嗜热性木质纤维素酶在纤维素乙醇生产中的研究进展

聚焦于嗜热性木质纤维素酶在纤维素乙醇生产中的应用,归纳了限制纤维素乙醇商业化的技术瓶颈,简单介绍了嗜热性木质纤维素酶的特点,重点介绍了嗜热性木质纤维素酶在筛选、修饰、固定化、异源表达、代谢调控以及协同作用中的研究进展,讨论了嗜热性木质纤维素酶在纤维素乙醇生产中发挥的作用。最后,提出了嗜热性木质纤维素酶在纤维素乙醇生产中面临的问题并展望了其应用前景。     

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。     

纤维素酶与木质纤维素生物降解转化的研究进展

利用纤维素酶将预处理后的秸秆降解成可发酵性单糖,然后发酵生产所需的液体燃料及化工产品的技术,对于我国解决能源、环境、人口就业等难题有着巨大的积极影响。在木质纤维素生物降解转化工艺中,减少纤维素酶用量及提高酶解效率是降低木质纤维素降解成本的关键。纤维素酶系和木质纤维素酶水解技术的改进需要深入了解纤维素酶系统的组成及其协同作用、纤维素酶的结构与功能以及纤维素酶的生产技术。将就以上几个方面的研究进展进行讨论,并深入探讨了纤维素酶糖化能力的评价方法。     

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。     

不同地区森林土壤降解天然木质纤维素能力的分析评价

目的:分析不同地区森林土壤样品的木质纤维素分解能力,为分离和挖掘新的土壤木质纤维素分解酶系及微生物奠定基础.方法:测定来源于不同气候类型和植被的土壤样品的纤维素酶和木聚糖酶活性的变化以及对天然木质纤维素的降解能力.结果:土壤样品具有较高的初始木聚糖酶活,相反许多样品的纤维素酶活未测到.富集后,除个别样品酶活性稍有下降之外其余均明显提高,其中木聚糖酶活增长最多达118.58 u·g~(-1),纤维素酶活涨幅最多达110.00 u·g~(-1).各样品木质纤维素的降解量从24.4mg到93.1mg不等,降解效率最高55.35%.结论:来源于不同气候条件和不同类型土壤样品在天然木质纤维素降解能力以及相关的纤维素酶和木聚糖酶活性上表现出了广泛的多样性差异.     

嗜热厌氧细菌Caldicellulosiruptor bescii降解木质纤维素研究进展

嗜热厌氧菌Caldicellulosiruptor bescii具有强大的木质纤维素降解能力,能以多种模式植物细胞壁多糖如微晶纤维素Avicel和木聚糖,甚至未经预处理的木质纤维素如柳枝稷作为唯一碳源快速生长,该菌还具有少见的厌氧降解木质素的能力。对基因组注释发现,该菌所编码的蛋白大多为多结构域双功能酶,即在多肽链的N端和C端分别是不同家族的糖苷水解酶,间隔以2-3个碳水化合物结合结构域。该菌降解纤维素相关的酶基因多集中于一个植物细胞壁多糖降解利用的基因簇,例如纤维素酶/木聚糖酶、纤维素酶/甘露聚糖酶和纤维素酶/木葡聚糖酶等。C.bescii的木聚糖酶主要属于GH10家族,该家族的酶底物特异性较为宽泛,氨基酸序列的同源性在18.7%-59.5%间。Caldicellulosiruptor属细菌进化出了一系列的机制使得糖苷水解酶和底物、细菌和木质纤维素能更好的吸附在一起,从而有利于木质纤维素的酶解。C.bescii有12个含SLH结构域的蛋白,以及新发现的黏附蛋白Tāpirin,可能参与了木质纤维素的吸附与利用。综述了近年来对C.bescii降解植物细胞壁的糖苷水解酶的基因资源挖掘方面和降解分子机制方面的研究进展,对高效、多功能高效木质纤维素降解酶的设计和优化具有积极的意义。     

