利用葡萄糖转苷酶制备纤维素酶可溶性诱导物的研究

在纤维素酶生产中,常用的诱导物纤维素是不溶性的固体高分子化合物,存在着传质阻力大、不易于流加培养、产酶效率低等问题.今以葡萄糖为原料,利用葡萄糖转苷酶的催化作用,定向合成纤维素酶的可溶性诱导物.经高效液相色谱分析,发现转糖苷产物中含有纤维素酶的强诱导物槐糖.在50℃下,转糖苷反应的适宜葡萄糖浓度为300～500 mg·mL-1,pH 3～4.5,反应100 h,产物中槐糖含量可达40 mg·mL-1.将葡萄糖经转苷酶作用后的复合物用于纤维素酶的生产,与直接采用葡萄糖相比,产酶时间提前25 h,滤纸酶活力提高14倍.该研究结果为酶法制备纤维素酶的高效可溶性诱导物探明了一条新途径,对于提高纤维素酶的生产效率、加速其工业化应用具有重要意义.     

丝状真菌产纤维素酶相关碳代谢阻遏因子的研究进展?

简述了酵母和丝状真菌中普遍存在的一种碳代谢抑制因子CRE(CreA、CreB、CreC、CreD和Cre1、Cre2、Cre3、Cre4);利用纤维素所必须的锌双核簇转录因子CLR-1,负责大部分纤维素酶基因表的CLR-2和CLR-1的活性阻遏者CLR-3;真菌响应外界环境有重要作用的VIB1以及协调初级代谢、碳代谢阻遏和真菌生长重要参与者cAMP依赖蛋白激酶A(PkaA)在丝状真菌纤维素酶生产中的调控方式及相关联系以及它们在不同的丝状真菌中的影响,进而综述它们对丝状真菌生产纤维素酶的影响。     

微生物降解纤维素机制的分子生物学研究进展

对纤维素酶的分子生物学，主要是该酶对天然纤维性底物的降解机制研究进展作了简要评述，包括纤维素酶的一、二、三级结构、酶分子的多形性、纤维素酶族、酶基因克隆方法及表达和分泌中存在的问题、新酶分子的构建等。并介绍对细胞融合、单克隆抗体、DNA体外定位诱变、活性中心测定和糖基化方法等在纤维素酶降解研究中的应用。     

真菌纤维素酶表观遗传修饰的研究进展

组蛋白乙酰化酶编码基因gcn5、蛋白甲基转移酶LaeA及其同源物Lae1、VeA等可通过作用于调节纤维素酶基因及其表达调控因子的表达,进而在转录水平上影响纤维素酶基因的表达。简述了真菌纤维素酶的种类与结构和真菌纤维素酶基因的表达调控机制,重点从DNA甲基化、蛋白甲基化和乙酰化以及非编码RNA等的角度,综述几种主要表观遗传修饰对真菌纤维素酶基因表达作用的研究进展。     

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。     

在植物病原真菌防治策略中的克隆化几丁质酶

几丁质酶所水解的几丁质——β-1,4-联乙酰基-D-葡萄糖胺的同聚物,为大部份真菌(包括子囊菌、担子菌和不完全真菌)细胞壁的主要成份。人们已从真菌、细菌和植物中分离得几丁质酶,它通过使菌丝顶端的几丁质合成退化以抑制真菌的生长。细菌(如沙雷氏菌属)和真菌(如木霉菌属)合成和分泌几丁质酶,则根据周围生长介质中所存在的几丁质的诱导而产生。在健康植物中,几丁质酶的含量很低,它们的表达方法随着诸如抗微生物化合物、蛋白酶抑制剂、β-1,3-葡聚糖酶等其他组份及受到病原体侵袭时,由植物防卫系统的诱导而增加。体内细胞中诱导产生几丁质酶的积累,其既不在细胞内,亦不在细胞外。迄今为     

美国合成生物异丁醇真菌细菌

<正>美国密歇根州密歇根州立大学和加州大学洛杉矶分校的研究团队日前宣布,已设计并合成出可用于从生物质直接生产异丁醇的真菌、细菌。真菌里氏木霉菌分泌的纤维素酶,使木质纤维素生物质水解成为可溶性糖类,大肠杆菌细菌可使可溶性糖类代谢成所需的产品。该项实验纤维素直接转化的理论最大产率高     

