选育纤维素乙醇生产的高效产酶工程菌技术的研究进展

综述了近年来选育高效产纤维素酶工程菌取得的最新理论研究及工艺进展,并就复合理化诱变获取高效产酶菌,以及利用原生质体融合、原生质体诱变、基因工程等技术对各种产纤维素酶、半纤维素酶菌株进行遗传改造,对纤维素酶高效产酶工程菌的选育进展进行了总结,进一步展望了选育高产纤维素酶菌株的研究方向。     

红树林环境产纤维素酶菌株的研究现状

红树林是自然分布于热带和亚热带位于陆地和海洋交界的滩涂地带,是陆地向海洋过度的特殊生态系统。由于红树林落叶等木质纤维素的沉积,产纤维素酶微生物也是该区的重要生物组成部分。文章综述了近几年来国内从红树林环境分离筛选产纤维素酶微生物的研究现状及应用前景。     

回转条件下微生物产酶表征

选定3株高产淀粉酶、蛋白酶、纤维素酶的细菌(枯草芽孢杆菌)、放线菌(链霉菌)和丝状真菌(里氏木霉)作为模式菌株,研究了在回转条件下单独发酵产酶及混合培养产酶过程及能力。结果表明,枯草芽孢杆菌、链霉菌和里氏木霉在pH值6.0～7.9之间都可生长。在回转条件下发酵30d,枯草芽孢杆菌维持一定产淀粉酶和蛋白酶能力;链霉菌能够产少量的蛋白酶和淀粉酶;里氏木霉除了具有一定的产纤维素酶能力外,也能产少量的蛋白酶和淀粉酶。将3株菌进行分步混合培养,发酵过程中均能保持较高的淀粉酶活力和蛋白酶活力,在20d和30d的发酵液中检测不到纤维素酶活力。     

一株产纤维素酶降香内生真菌的分离鉴定及产酶活性检测

从降香中分离纯化获得一株产纤维素酶内生真菌JXP2-2,并对其进行了产酶活性测定和分子生物学鉴定.结果表明,菌株JXP2-2在PDB液体培养基中培养4.5 d时的产酶活性最强,纤维素酶活力达到84.50 IU·mL-1;菌株JXP2-2与尖孢炭疽菌(Colletotrichum acutatum)KX347475的碱基...     

一株高产低温纤维素酶南极细菌的筛选、鉴定及酶学性质初步研究

从82株南极细菌中筛选出1株高产低温纤维素酶菌株NJ64,通过16SrDNA序列分析对其进行鉴定,并对其产酶条件及酶学性质进行了初步研究。结果表明,NJ64菌株为假交替单胞菌属（Pseudoalteromonassp．）;最适产酶条件为：培养时间72h、初始pH值7．5、培养温度10℃;所产纤维素酶在pH值9．0左右、40℃条件下的酶活性最高,属于低温酶。     

一株产纤维素酶镰刀菌的筛选、鉴定与酶学性质

根据菌株菌落、菌丝体、孢子等形态特征及其生理特性,初步鉴定高产纤维素酶的丝状真菌为尖孢镰刀菌(Fusarium oxysporum),命名为XA-1。考察了不同碳源及氮源、培养温度、初始pH等因素对XA-1产酶的影响,并研究了该菌所产纤维素酶酶学性质及酶解性能。该菌的最适产酶条件为:分别以水葫芦和硫酸铵为碳、氮源,30℃,pH 5.0,培养6 d后,内切葡聚糖酶(CMCase)、β-葡萄糖苷酶(β-Gluase)和滤纸酶活力(FPA)分别达到4 083.2、3 258.8 U/g和773.2 U/g(成熟曲)。CMCase、β-Gluase最适反应温度为45℃,FPA则为55℃;CMCase、β-Gluase和FPA的最适反应pH分别为5.0、4.5和5.0。菌株XA-1纤维素酶酶解香蕉秆或水葫芦32 h后,酶解得率分别达到27.3%和29.8%。菌株XA-1在纤维素酶开发及转化秸秆类纤维素为可发酵糖方面显示出较好的应用前景。     