裂解性多糖单加氧酶HcLPMO与纤维素酶协同降解纤维素

【目的】为研究HcLPMO的活性测定方法及其与纤维素酶的协同降解特性。【方法】利用大肠杆菌表达系统进行HcLPMO异源表达,研究以AmplexTM Ultra Red为荧光底物的LPMOs活性检测条件;研究HcLPMO与纤维素酶最优配比协同降解微晶纤维素及其他多种生物质底物的能力。【结果】表达条件确定最适装液量为20%,最适诱导温度为20°C。活性测定研究结果表明HcLPMO需先与铜离子结合才具有活性,电子供体抗坏血酸钠(ASC)最适浓度为10–4 mol/L,并发现AmplexTM Ultra Red浓度以及辣根过氧化物酶浓度对酶活的检测影响较小。HcLPMO与纤维素酶协同降解微晶纤维素研究确定HcLPMO与纤维素酶最优配比为2:3,葡萄糖产量相较纤维素酶单独作用提高了99.48%。此外,针对多种生物质底物,发现该酶与纤维素酶的复配体系对汽爆玉米秸秆和微晶纤维素的协同降解效果较好,相较于单独用纤维素水解酶,葡萄糖产量分别提高了63.81%和59.43%,而对碱处理玉米芯和木薯渣降解效果次之,葡萄糖产量仅分别提高35.41%和11.06%。【结论】HcLPMO与纤维素酶复配能够有效提高酶法降解纤维素效率;而底物前处理如蒸汽爆破或碱处理对于HcLPMO与纤维素酶协同降解木质纤维素影响较大。     

丝状真菌溶解性多糖单加氧酶的研究进展

溶解性多糖单加氧酶(lytic polysaccharide monooxygenases, LPMOs)是近些年才发现的一种蛋白,能够催化多糖葡萄糖苷键的氧化裂解,显著促进丝状真菌纤维素酶系对木质纤维素的降解作用。本综述对溶解性多糖单加氧酶发现过程、晶体结构、反应机制、活性位点和区域选择性、与纤维素酶的协同作用等方面分别进行了阐述,并对溶解性多糖单加氧酶对于木质纤维素降解方面的应用进行了论述。     

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...     

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。     

Research advances in expansins and expansion-like proteins involved in lignocellulose degradation

Biofuel derived from lignocellulosic biomass has attracted considerable attention as a renewable energy source. Nevertheless, the conversion of lignocellulose into fermentable sugars is inherently difficult because of the complex structures of lignocelluloses. Accessory proteins, like expansins, have a non-hydrolytic disruptive effect on crystalline cellulose and can synergistically cooperate with cellulase to improve hydrolysis efficiency. This review summarizes recent studies on expansins and expansin-like proteins, in terms of their expression and purification, synergism in lignocellulose hydrolysis, structure–function studies and binding characteristics. Future research prospects are also presented. This review provides a discussion of expansins in the context of lignocellulose hydrolysis.     

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。     

Effect of adding surfactant for transforming lignocellulose into fermentable sugars during biocatalysing

Fuel ethanol is one of the most important alternative fuels used as a substitute for fossil fuel. Lignocellulose is the most abundant biomass resource for the production of fuel ethanol. However, the hydrolysis of lignocellulose requires high enzyme loading. In order to strengthen the process of enzyme hydrolysis of lignocellulose, surfactant-polyethylene glycol (PEG) was applied to the catalysis of lignocellulose into fermentable sugars. The effect of PEG on both the enzymatic hydrolysis and adsorption of cellulose were investigated. The addition of surfactant obviously facilitated enzymatic hydrolysis. In particular, upon addition of PEG4000, the enzyme catalytic efficiency increased by 51.06%. Meanwhile, the adsorption quantity of cellulase decreased by 11.25%. In addition, the mechanism of the effect of PEG on enzymatic hydrolysis and cellulase adsorption is discussed.     

