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。     

木质纤维素原料酶水解产乙醇工艺的研究进展

木质纤维素原料预处理后,经水解、发酵等过程,可生产乙醇作为清洁燃料,这大大提高了农业和林业废弃物的利用率,减轻了环境污染,并为经济的可持续发展提供了保证。目前木质纤维素酶水解因其具有明显优势而受到重视,被普遍研究和采用。综述了近年来木质纤维素原料的预处理方法、酶与水解技术、发酵工艺以及发酵耦合分离技术的最新研究成果。     

提高木质纤维素酶解糖化效率的研究进展

木质纤维素高效水解为可发酵糖是其在生物燃料及高附加值化学品转化过程中至关重要的环节。从环保的角度出发,水解方法中的酶解是木质纤维素被彻底降解而无环境污染的有效途径,并且酶水解反应糖损耗低、副产物少、条件温和,因此受到广泛关注。但木质纤维素的组成与结构极为复杂,加之纤维素酶存在稳定性差、寿命短、活性低等缺陷,致使酶解效率较低,酶解糖化成本过高,为此国内外学者对如何提高木质纤维素酶解效率开展诸多方面的研究,综述了近年来提高木质纤维素酶解效率研究取得的最新理论研究及工艺进展,并就木质纤维素的预处理、产酶菌株/技术、酶复配/重组、酶解助剂、酶固定、外场作用、酶回收重利用及酶解反应器多方面的研究情况进行了总结,进一步展望了木质纤维酶解糖化的发展方向。     

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。     

纤维小体在燃料乙醇中的应用

纤维小体在木质纤维素的降解中起着重要作用。它不仅含有降解纤维素所需的各种纤维素酶系,而且组装成具有高效催化活性的多酶复合体形式。介绍了纤维小体基本结构与功能,重点概述了其在生物燃料乙醇中的应用并对纤维小体的研究提出了展望。     

木质纤维素酶基因资源挖掘及真菌酶系改造简

简述了木质纤维素酶基因资源挖掘的策略和方法及其在丝状真菌酶系改造中的应用。从候选基因的获取（木质纤维素酶基因资源的挖掘和高效利用）、外源基因的表达、酶系的复配和重构等方面综述了丝状真菌酶系改造的最新进展,并提出了丝状真菌酶系改造中亟须解决的关键问题。     

木质纤维素酶基因资源挖掘及真菌酶系改造

简述了木质纤维素酶基因资源挖掘的策略和方法及其在丝状真菌酶系改造中的应用。从候选基因的获取(木质纤维素酶基因资源的挖掘和高效利用)、外源基因的表达、酶系的复配和重构等方面综述了丝状真菌酶系改造的最新进展,并提出了丝状真菌酶系改造中亟须解决的关键问题。     

木质纤维素预处理技术研究进展

详细评述了木质纤维素的预处理工艺研究进展,特别是浓酸低温水解-酸回收工艺、稀酸二阶段水解工艺、金属离子在稀酸水解过程中的助催化作用以及水蒸汽爆裂、氨纤维爆裂、CO2爆裂、酶催化水解等方法的研究进展情况。木质纤维素原料预处理技术发展为发酵生产乙醇技术的研究开发奠定了坚实基础。     

纤维素酶在生物质转化中的应用研究进展

纤维素酶是木质纤维素转化的重要酶系,主要是由内切β-1,4-葡聚糖酶、外切β-1,4-葡聚糖酶和β-葡萄糖苷酶组成,水解不同位置的糖苷键从而形成葡萄糖。本文总结了纤维素酶近几年来的研究开发与应用研究进展,包含产酶菌和菌种选育、辅助蛋白,介绍了纤维素酶来源和组成、纤维素酶工业化生产的最新研究进展以及纤维素酶在生物质转化方面的应用,纤维素酶在1.5代、2代纤维素燃料乙醇及生物质制气中都有重要的应用。     

阿魏酸酯酶和纤维素酶在水解汽爆稻草中的协同作用

利用阿魏酸酯酶, 水解天然木质纤维素原料中半纤维素与木质素之间的阿魏酸酯键, 从破坏两者共价键连接的角度, 探索阿魏酸酯酶促进纤维素酶水解汽爆稻草中纤维素的可行性。结果显示, 当阿魏酸酯酶加入量为240 mu/g底物、水解72 h时, 汽爆稻草纤维素的酶解率、不溶性底物失重率较不加阿魏酸酯酶分别增加了32.00%、32.77%; 阿魏酸酯酶(300 mu/g底物)作用120 min后, 纤维素酶对汽爆稻草纤维素的酶解率、不溶性底物失重率分别增加了29.85%、32.48%。通过比较不同酶法处理后的汽爆稻草的可及度和红外光谱图发现, 阿魏酸酯酶能有效地水解原料中的酯键, 提高原料可及度50%以上。由此表明, 阿魏酸酯酶和纤维素酶之间存在较大的协同作用, 添加阿魏酸酯酶能够提高纤维素酶对天然木质纤维素的酶解效率。     