腐生丝状嗜热真菌分泌的嗜热纤维素水解酶研究进展

腐生丝状嗜热真菌是微生物中极重要的一个类群,大多数嗜热真菌能在常温及高温条件下生存.腐生丝状嗜热真菌分泌的嗜热酶具有优良的热稳定性以及酶活力,在工业生产中具有广阔的应用前景.本文对腐生丝状嗜热真菌分泌的三种主要嗜热纤维素水解酶在工业方面的应用、分子领域的研究现状予以综述,为嗜热纤维素水解酶在生物技术上的应用提供基础.     

果糖补料与真菌诱导对红豆杉细胞悬浮培养体系的协同效应

研究了果糖补料和真菌诱导对南方红豆杉细胞悬浮培养体系中细胞生长状态和紫杉烷合成的协同作用。结果表明果糖补料和真菌诱导子的协同作用引起细胞的生理状态发生改变 ,培养基碱化 ,细胞的生物量有一定程度的降低 ,而表征酶含量的蛋白质含量有明显的提高 ,酚类物质大量积累 ,目的产物紫杉醇 (Taxol)被大量合成 ,其产量在摇瓶和反应器体系中最高分别达到 81.0mg/L和 71.7mg/L。     

碳源对固定化里氏木霉合成纤维素酶的影响

本文研究了固定化里氏木霉（TrichodermareeseiRutC30）在不同碳源条件下合成纤维素酶的时间进程及酶系组成特征。试验结果表明，碳源的性质对纤维素酶复合体系的组成有明显影响。在以纸浆为碳源制备的酶液中C_1／FPA和C_x／FPA的比值较大，分别为39．32和107．41，而CB／FPA的比值较小（0．08）；在以淀粉水解液为碳源所制备的纤维素酶液中C_1／FPA和C_x／FPA的比值较小，分别为28.54和66．44，但CB／FPA的比值明显较大（0．14）。当采用复合碳源（淀粉水解液和纸浆）时，发酵液中C_1、C_x：和纤维二糖酶的三种组分的活力均可达到较高水平，C_1／FPA和C_x／FPA分别可达36．2和95．10，与单独采用可溶性碳源时相比有了明显提高，CB／FPA也可达到0．12，与碳源为纸浆时相比提高了50％。本研究结果在走向调节纤维素酶复合体系的构成，对进一步提高酶制剂的实际应用效能方面将具有重要的参考价值。     

Green tissue‐specific production of a microbial endo‐cellulase in maize (Zea mays L.) endoplasmic‐reticulum and mitochondria converts cellulose into fermentable sugars

BACKGROUND: Commercial conversion of lignocellulosic biomass to fermentable sugars for biofuels and chemical byproducts uses relatively expensive bulk production of biologically active cellulase enzymes, which could alternatively be achieved by using solar energy for direct production of these enzymes within feedstock crop cellulosic biomass. RESULTS: The Acidothermus cellulolyticus endo‐cellulase E1 has been produced in transgenic maize plants. This heterologous enzyme was specifically targeted for accumulation into two sub‐cellular compartments, endoplasmic reticulum (ER) or mitochondria of plant leaves and stalks. Furthermore, successful use of this maize‐produced heterologous cellulase in converting cellulose into fermentable sugars for biofuels, has been confirmed. CONCLUSIONS: Green‐specific expression of cellulases in maize plants can avoid public controversies associated with production of transgene products in maize seeds and/or pollen. Sub‐cellular targeting of cellulases may result in better expression of transgene products because these compartments, specially ER, normally contain molecular chaperones that enhance protein folding and there the biological activity. Also, using solar energy to produce cellulases within crop cellulosic biomass can replace the costly process of cellulase production in microbial bioreactors, and therefore, save costs. Copyright © 2008 Society of Chemical Industry     

Induction of xylose reductase and xylitol dehydrogenase activities on mixed sugars in Candida guilliermondii