Fractionating recalcitrant lignocellulose at modest reaction conditions

Effectively releasing the locked polysaccharides from recalcitrant lignocellulose to fermentable sugars is among the greatest technical and economic barriers to the realization of lignocellulose biorefineries because leading lignocellulose pre-treatment technologies suffer from low sugar yields, and/or severe reaction conditions, and/or high cellulase use, narrow substrate applicability, and high capital investment, etc. A new lignocellulose pre-treatment featuring modest reaction conditions (50 degrees C and atmospheric pressure) was demonstrated to fractionate lignocellulose to amorphous cellulose, hemicellulose, lignin, and acetic acid by using a non-volatile cellulose solvent (concentrated phosphoric acid), a highly volatile organic solvent (acetone), and water. The highest sugar yields after enzymatic hydrolysis were attributed to no sugar degradation during the fractionation and the highest enzymatic cellulose digestibility ( approximately 97% in 24 h) during the hydrolysis step at the enzyme loading of 15 filter paper units of cellulase and 60 IU of beta-glucosidase per gram of glucan. Isolation of high-value lignocellulose components (lignin, acetic acid, and hemicellulose) would greatly increase potential revenues of a lignocellulose biorefinery.     

Evaluating kinetics of enzymatic saccharification of lignocellulose by fractal kinetic analysis

The effects of impacting factors, including cellulase loading, operation temperature, product glucose inhibition, and high solid pretreated biomass loading were examined systemically on the enzymatic saccharification of lignocellulose (dilute acid pretreated corn stover) in the presence and absence of tri-block copolymer L64 (also referred to polymeric nonionic surfactant). The complex kinetics of enzymatic saccharification of cellulose were subjected to fractal kinetic analysis based on a fractal kinetic model, which is described with fractal kinetic parameters of the rate constant and fractal exponent. The results indicate that glucose inhibition including high lignocellulose loading is indexed by decreasing rate constant while lignin inhibition and high operation temperature is indexed by increasing fractal exponent. The effect of a nonionic surfactant on the enzymatic saccharification of lignocellulose mainly contributed to the elimination of lignin inhibition by decreasing the corresponding fractal exponent. However, the effect of the nonionic surfactant on cellulase activity and stability was very limited.     

Lignocellulose biotransformation with immobilized cellulase,d-glucose oxidase and fungal peroxidases

Three enzymes, cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4], d-glucose oxidase (β-d-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) and peroxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) immobilized on glass beads, have been incubated with lignocellulose. Fungal peroxidases from Trametes versicolor and Inonotus radiatus when mixed with cellulase and d-glucose oxidase were able to liberate phenolic compounds and d-glucose from lignocellulose. Three lignin monomers were identified. When the immobilized enzymes were incubated individually with lignocellulose they did not degrade lignin.     

Enteric bacterial catalysts for fuel ethanol production.

The technology is available to produce fuel ethanol from renewable lignocellulosic biomass. The current challenge is to assemble the various process options into a commercial venture and begin the task of incremental improvement. Current process designs for lignocellulose are far more complex than grain to ethanol processes. This complexity results in part from the complexity of the substrate and the biological limitations of the catalyst. Our work at the University of Florida has focused primarily on the genetic engineering of Enteric bacteria using genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase. These two genes have been assembled into a portable ethanol production cassette, the PET operon, and integrated into the chromosome of Escherichia coli B for use with hemicellulose-derived syrups. The resulting strain, KO11, produces ethanol efficiently from all hexose and pentose sugars present in the polymers of hemicellulose. By using the same approach, we integrated the PET operon into the chromosome of Klebsiella oxytoca to produce strain P2 for use in the simultaneous saccharification and fermentation (SSF) process for cellulose. Strain P2 has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. Recently, the ability to produce and secrete high levels of endoglucanase has also been added to strain P2, further reducing the requirement for fungal cellulase. The general approach for the genetic engineering of new biocatalysts using the PET operon has been most successful with Enteric bacteria but was also extended to Gram positive bacteria, which have other useful traits for lignocellulose conversion. Many opportunities remain for further improvements in these biocatalysts as we proceed toward the development of single organisms that can be used for the efficient fermentation of both hemicellulosic and cellulosic substrates.