纤维素改性研究进展

综述了近年来纤维素改性的进展情况。纤维素预处理往往是纤维素改性的第一步,包括物理化学等方法。纤维素改性物主要包括纤维素酯类、醚类及接枝共聚物,介绍了近年来其化学改性的方法进展。纤维素的生物改性主要应用于造纸行业,利用纤维素酶、半纤维素酶等处理纸浆。细菌纤维素的改性方法包括细菌发酵时的生物改性及纤维素提纯之后的化学改性。最后展望了纤维素改性的应用前景。     

桦剥管孔菌固态发酵纤维素酶及发酵基质酶解

建立纤维素酶固态发酵与生物预处理相耦合工艺。实验优化固态发酵条件,测定发酵基质结晶度及酶解糖化得率。结果表明3 g稻草粉为基质,0.5%淀粉为碳源,1%蛋白胨为氮源,0.5%芦丁,初始pH值为5,发酵14d,褐腐真菌Piptoporus betulinus产CMC酶活力达到76.46 U/g,滤纸酶活力达到7.75 U/g;酶解糖化阶段减少外源纤维素酶量36.05%;P.betulinus降解固态基质中的无定型纤维素,暴露结晶纤维素,提高发酵基质的酶解糖化得率184%。     

紫外线诱变提高细菌产纤维素酶活力的研究

从南京红山动物园土样及食草动物的粪便中分离出14种产纤维素酶菌株,其中F1表现出较高的酶活力.以F1为出发菌株,通过紫外线诱变处理,采用透明圈法初筛和摇瓶培养复筛,获得了10株高产纤维素酶的突变株Q1～Q10.经紫外线诱变处理的Q3突变株产酶活力最高,与出发菌株相比酶活力提高了15.8倍.     

联合生物加工产纤维素乙醇中真菌的开发与应用

联合生物加工（consolidated bioprocessing,CBP）是在单一或组合微生物作用下,将纤维素酶生产、纤维素水解糖化、戊糖和己糖发酵产醇整合于单一步骤的生物加工过程。本文从真菌在CBP产纤维素乙醇中的开发历程着眼,回顾了纤维素乙醇产业化的发展进程,介绍了CBP产纤维素乙醇的作用机理,系统总结了目前国内外文献中报道的CBP底盘真菌的主要种类及优缺点,并综述了CBP真菌的开发策略,包括工程化策略和共培养策略,着重阐述了工程化策略的技术路线和研究进展。指出综合运用先进生物技术和基于代谢分析数据的计算机模拟系统开发CBP目标微生物,设计新型高效的生物反应器以及将CBP技术与现有生物工业整合,是未来将CBP技术应用于纤维素乙醇产业的关键。     

Enzymatic Hydrolysis of Carboxymethylcellulose and Filter Paper by Immobilized Cellulases on Lignophenols

The cost of cellulase is a major factor limiting the enzymatic hydrolysis of cellulosic biomass. Thus, immobilization of cellulase would be an important advancement. Lignophenol is a lignin-based functional phenolic polymer synthesized from a lignocellulosic material and a phenol derivative at ambient temperature. Cellulase derived from Trichoderma reesei is easily immobilized by softwood and hardwood lignocresol simply by mixing to produce a water-insoluble lignophenol-cellulase complex. Enzymatic hydrolysis performances of cellulases immobilized on lignocresols are approximately 80–90% and 30–50% relative to that of free cellulase in the hydrolysis of carboxymethylcellulose (CMC) and filter paper, respectively. Cellulase was active enough even after adsorption on lignocresols. Limited physical contact between solid substrates and immobilized cellulase due to the presence of lignocresol seems to lead to lower enzymatic activity for solid substrates. Hardwood lignocresol-immobilized cellulase exhibits slightly higher activity than softwood lignocresol-immobilized cellulase when the same amount of cellulase is used per gram lignocresol. Although cellulase activity gradually decreases with recycling, sufficient enzymatic activity, at least for hydrolyzing soluble substrates, remains that it can be reused.     

