在酿酒酵母中表达研究进展及利用酿酒酵母统合加工纤维素乙醇的策略。     

同步分泌高效纤维素酶和木聚糖酶菌株YB的鉴定及其酶学性质研究

筛选和鉴定可降解木质纤维素的真菌,并研究其产酶特征。采用刚果红平板涂布法,从荔枝腐叶中筛选具有木质纤维素降解能力的真菌,结合ITS-rDNA序列分析进行鉴定,初步测定其产酶条件,然后采用DEAE Sepharose Fast Flow阴离子交换层析与Sephadex G-100凝胶层析对硫酸铵沉淀的粗酶液进行分离纯化,对其开展酶学性质研究。结果显示,筛选出一株可降解木质纤维素降解的菌株YB,鉴定为绿木霉(Trichoderma virens)。在发酵过程中,纤维素酶和木聚糖酶的最大活力分别为313.53±26.78 U/mL和18 120.87±500.37 U/mL。分离纯化得到纤维素酶(CMC酶)Ⅰb、Ⅳ和木聚糖酶Ⅰa;通过SDS-PAGE检测,其分子量分别为58.5 kD、22.8 kD和44.5 kD。3种酶的最适酶促反应条件均为:50℃,pH 5.0。其中,木聚糖酶能有效降解玉米芯木聚糖为木糖和多种木寡糖。菌株Trichoderma virens YB可分泌高效木质纤维素降解酶,具有应用于木聚糖酶和木寡糖生产的潜力。     

鸡尾酒δ整合策略构建表达三类纤维素酶的酿酒酵母工程菌株及初步评价

木质纤维素乙醇具有替代化石燃料的潜力,其生产过程包括生物质预处理、纤维素酶生产、水解和发酵等多个步骤。将纤维素酶生产、水解和发酵组合在一起的统合生物加工过程（consolidated bioprocessing,CBP）由于能降低水解和发酵成本而具有应用于纤维素乙醇生产的潜力,该技术的关键是构建能有效降解纤维素的工程菌株,而构建表达纤维素酶的酿酒酵母即是其中一种选择。采用鸡尾酒多拷贝δ整合的策略将7种纤维素酶基因（Trichoderma reesei cbh1、cbh2和egl2,Aspergillus aculeatus cbh1、egl1和bgl1）表达盒整合至酿酒酵母W303-1A染色体上,经4轮整合筛选得到菌株LA1、LA2、LA3和LA4。对这4个菌株进行纤维素酶活性测定,结果表明从LA1到LA3各种纤维素酶活性呈递增趋势,而LA4的酶活性与LA3的酶活水平相当。对菌株LA3进行酸碱预处理玉米芯料的发酵评价,结果表明：①在外加商品化纤维素酶的情况下,与对照菌株W303-1A和AADY相比,LA3能有效利用纤维素料发酵产醇;②与分步整合的菌株W3相比,发酵性能更优;③培养基中的营养成分影响菌株发酵性能。这些结果表明,鸡尾酒δ整合是一种有效的构建酿酒酵母CBP菌株的方法。     

生物质生物预处理研究进展与展望

木质纤维素生物质分布广、产量大、可再生,用于制备生物基能源、生物基材料和生物基化学品。木质纤维素生物质组成复杂,包含纤维素、半纤维素和木质素等,木质素与半纤维素通过共价键、氢键交联形成独特的“包裹结构”,纤维素含有复杂的分子内与分子间氢键,上述因素制约着其资源化利用。生物预处理以其独特优越性成为生物质研究的重要方面。系统阐述了生物预处理过程中木质素降解和基团修饰对纤维素酶解的影响,纤维素含量及结晶区变化,半纤维素五碳糖利用,微观物理结构的改变。进一步提出了以生物预处理为核心的组合预处理、基于不同功能的多酶协同催化体系、木质纤维素组分分级利用和新型高效细菌预处理工艺是生物预处理未来发展的重要趋势。     

Endowing non-cellulolytic microorganisms with cellulolytic activity aiming for consolidated bioprocessing

With the exhaustion of fossil fuels and with the environmental issues they pose, utilization of abundant lignocellulosic biomass as a feedstock for biofuels and bio-based chemicals has recently become an attractive option. Lignocellulosic biomass is primarily composed of cellulose, hemicellulose, and lignin and has a very rigid and complex structure. It is accordingly much more expensive to process than starchy grains because of the need for extensive pretreatment and relatively large amounts of cellulases for efficient hydrolysis. Efficient and cost-effective methods for the production of biofuels and chemicals from lignocellulose are required. A consolidated bioprocess (CBP), which integrates all biological steps consisting of enzyme production, saccharification, and fermentation, is considered a promising strategy for reducing production costs.     

