Fluid immobilized cellulase

Summary Fluid immobilized cellulase was prepared using polyethyleneglycols and hexamethylene diisocyanate, and its properties studied. The cellulase activity of the immobilized enzymes varied with monomer composition and molecular weight of polyethyleneglycols. The enzyme activity was affected by the viscosity of the carrier. A solid substrate (cellulose powder) can be hydrolyzed with the fluid immobilized enzyme.     

纤维素酶的底物专一性

天然纤维素的有效酶解取决于外切葡聚糖纤维二糖水解酶（ＣＢＨ）和内切葡聚糖水解酶（ＥＧ）的协同作用。ＥＧ随机水解纤维素无定形区分子链内的β－１,４－糖苷键;ＣＢＨ则由分子链的还原性末端水解出纤维二糖。这种底物专一性差别的原因在于ＣＢＨ呈“桶状”的活性部痊表面存在２个“ｌｏｏｐ”结构,只能容许纤维素分子链的末端伸入到活性裂隙中。ＥＧ无“ｌｏｏｐ”结构在存在,对底物是充分可及的。ＥＧ催化结构域中底物结合     

Visualizing cellulase activity

Commercial exploitation of lignocellulose for biotechnological production of fuels and commodity chemicals requires efficient—usually enzymatic—saccharification of the highly recalcitrant insoluble substrate. A key characteristic of cellulose conversion is that the actual hydrolysis of the polysaccharide chains is intrinsically entangled with physical disruption of substrate morphology and structure. This “substrate deconstruction” by cellulase activity is a slow, yet markedly dynamic process that occurs at different length scales from and above the nanometer range. Little is currently known about the role of progressive substrate deconstruction on hydrolysis efficiency. Application of advanced visualization techniques to the characterization of enzymatic degradation of different celluloses has provided important new insights, at the requisite nano‐scale resolution and down to the level of single enzyme molecules, into cellulase activity on the cellulose surface. Using true in situ imaging, dynamic features of enzyme action and substrate deconstruction were portrayed at different morphological levels of the cellulose, thus providing new suggestions and interpretations of rate‐determining factors. Here, we review the milestones achieved through visualization, the methods which significantly promoted the field, compare suitable (model) substrates, and identify limiting factors, challenges and future tasks. Biotechnol. Bioeng. 2013; 110: 1529–1549. © 2013 Wiley Periodicals, Inc.     

Measurement of saccharifying cellulase

This article sets forth a simple cellulase assay procedure. Cellulose is variable in nature, insoluble and resistant to enzymatic attack. As a result there have been a bevy of bewildering cellulase assays published that yielded irrational results. Certain protocols focused on the rapidity of the assay while ignoring that only the most readily susceptible cellulose regions were being hydrolyzed. Other assays simplified the system by using modified soluble substrates and yielded results that bore no relationship to the real world hydrolysis of insoluble cellulose. In this study Mandels, Andreotti and Roche utilized a common substrate, Whatman filter paper. Hydrolysis of a 50 mg sample of the paper was followed to roughly 4% degradation, which circumvented the problems of attack of only the most susceptible zones. This common hydrolysis target range also resulted in some balance with regard to the interaction of the several cellulase components. The method was subsequently widely adopted.     

Abscission: role of cellulase

Cellulase (β-1,4-glucan-glucanohydrolase EC 3.2.1.4) activity increased during abscission and was localized in the cell separation layer of Phaseolus vulgaris L. cv. Red Kidney (bean), Gossypium hirsutum L. cv. Acala 4-42 (Cotton) and Coleus blumei Benth. Princeton strain (Coleus) abscission zone explants. Cellulase activity was optimum at pH 7, was reduced by one-half after heating to 55° for 10 min, and was associated with the soluble components of the cell. Explants treated with aging retardants (indoleacetic acid, ⁶N-benzyladenine, and coumarin), CO₂, actinomycin D or cycloheximide had less cellulase activity than untreated controls. Ethylene increased cellulase activity of aged explants after a 3-hr lag period but had no effect on cellulase activity of freshly excised explants. It was concluded that 1 of the roles of ethylene in abscission is to regulate the production of cellulase which in turn is required for cell separation.     

纤维素酶的研究进展

纤维素酶是糖苷水解酶的一种,它可以将纤维素物质水解成简单糖,进而发酵产生乙醇,从而解决农业、再生能源以及环境污染等问题。初期的研究主要集中在对微生物纤维素酶的研究,随着对纤维素酶研究的不断深入,“动物自身不含纤维素酶”这一传统理论被推翻,动物纤维素酶成为纤维素酶研究的热点。另外,生物化学、分子生物学以及基因工程等多种交叉学科的快速发展,获得适合工业化的高比活力的纤维素酶已指日可待。     

宏基因组学在纤维素酶研究中的应用进展

纤维素酶能降解纤维素,被广泛应用于生物修复、食品加工、化工合成等领域,开发高活力、广底物、耐高温高碱等极端条件的新型纤维素酶具有重要意义。宏基因组学以特定环境样品中微生物的基因组总和为研究对象,避开传统的微生物分离培养过程,为基因资源的开发、利用提供了新技术。文中结合本课题组的研究工作,综述了利用宏基因组学获取纤维素酶的策略,同时着重介绍利用宏基因组学从动物胃肠道、土壤等环境中获取纤维素酶的研究。     

Properties of cellulase fromTrichoderma viride

Cellulase produced by Trichoderma viride acted on carboxymethyl cellulose with a Km of 4.9 g substrate per litre, showing a pH optimum at 4.5 and a temperature optimum at 55 degrees C. Ag+, Hg2+, Zn2+, Cu2+ and N3- were inhibitory..     

纤维素酶的固体培养方法

通过对菌株产酶能力的测定,确定了绿色木霉（Trichoderma viride)HWO7作为生产菌株。研究了培养基组成、培养条件和培养过程对产酶能力的影响,确定了固体培养生产纤维素酶的生产工艺。研究了纤维素酶的酶学特性。在确定工艺条件下,曲料的绝干酶活力（CMC－Na）达2605．1u/g,产品收率79．0％。     

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。     

Production of cellulase by Trichoderma.

The cellulase complex in T. viride is inducible. For large-scale enzyme production the fungus should be cultured on media containing cellulose. The cellulase enzymes are respressible. To produce and maintain best cellulase yields cultural conditions which lead to carbohydrate consumption in excess of cellular needs should be avoided. With the present mutant (QM9414) extracellular enzyme preparations having 1.6 FP units/ml and 1.6 mg protein/ml have been obtained within four to five days in submerged fermentation. Such preparations are capable of producing a 5% sugar solution when mixed with 10% ball milled cellulose and incubated 24 hr at 50 degrees C. Further improvements of cellulase yields are being sought by continued mutagenesis and increased nutrient levels in the growth medium.