Regulatory aspects of cellulase biosynthesis and secretion

The cellulase enzyme system consists of cellobiohydrolase, endoglucanase, and beta-glucosidase and has been extensively studied with respect to its biosynthesis, properties, mode of action, application, and, most recently, secretion mechanisms. A knowledge of the factors governing the biosynthesis and secretion of these enzymes at the molecular level will be useful in maximizing enzyme productivity in extracellular fluid. Among other topics, the regulatory effects of sorbose (a noninducing sugar which is not a product of cellulose hydrolysis) on cellulase synthesis and release are described. Cellulase genes have recently been cloned into a number of microorganisms with a view to understanding the gene structure and expression and to obtaining the enzyme components in pure form. The factors governing biosynthesis and secretion of cellulases in recombinant cells are also discussed. Cellulases are known to be glycoproteins, therefore, the role of O- and N-linked glycosylation on enzyme stability and secretion is also detailed.     

一株降解纤维素放线菌的产纤维素酶基因克隆与表达

【背景】纤维素在自然界中储量丰富,但天然纤维素的难降解性成为广泛应用纤维素资源的壁垒,近年来利用微生物来降解纤维素成为热点研究。【目的】筛选分离得到一株具有降解纤维素功能的放线菌菌株Lb1,通过全基因组测序确定其产纤维素酶关键基因5676,对基因5676进行克隆转化,使其在大肠杆菌中进行表达。【方法】通过基因工程技术将产纤维素基因连接到表达质粒上并导入表达菌株,对其降解纤维素生成葡萄糖的能力进行探究。【结果】将Lb1菌株的16S rRNA基因进行比对,确定菌株Lb1属于链霉菌属,命名为Streptomyces sp. Lb1。成功构建出纤维素酶表达载体,并且导入表达菌株大肠杆菌BL21(DE3),重组菌株的产纤维素酶能力大于空载菌株。【结论】通过基因工程技术成功克隆出产纤维素酶基因,从而表达纤维素酶,为今后利用微生物降解纤维素的大规模应用提供参考。     

入侵动物福寿螺内生纤维素酶特性及应用研究进展

纤维素酶高效降解纤维素生产单糖,在纺织、造纸、食品、饲料等行业有重要应用价值。近年来,动物源的纤维素酶展现出优良的降解能力,其应用受到广泛关注。软体动物福寿螺是入侵我国华南地区的恶性外来物种,已经对水稻生产和湿地生物多样性造成了严重危害。福寿螺对多种植物的强消化能力与其体内的内生纤维素酶有密切关系。本文聚焦于福寿螺组织的内生纤维素酶,综述了该酶的基因特征、活性因素、重组表达及生产应用。各种福寿螺内生纤维素酶的编码基因在分子质量、基因长度、开放阅读框长度、同源性等方面存在明显差异,环境因素pH、温度、金属离子、阴离子及螯合剂显著影响其活性。采用基因工程和优化表达菌株培养可以提高酶的生产效率,福寿螺内生纤维素酶的转化表达能提高食用菌生物效率和产量,提升动物饲料利用率,增强果蔬汁液提取,其在啤酒酵母中的表达有助于提高乙醇生产率。基于福寿螺内生纤维素酶应用中面临的问题,提出了加快福寿螺内生纤维素酶资源利用并强化对福寿螺属的其他螺类内生纤维素酶研究的建议。     

Expression and transport of cellulase in avocado mesocarp during ripening

Summary Cellulase localization in the mesocarp of ripening avocado fruits (Persea americana Mill. cv. Hass) was studied by a variety of immunological methods. As localized by immunodetection on whole fruit tissue blots, cellulase first appeared near the stylar end of the fruit during the late portion of the rise in climacteric respiration. Cellulase appearance subsequently expanded outward and upward, reaching the peduncle end of the fruit by the day after the climacteric peak. Cellulase expression in cells surrounding vascular bundles was delayed relative to expression in the adjacent mesocarp. Immuno-labeled frozen sections, viewed with the light microscope, showed that cellulase appeared in both parenchyma and oil cells concom-mitantly with wall breakdown. Immunogold detection of cellulase by electron microscopy revealed labeling associated with the endoplasmic reticulum, plasmodesmata, and cell wall during the period between the late portion of the climacteric rise and the day after the climacteric peak. Cellulase appeared in the nucleus during all stages of ripening after the early portion of the climacteric rise. Immunoblot analysis of organelle fractions, isolated from avocado fruit at the climacteric peak of respiration, revealed three molecular weight forms of cellulase; a light and a heavy form found in endoplasmic reticulum-enriched fractions, and an intermediate form found in Golgi and plasma membrane-enriched fractions.Abbreviations Endo-H endoglyosidase H - Tris tris(hydroxymethyl)-aminomethane - MeOH methyl alcohol - EtOH ethyl alcohol - BSA bovine serum albumin - PM plasmamembrane - ATPase adenosine 5-triphosphatase - Pi inorganic phosphate - IDPase inosine 5-di-phosphatase - ER endoplasmic reticulum The work presented here has been submitted in partial fulfillment of the requirements for the Ph.D. degree.     

