外切纤维素酶的研究与应用进展

纤维素酶主要包括三大类：外切纤维素酶、内切纤维素酶和β-葡萄糖苷酶。其中,外切纤维素酶因具有活性高、耐受性好、来源广等特点而被广泛应用于各种工业生产中。基于此,主要阐述了外切纤维素酶的分类、来源以及生化特性,介绍了外切纤维素酶的筛选及其水解产物分析的相关新技术,并综述了其在造纸业、食品加工业、制药业、纺织业、能源再生等工业领域中的应用进展,以期推动外切纤维素酶的深入研究及工业化应用。     

木质纤维素降解酶生产菌株的遗传改造及应用

通过纤维素酶将木质纤维素向生物新能源的转化对经济社会的可持续发展具有重要意义,被用于纤维素酶制剂工业化生产的微生物大多属于丝状真菌,但丝状真菌的遗传操作困难,且纤维素酶诱导机制尚未阐明,严重制约了纤维素酶高产菌株选育与应用。本文综述了近年来纤维素酶高产菌株遗传操作方法的进展,重点论述了丝状真菌合成纤维素酶过程中的信号感应、信号传导、转录调控的研究,通过理性改造以提高纤维素酶生产菌株的产酶能力,并且总结展望了丝状真菌在工业生产中的应用。     

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。     

微生物纤维素酶的研究现状

主要探讨了近年来微生物纤维素酶的研究进展。重点概述了纤维素酶的分子结构、功能、作用机制以及产纤维素酶微生物种类的研究现状,并对该领域的研究问题和前景进行讨论。     

丝状真菌中纤维素酶与半纤维素酶的合成调控

综述了丝状真菌合成纤维素酶和半纤维素酶的相关调控研究进展。对最近研究文章分析发现:细胞外信号分子(C源、光信号),转录因子以及染色质重建等对丝状真菌合成调控纤维素酶及半纤维素酶有重要影响。同时解析丝状真菌合成纤维素酶和半纤维素酶调控网络,以期为利用基因工程改造纤维素酶和半纤维素酶生产工业菌株提供理论指导。     

碳纳米管固定化纤维素酶的最佳工艺研究

纤维素酶在环保、医药、食品等领域都具有广泛的应用前景,但由于纤维素酶的生产成本较高,生物活性较低,使得纤维素酶的应用受到了限制。为了寻找一种固定化纤维素酶的方法,使酶可以重复多次使用,首次以多壁碳纳米管为载体固定化纤维素酶,研究功能化的多壁碳纳米管固定化纤维素酶的固定化条件,采用正交试验对酶固定化中的主要条件进行优化,并通过傅里叶变换红外光谱仪对多壁碳纳米管（multiwalled carbon nanotube,MWCNTs）、纤维素酶及固定化纤维素酶的结构进行表征。结果表明,固定化纤维素酶的最佳工艺条件为：酶浓度5 mg·mL-1,温度40 ℃,pH 5.0,固定化时间3 h;通过傅里叶变换红外光谱证实纤维素酶成功固定到多壁碳纳米管上。     

碱性纤维素酶及其应用的研究进展

碱性纤维素酶在碱性条件下具有内切β-1,4葡萄糖苷酶活力,在加酶洗涤剂等行业具有重要的应用价值。目前已发现由芽孢杆菌和链霉菌产生的多种碱性纤维素酶,其中大部分的基因已被克隆、测序和表达,其催化反应特性、反应机理和应用也得到了较深入的研究。从碱性纤维素酶的产生菌、酶反应特性和反应机理、碱性纤维素酶的基因克隆和表达,以及碱性纤维素酶的应用等几个方面,介绍了有关碱性纤维素酶的最新研究进展。     

丝状真菌中纤维素酶与半纤维素酶的合成调控简

综述了丝状真菌合成纤维素酶和半纤维素酶的相关调控研究进展。对最近研究文章分析发现：细胞外信号分子（C源、光信号）,转录因子以及染色质重建等对丝状真菌合成调控纤维素酶及半纤维素酶有重要影响。同时解析丝状真菌合成纤维素酶和半纤维素酶调控网络,以期为利用基因工程改造纤维素酶和半纤维素酶生产工业菌株提供理论指导。     

微生物纤维素酶分子生物学研究进展

概述了近二十年来纤维素酶分子生物学的研究情况,包括纤维素酶合成调控,纤维素酶基因结构,基因复制及纤维素酶合成调控。     

斜卧青霉去泛素化蛋白酶CREB的缺失提高纤维素酶的生产

斜卧青霉Penicillium decumbens T.是1种重要的产纤维素酶丝状真菌,能有效地降解利用木质纤维素生产第2代生物燃料。为了提高斜卧青霉纤维素酶的产量,构建了去泛素化酶基因creB的敲除盒,并通过同源双交换重组的方法,获得了creB基因缺失突变株ΔcreB。该突变株呈现明显的纤维素酶表达分泌抗葡萄糖代谢阻遏效应,ΔcreB菌株的滤纸酶活、内切纤维素酶活、木聚糖酶活以及外切纤维素酶活分别提高1.8倍、1.71倍、2.06倍以及2.04倍,其胞外蛋白质含量提高了2.68倍。确定了creB基因缺失突变株具有抗碳源代谢物阻遏的生理现象,CREB对斜卧青霉生产纤维素酶的能力具有显著影响,为系统改造丝状真菌高产纤维素酶菌株提供了理论指导。     