A Comparison of the Activity Reduction Occurring in Two Detergent‐Assisted Protein (Cellulase and Lysozyme) Foam Fractionation Processes

Abstract

Foam fractionation has the potential to be an inexpensive alternative to current protein drug concentration or separation methods; however, it has a few drawbacks. One is the fact that not all proteins form a foam layer when aerated at low concentrations. The other is the possible protein denaturation caused during the foaming process. Adding a detergent to the nonfoaming protein solution causes it to foam when aerated. Here, cellulase and lysozyme are studied as model proteins in this process. By themselves, both cellulase and lysozyme solutions hardly form a foam layer when aerated at concentrations below 1000 mg/L (1000 ppm). The addition of 100 mg/L of cetyltrimethylammonium bromide (CTAB) to a 200 mg/L cellulase solution increases the foam volume and makes it possible to almost quadruple (relative to the initial bulk concentration) the concentration of the resulting cellulase foam solution. The foaming, however, reduces the cellulase activity. Diluting the foam with β‐cyclodextrin regains some of the lost activity because β‐cyclodextrin strips CTAB away from the cellulase, which allows the cellulase to refold to its native state. CTAB detergent does not work well with lysozyme, but the addition of SDS detergent leads to a tripling of the concentration of lysozyme solution without any reduction in enzymatic activity.     

纤维素酶水解及其在能源与环境保护中的应用

纤维素酶的利用是扩大纤维素应用领域和高值化利用纤维素的一条可行的新途径。本文综述了纤维素酶的来源、纤维素酶的结构和组成、纤维素酶解机理及影响因素、纤维素酶在能源及环境保护方面的应用。     

生物技术生产纤维素酶及其应用研究进展

简要介绍纤维素酶的酶学性质、降解机制、生产工程菌的选育、纤维素酶的应用情况,以及对纤维素酶生产与应用方面存在的问题和未来发展趋势进行了分析与探讨。纤维素酶在食品、酿造行业、农副产品深加工、饲料、医药、环境保护和化工等领域有着非常广阔的应用前景和应用潜力。我国纤维素酶的生产及应用研究近年来取得了很大进展,今后必将在应用深度和广度上进一步扩展。     

Improvement of cotton cloth soil removal by inclusion of alkaline cellulase from Bacillus sp. KSM-635 in detergents

Alkaline cellulase from Bacillus sp. KSM-635 (EC 3.2.1.4) had reproducible detergent effects on cotton cloth that was artificially or naturally soiled with oily and/or particulate matter, under European washing conditions. The detergent effects of the cellulase, in combination with surfactants, apparently were the result of enzymatic action on amorphous regions of cotton fibers in which soil was trapped. The contribution of cellulase to soil removal increased as (i) the amount of soil in the amorphous regions of fibers in test fabrics was increased and (ii) the inhibition, by soil that adhered to the fibers' surfaces, of the action of the cellulase on fibers was reduced. Alkaline cellulase had the potential to replace, in part, both surfactants and zeolite in detergents, and it reduced washing time and allowed washing at lower temperatures under European washing conditions. The marked detergent effect of cellulase on naturally soiled cotton fabric was visually apparent, and it inhibited the accumulation of ash, calcium, and other inorganic components on cotton fibers during wash-and-wear cycles. These contributions of cellulase to the cleanliness of cotton fabrics were clearly increased by repeated wash-and-wear cycles. Cotton fabrics were not degraded by washing with the cellulase because effective hydrolysis by the cellulase occurred only in the amorphous regions of cotton fibers.     