Failed expression of an endo-beta-1,4-glucanhydrolase (cellulase) in a non-abscinding mutant of Lupinus angustifolius cv Danja.

Cellulase expressions in a normal shedding wild-type and a non-abscinding single gene mutant of Lupinus angustifolius have been studied during ethylene treatments of leaf abscission zone explants. Of the range of different glycohydrolases investigated only the abscission cell-specific beta-1,4-glucanhydrolase (cellulase) was not produced in the non-abscinding mutant. An endo-polygalacturonase was induced equally in both wild-type and mutant and other glycohydrolases were equally up-regulated. The abscission cell-specific cellulase induced at shedding of wild-type is antigenically similar to the Phaseolus vulgaris induced leaf abscission pI 9.5 cellulase but with a higher molecular mass (50 kD compared with 48 kD) and like the bean abscission-specific cellulase that of lupin is not glycosylated. Causes of the loss of function of cellulase expression in the non-shedding mutant are discussed.     

Correlation of cellulase gene expression and cellulolytic activity throughout the gut of the termite Reticulitermes flavipes

Termites have developed cellulose digestion capabilities that allow them to obtain energy and nutrition from nutritionally poor food sources, such as lignocellulosic plant material and residues derived from it (e.g., wood and humus). Lower termites, which are equipped with both endogenous (i.e., of termite origin) and symbiotic cellulases, feed primarily on wood and wood-related materials. This study investigated cellulase gene diversity, structure, and activity in the lower termite, Reticulitermes flavipes (Kollar). We initially used a metagenomics approach to identify four genes encoding one endogenous and three symbiotic cellulases, which we refer to as Cell-1, -2, -3 and -4. These four genes encode proteins that share significant sequence similarity with known endoglucanases, exoglucanases and xylanases. Phylogenetic analyses further supported these inferred relationships by showing that each of the four cellulase proteins clusters tightly with respective termite, protozoan or fungal cellulases. Gene structure studies revealed that Cell-1, -3 and -4 are intron-free, while Cell-2 contains the first intron sequence to be identified from a termite symbiont cellulase. Quantitative real-time PCR (qRT-PCR) revealed that the endogenous Cell-1 gene is expressed exclusively in the salivary gland/foregut, whereas symbiotic Cell-2, -3, and -4 are highly expressed in the hindgut (where cellulolytic protists are harbored). Cellulase activity assays mapped the distribution pattern of endoglucanase, exoglucanase and xylanase activity throughout the R. flavipes digestive tract. Cellulase gene expression correlated well with the specific types of cellulolytic activities observed in each gut region (foregut+salivary gland, midgut and hindgut). These results suggest the presence of a single unified cellulose digestion system, whereby endogenous and symbiotic cellulases work sequentially and collaboratively across the entire digestive tract of R. flavipes.     

Development and characterization of membrane surface display system using molecular chaperon, prsA, of Bacillus subtilis

We report a new membrane surface display system based on molecular chaperon, prsA, of Bacillus subtilis. Clostridium thermocellum cellulase, celA, was fused to C-terminal end of PrsA. Cellulase activity of B. subtilis protoplast, which expressed PrsA-CelA was 15 times higher compared to control strain. More than 85% of total cellulase activity was observed in surface displayed format and less than 15% of total cellulase activity was found in supernatant. Flow cytometric analysis of protoplast of PrsA-CelA fusion expressing bacteria provided another proof of uniform expression of fusion protein onto cytoplasmic membrane of B. subtilis. Without lysozyme treatment, only part of cellulase activity (10%) was observed in whole cell fraction.     

Development of effective modified cellulase for cellulose hydrolysis process

Cellulase was modified with amphilic copolymers made of alpha-allyl-omega-methoxy polyoxyalkylene (POA) and maleic acid anhydride (MAA) to improve the cellulose hydrolytic reactivity and cellulase separation. Amino groups of the cellulase molecule are covalently coupled with the MAA functional groups of the copolymer. At the maximum degree of modification (DM) of 55%, the modified cellulase activity retained more than 80% of the unmodified native cellulase activity. The modified cellulase shows greater stability against temperature, pH, and organic solvents, and demonstrated greater conversion of substrate than native cellulase does. Cellulase modification is also useful for controlling strong adsorption of cellulase onto substrate. Moreover, cellulase modified with the amphiphilic copolymer displays different separation characteristics which are new. One is a reactive two-phase partition and another is solubility in organic solvents. It appears that these characteristics of modified cellulase work very effectively in the hydrolysis of cellulose as a total system, which constitutes the purification of cellulase from culture broth, hydrolysis of cellulose, and recovery of cellulase from the reaction mixture. (c) 1995 John Wiley & Sons, Inc.