Characterization and immobilization of liposome-bound cellulase for hydrolysis of insoluble cellulose

The liposome-bound cellulase was prepared by covalently coupling cellulase with the enzyme-free liposomes bearing aldehyde groups so that cellulase was located solely on the outer membrane of liposomes. The modified cellulase possessed the higher activity efficiency and lipid-based specific activity than the cellulase-containing liposomes reported previously. The enzyme-free liposomes bearing aldehyde groups were covalently immobilized with the chitosan gel beads and the free cellulase was coupled with the treated gel beads to prepare the immobilized liposome-bound cellulase. The activity efficiency of the immobilized liposome-bound cellulase was much higher than that of the conventionally immobilized cellulase. The results on reusability of the immobilized liposome-bound cellulase in the hydrolysis of either soluble or insoluble cellulose showed that the immobilized liposome-bound cellulase had the higher remaining cellulase activity and reusability than the conventionally immobilized cellulase for the hydrolysis of either type of cellulose. The liposomal membrane was suggested to be efficient in maintaining the cellulase activity during the hydrolysis.     

2种纤维素酶水解效果比较及酶蛋白组分分析

比较了自产纤维素酶和商品纤维素酶的水解效果,并采用超滤、层析、SDS-PAGE相结合的方法分析2种纤维素酶蛋白组分的差异。里氏木霉以纸浆为C源合成的自产纤维素酶的水解得率高于商品纤维素酶,自产纤维素酶水解48h的得率为66.24%,商品纤维素酶的得率为52.19%。自产纤维素酶中存在着Cel6A酶组分和XYNⅡ酶组分,而商品纤维素酶中没有检测到这2种酶组分。自产纤维素酶和商品纤维素酶的Cel1A酶组分和Cel7A酶组分间存在着分布和含量上的差异。自产纤维素酶在相对分子质量(2.5～3.5)×104范围内存在着几条蛋白条带,而商品纤维素酶则是在相对分子质量3.5×104附近存在着几条蛋白条带。     

Changes in Two Forms of Membrane-Associated Cellulase during Ethylene-Induced Abscission

Only one form of membrane-associated cellulase was found previously in the lower petiolar pulvinus of Phaseolus vulgaris (cv Red Kidney). The cellulase has an isoelectric point (pI) of 4.5 (DE Koehler, LN Lewis 1979 Plant Physiol 63: 677-679). This enzyme was detected in abscission zones collected before the onset of abscission (control tissue), and was thought to represent a pre-secretory form of another cellulase, the abscission cellulase, which has a basic pI and is secreted during abscission. We now show that this acidic, membrane-associated cellulase is a glycoprotein, tightly bound to the membrane, with maximum activity at pH 5.1, and that it is not immunologically related to the abscission cellulase. Furthermore, when bean explants are induced to abscise with ethylene, the activity of the acidic cellulase declines rapidly to 50% of control levels in the first day. When abscission is fully developed, the membranes contain a basic form of cellulase with a pI of 8.0 to 9.0 and only trace levels of the acidic cellulase. The basic form is not a high mannose glycoprotein; it has maximum activity in a broad pH range (4.0-8.0) and is antigenically related to the abscission cellulase, which is induced during abscission and transported to the cell wall. Antibody raised against the abscission cellulase recognized two proteins in a crude membrane fraction from abscising tissue. One of those proteins comigrated with the abscission cellulase, and the other was 1 to 2 kilodaltons larger. Thus, during abscission, the acidic membrane-associated cellulase rapidly declines before the appearance of the abscission cellulase. We conclude that there is no conversion from the acidic cellulase to the basic cellulase and suggest that the acidic and basic cellulase isoenzymes are proteins derived from two different genes.     

Saccharification and adsorption characteristics of modified cellulases with hydrophilic/hydrophobic copolymers.

Saccharification and adsorption characteristics of native and modified cellulases were investigated. Copolymers, containing polyoxyalkylene and maleic anhydride (MA) were used to modify cellulase. Amino groups of the cellulase were covalently coupled with the MA. As the degree of modification (DM) increased, the activity of modified cellulase slightly decreased. At the maximum DM, the modified cellulase activity retained more than 75% of the unmodified native cellulase activity. In saccharification, native cellulase rapidly adsorbed onto the substrate at initial reaction time. Native cellulase adsorbed tightly onto the substrate surface and did not desorb as reaction time proceeded. The strong adsorption of cellulase onto the substrate can, however, be controlled by the modification. As the hydrophilicity of modified cellulase increased, free modified enzyme concentration also increased. As a result, the conversion rate of modified cellulase was higher than the native one.     

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.     