Effects of natural oils and surfactants on cellulase production and activity

Emulsification of natural oils by a surfactant increases their efficiency as chemical antifoams. The presence of low concentrations of fatty acids or other surfactants have been reported to inhibit or stimulate microbial growth and/or product formation. The effects of different natural oils, Tween 80 and saponin on growth and cellulase production by T. reesei and S. pulverulentum have been investigated. It was found that in general, emulsification leads to higher cellulase activities in both cultures, though there are variations in enzyme levels depending on the presence or absence of Tween 80 and of different oils in growth media as well as the substrates used for cellulase assay.     

Characterization of cellulase under various intensities of static magnetic fields

The activity and secondary conformation of cellulase were investigated under static magnetic fields (0.15, 0.30, and 0.45 T). Compared to control group, activity and conformational change of cellulase were observed by means of UV spectrophotometer and far-UV circular dichroism detection after exposed in various static magnetic fields. The cellulase exhibited its maximal specific activity in the pH range of 4.0–4.6, and the optimal temperature was 60 °C. In addition, the circular dichroism spectra and kinetic parameters of cellulase have also been changed by magnetic fields.     

Immobilization of cellulase on functionalized cobalt ferrite nanoparticles

Amine functionalized cobalt ferrite (AF-CoFe₂O₄) magnetic nanoparticles (MNPs) were used for immobilization of cellulase enzyme via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDS) and N-hydroxy-succinimide (NHS) coupling reaction. The structural, morphological and magnetic properties of AF-CoFe₂O₄ were determined. TEM micrograph revealed a mean diameter of ～8 nm and showed that the AF-CoFe₂O₄ remain distinct with no significant change in size after binding with cellulase. Fourier transform infrared (FT-IR) spectroscopy confirmed the binding of cellulase to AF-CoFe₂O₄. The properties of immobilized cellulase were investigated by optimizing binding efficiency, pH, temperature and reusability. The results showed that the immobilized cellulase has higher thermal stability than free cellulase, which might be due to covalent interaction between cellulase and AF-CoFe₂O₄ surface. The immobilized cellulase also showed good reusability after recovery. Therefore, AF-CoFe₂O₄ MNPs can be considered as promising candidate for enzyme immobilization.     

Cleanability Improvement of Cotton Fabrics Through Their Topographical Changes Due to the Conditioning with Cellulase Enzyme

In this study, topographical changes of woven cotton fabrics conditioned with a cellulase enzyme during several wash–dry cycles are systematically studied. A recent study of cellulase enzyme effect on cellulose films has proven that this substance selectively attacks amorphous regions of cellulose, consisting of small hills in a matrix of flat crystalline regions. In another study, topographical changes caused by cotton treatment with cellulase by conditioning while washing were analysed on three different length scales in order to interpret their cooperation on water and oil absorption mechanisms and, hence, on the cleanability of cotton fabrics stained with liquid–solid, liquid and solid soils. In the present study, we emphasise the micro-topographical changes resulting from several wash–dry cycles by the application of mathematical methods to quantify the changes of yarn micro-surfaces. As a result, we present a conceptual model that describes how the topographical effect of washing and conditioning by cellulase enzyme improves the cleanability of woven plain cotton fabrics.     

Tween 80 enhancing cellulase recyclability during activation of dissolving pulp

Abstract

Enhancing cellulase recyclability is a promising approach to reducing the high enzyme cost for activation of dissolving pulp. For this purpose, Tween 80, an amphiphilic surfactant, was employed to facilitate cellulase recovery from fibers. Results indicated that the Cellulase–Tween complex was formed (with a particle size increase of 140%) through a hydrophobic interaction. A much lower maximum binding capacity and affinity (i.e., absorption ability) of cellulase onto fibers (6.03 versus 24.3?mg protein/g cellulose and 7.45 versus 13.8?ml/mg protein) were achieved for the complex in comparison with the control. The recovered cellulase activity was increased up to 65.7% from the control one in five consecutive recycle rounds with Tween 80 addition. As a result, the recovered cellulase efficiency was improved in terms of viscosity and reactivity. Other properties, such as α-cellulose content, alkaline solubility, molecular weight distribution, morphology, and crystalline structure, were all supported by the positive effect of Tween 80 in cellulase recycling.