Hydrolysis of lignocellulosic materials for ethanol production: a review

Lignocellulosic biomass can be utilized to produce ethanol, a promising alternative energy source for the limited crude oil. There are mainly two processes involved in the conversion: hydrolysis of cellulose in the lignocellulosic biomass to produce reducing sugars, and fermentation of the sugars to ethanol. The cost of ethanol production from lignocellulosic materials is relatively high based on current technologies, and the main challenges are the low yield and high cost of the hydrolysis process. Considerable research efforts have been made to improve the hydrolysis of lignocellulosic materials. Pretreatment of lignocellulosic materials to remove lignin and hemicellulose can significantly enhance the hydrolysis of cellulose. Optimization of the cellulase enzymes and the enzyme loading can also improve the hydrolysis. Simultaneous saccharification and fermentation effectively removes glucose, which is an inhibitor to cellulase activity, thus increasing the yield and rate of cellulose hydrolysis.     

Combining hot-compressed water and ball milling pretreatments to improve the efficiency of the enzymatic hydrolysis of eucalyptus

Background  

Lignocellulosic biomass such as wood is an attractive material for fuel ethanol production. Pretreatment technologies that increase the digestibility of cellulose and hemicellulose in the lignocellulosic biomass have a major influence on the cost of the subsequent enzymatic hydrolysis and ethanol fermentation processes. Pretreatments without chemicals such as acids, bases or organic solvents are less effective for an enzymatic hydrolysis process than those with chemicals, but they have a less negative effect on the environment.     

Pretreatments for Enhanced Enzymatic Hydrolysis of Pinewood: a Review

Pinewood is an abundant source of lignocellulosic biomass that has potential to be used as renewable feedstock in biorefineries for conversion into advanced biofuels and other value-added chemicals. However, its structural recalcitrance, due to the compact packing of its major components, viz. cellulose, hemicellulose and lignin, high lignin content, and high cellulose crystallinity, is a major bottleneck in its widespread use as a biorefinery feedstock. Typical chemical, thermal, and biological pretreatment technologies are aimed at removing lignin and hemicellulose fractions for improving enzyme accessibility and digestibility of cellulose. This review highlights common pine pretreatment procedures, associated key parameters and resulting enzymatic hydrolysis yields. The challenges and limitations are also discussed as well as potential strategies to overcome them, providing an essential source of information to realize pine as a compelling biorefinery biomass source.     

Preparation of cellulase concoction using differential adsorption phenomenon

Controlled depolymerization of cellulose is essential for the production of valuable cellooligosaccharides and cellobiose from lignocellulosic biomass. However, enzymatic cellulose hydrolysis involves multiple synergistically acting enzymes, making difficult to control the depolymerization process and generate desired product. This work exploits the varying adsorption properties of the cellulase components to the cellulosic substrate and aims to control the enzyme activity. Cellulase adsorption was favored on pretreated cellulosic biomass as compared to synthetic cellulose. Preferential adsorption of exocellulases was observed over endocellulase, while β-glucosidases remained unadsorbed. Adsorbed enzyme fraction with bound exocellulases when used for hydrolysis generated cellobiose predominantly, while the unadsorbed enzymes in the liquid fraction produced cellooligosaccharides majorly, owing to its high endocellulases activity. Thus, the differential adsorption phenomenon of the cellulase components can be used for the controlling cellulose hydrolysis for the production of an array of sugars.     