The effect of pH on the foam fractionation of beta-glucosidase and cellulase

The surface tension-pH profile of beta-glucosidase was established to determine its relationship to the corresponding profile of cellulase and to the foam fractionation of that cellulase. The goal of this work was to determine the optimal foaming points for both cellulase and cellobiase. This data may prove useful in the separation of certain components of cellulase, since the non-foaming hydrophilic beta-glucosidase does not foam as well as the hydrophobic components of cellulase at low concentrations. A key finding from these experiments was that there are two local minima in the surface tension-pH trajectory for Trichoderma reesei cellulase, as contrasted to the usual single minimum. The lower of these minimum points corresponds to the cellulase isoelectric point. The double minimum surface tension-pH profile was also observed for cellobiase alone. The optimal foaming pH for cellobiase alone was determined to be around 10.5, while for cellulase it was between 6 and 9.     

Competitive adsorption of cellulase components and its significance in a synergistic mechanism

Some studies on the adsorption of cellulase on cellulose revealed part of the mechanisms involved in the enzymatic hydrolysis of cellulose and provided some clues to the synergistic mechanism of cellulase complex. The adsorption of cellulase was significantly affected by the reaction conditions and physical chemical characteristics of cellulose. Endoglucanase consisted of adsorbable and nonadsorbable components. Cellobiohydrolase had the strongest adsorption affinity. Each cellulase component is postulated to have distinctly different adsorption sites on cellulose, corresponding to the active sites in the hydrolysis reaction. Competitive adsorption kinetics between cellulase components were also observed during the adsorption process. The degree of competitive adsorption was most remarkable when the composition of cellulase components was nearly the same as that in the crude cellulase complex. This seems to show the optimal relative composition of cellulase components. The synergism between cellobiohydrolase and endoglucananse could be elucidated more clearly by this competitive adsorption model of the reaction mechanism.     

Screening of cellulases for biofuel production: online monitoring of the enzymatic hydrolysis of insoluble cellulose using high-throughput scattered light detection

A new prospective cellulase assay simultaneously combining high-throughput, online analysis and insoluble cellulosic substrates is described. The hydrolysis of three different insoluble cellulosic substrates, catalysed by a commercial cellulase preparation from Trichoderma reesei (Celluclast), was monitored using the BioLector - allowing online monitoring of scattered light intensities in a continuously shaken microtiter plate. Cellulase activities could be quantitatively assayed using the BioLector. At low cellulase/cellulose ratios, the Michaelis-Menten parameters of the cellulase mixture were mainly affected by the crystallinity index of the cellulose. Here, the apparent maximum cellulase activities inversely correlated with the crystallinity index of the cellulose. At high cellulase/cellulose ratios the particle size of the cellulose, defining the external surface area accessible to the cellulases, was the key determining factor for cellulase activity. The developed technique was also successfully applied to evaluate the pH optimum of cellulases. Moreover, the non-hydrolytic deagglomeration of cellulose particles was investigated, for the first time, using high-throughput scattered light detection. In conclusion, this cellulase assay ideally links high-throughput, online analysis and realistic insoluble cellulosic substrates in one simple system. It will considerably simplify and accelerate fundamental research on cellulase screening.     

Molecular cloning and nucleotide sequence of the alkaline cellulase gene from the alkalophilic Bacillus sp. strain 1139

The cellulase gene from the alkalophilic Bacillus sp. strain 1139 was cloned in Escherichia coli using pBR322. Plasmid pFK1 was isolated from transformants producing cellulase, and the cloned cellulase gene was found to be in a 4 X 6 kb HindIII fragment. The cellulase gene was subcloned in a functional state on a 2 X 9 kb DNA fragment and its nucleotide sequence was determined. The coding sequence showed an open reading frame encoding 800 amino acids. The pFK1-encoded cellulase had the same enzymic properties as the extracellular cellulase produced by the alkalophilic Bacillus sp. strain 1139, but its Mr was slightly higher.     

Occurrence and Localization of 9.5 Cellulase in Abscising and Nonabscising Tissues

Nitrocellulose tissue prints immunoblotted with 9.5 cellulase antibody were used to demonstrate areas of cellulase localization within Phaseolus vulgaris explants on exposure to ethylene. The 9.5 cellulase was induced in the distal and proximal abscission zone and in the stem. In both abscission zones, the 9.5 cellulase was found in the cortical cells of the separation layer, which develops as a narrow band of cells at the place where fracture occurs. The enzyme was also found associated with the vascular traces of the tissues adjacent to the separation layer extending through the first few millimeters at each side of the separation layer. The two abscission zones differed in the way that cellulase distributed through the separation layer as abscission proceeded. In the distal zone, cellulase appeared first in the cells of the separation layer adjacent to vascular traces and extended toward the periphery. In the proximal zone, 9.5 cellulase accumulated first in the cortical cells that lie in the adaxial side and then extended to the abaxial side. In response to ethylene, 9.5 cellulase was also induced in the vascular traces of the stem and the pulvinus without developing a separation layer. The role of 9.5 cellulase in the vascular traces is unknown. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified the same 51-kilodalton protein in both abscising and nonabscising tissues. Therefore, the determinant characteristic of the abscission process is the induction of 9.5 cellulase by cortical cells in the separation layer, and this implies that these cells have a unique mechanism for initiating 9.5 cellulase synthesis.