High-throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass

Several factors will influence the viability of a biochemical platform for manufacturing lignocellulosic based fuels and chemicals, for example, genetically engineering energy crops, reducing pre-treatment severity, and minimizing enzyme loading. Past research on biomass conversion has focused largely on acid based pre-treatment technologies that fractionate lignin and hemicellulose from cellulose. However, for alkaline based (e.g., AFEX) and other lower severity pre-treatments it becomes critical to co-hydrolyze cellulose and hemicellulose using an optimized enzyme cocktail. Lignocellulosics are appropriate substrates to assess hydrolytic activity of enzyme mixtures compared to conventional unrealistic substrates (e.g., filter paper, chromogenic, and fluorigenic compounds) for studying synergistic hydrolysis. However, there are few, if any, high-throughput lignocellulosic digestibility analytical platforms for optimizing biomass conversion. The 96-well Biomass Conversion Research Lab (BCRL) microplate method is a high-throughput assay to study digestibility of lignocellulosic biomass as a function of biomass composition, pre-treatment severity, and enzyme composition. The most suitable method for delivering milled biomass to the microplate was through multi-pipetting slurry suspensions. A rapid bio-enzymatic, spectrophotometric assay was used to determine fermentable sugars. The entire procedure was automated using a robotic pipetting workstation. Several parameters that affect hydrolysis in the microplate were studied and optimized (i.e., particle size reduction, slurry solids concentration, glucan loading, mass transfer issues, and time period for hydrolysis). The microplate method was optimized for crystalline cellulose (Avicel) and ammonia fiber expansion (AFEX) pre-treated corn stover.     

Assessing the Performance of Bacterial Cellulases: the Use of <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Paenibacillus</Emphasis> Strains as Enzyme Sources for Lignocellulose Saccharification

Plant biomass offers a renewable and environmentally favorable source of sugars that can be converted to different chemicals, second-generation ethanol, and other liquid fuels. Cellulose makes up approximately 45 % of the dry weight of lignocellulosic biomass. Prior to the enzymatic hydrolysis of cellulose, lignin and hemicellulose must be structurally altered or removed, at least in part, by chemical and/or physical pretreatments. However, the high cost and low efficiency of the enzymatic hydrolysis prevent the process from being economically competitive. For this reason, it is necessary to find enzymes suitable for this type of process, with higher specific activities and greater efficiency. Members of the Bacillus and Paenibacillus genera have been traditionally used for the production of many enzymes for industrial applications. Cellulases produced by both genera have shown activity on soluble and crystalline cellulose and high thermostability and/or activity over a wide pH spectrum. In this review, the most recent information about the characterization of cellulolytic enzymes obtained from new strains of the Bacillus and Paenibacillus genera are reviewed. We focused on the variety of isoenzymes produced by these cellulolytic strains, their optimal production and reaction conditions, and their kinetic parameters and biotechnological potential.     

木质纤维生物质资源高效利用分子技术的开发

木质纤维生物质是地球上最丰富的可再生生物质资源,可为造纸、化工、纺织和生物能源等工业提供重要的原材料。木质纤维生物质主要包括木质素、纤维素和半纤维素三种生物多聚物成分。如何利用分子手段改造这些生物聚合物,提高它们的工业利用率是目前高度关注的问题。综述了近年来木质纤维多聚物在生物合成与改造方面的研究进展,展望了利用分子技术改造植物木质纤维生物质实现其高效利用的前景。     

Recent trends in ionic liquid (IL) tolerant enzymes and microorganisms for biomass conversion

Second generation biofuel production depends on lignocellulosic (LC) biomass transformation into simple sugars and their subsequent fermentation into alcohols. However, the main obstacle in this process is the efficient breakdown of the recalcitrant cellulose to sugar monomers. Hence, efficient feedstock pretreatment and hydrolysis are necessary to produce a cost effective biofuel. Recently, ionic liquids (ILs) have been recognized as a promising solvent able to dissolve different biomass feedstocks, providing higher sugar yields. However, most of the hydrolytic enzymes and microorganisms are inactivated, completely or partially, in the presence of even low concentrations of IL, making necessary the discovery of novel hydrolytic enzymes and fermentative microorganisms that are tolerant to ILs. In this review, the current state and the challenges of using ILs as a pretreatment of LC biomass was evaluated, underlining the advances in the discovery and identification of new IL-tolerant enzymes and microorganisms that could improve the bioprocessing of biomass to fuels and chemicals.