The ability of various sugars to induce xylose reductase (aldose reductase, EC 1.1.1.21) and xylitol dehydrogenase (D -xylulose reductase, EC 1.1.1.9) activities in Candida guilliermondii was studied. D -Xylose was found to be the best inducer of activities of both enzymes, followed closely by L -arabinose. Very low xylose reductase activity was induced by cellobiose, D -mannose, D -glucose, D -galactose, D -fructose or glycerol. With xylitol dehydrogenase, cellobiose and D -fructose caused partial induction of enzyme activity, while negligible activity was induced by D -mannose, D -glucose, D -galactose or glycerol. Several sugars were tested for the ability to repress the induction by D -xylose of xylose reductase and xylitol dehydrogenase activities in C. guilliermondii. Enzyme activities induced on D -xylose served as controls. L -Arabinose, cellobiose and D -galactose did not repress enzyme induction by D -xylose, while D -mannose, D -glucose and D -fructose repressed enzyme induction to varying extents. Results from enzyme induction generally correlated with patterns of mixed sugar utilization, with some anomalies associated with the utilization of D -galactose and D -fructose in the presence of D -xylose. The results show that the utilization of D -xylose by C. guilliermondii is subject to regulation by induction and catabolite repression.     

Saccharification of microcrystalline cellulose by cellulase of Morchella conica mushroom mycelium in comparison with a commercial cellulase

A comparison was made of the hydrolysis of microcrystalline cellulose Avicel using the cellulolytic complex of the ascomycete Morchella conica and a commercial cellulase. An enzyme concentration of 1 U AVase mg^?1 substrate gave the best results during the hydrolytic processes. At 24 h, the M. conica enzyme complex achieved 35.5% and the commercial cellulase 31·2% saccharification, with glucose 84·2% and 52% of the total reducing sugars liberated, respectively. The specific rates of hydrolyses were 0·77 and 0·14 h^?1 for reducing sugars and 0·54 and 0·12 h^?1 for glucose formation with M. conica and the commercial cellulase, respectively. At 96 h, the degree of saccharification reached 46% for M. conica and 49% for the commercial cellulase, with glucose 76·5% and 65·9% of the total reducing sugars liberated, respectively. Both the complexes were quite stable with a residual activity of 62% CMCase and 47% AVase for M. conica, and 74% CMCase and 57% AVase for the commercial cellulase at 96 h of hydrolysis. The qualitative analysis of the hydrolysis products by TLC indicated, for M. conica, an earlier appearance of cellobiose, which was quickly hydrolyzed to glucose.     

Simultaneous hydrolysis and fermentation of pulp mill primary clarifier sludge

Bioconversion of sludge from the primary clarifier of a sulphite pulping operation to ethanol offers a number of advantages over conventional disposal options. The amount of material which must be disposed of is reduced while, at the same time, salable and environmentally friendly fuel-ethanol is produced. In this study, primary clarifier sludge (PCS) was shown to be hydrolysed to produce fermentable sugars at a rate proportional to enzyme loading. Initial (1 hour) hydrolysis rates as high as 12.6 g reducing sugar/L · h were observed at an initial enzyme loading of 10 filter paper units (FPU)/g. Hydrolysis was inhibited by spent sulphite liquor (SSL), an inhibition which could be completely overcome by fermenting the SSL to remove sugars. Surfactants were found to only marginally improve the production of sugars. To reduce the deleterious effects of end product inhibition, single stage simultaneous hydrolysis and fermentation (SHF) was carried out using cellulase enzymes and Saccharomyces cerevisiae.     

Growth rate dependence of enzyme synthesis in chemostat cultures: α-amylase, β-galactosidase,acid phosphatase and β-fructosidase

Specific rate of enzyme synthesis in microbial cells was correlated to specific growth rate in chemostat culture using three strains of microorganism. α-Amylase production by Bacillus subtilis was linearly associated with growth rate in a glucose-limited chemostat culture. β-Galactosidase production by Saccharomyces fragilis was rather invariable with growth rate in a galactose medium. β-Fructosidase production by Saccharomyces carlsbergensis in a phosphate-poor glucose medium was independent on growth rate, except at low dilution rates where it increased with growth rate. Production of acid phosphatase by the same microbe showed a maximum at a particular intermediate dilution rate. These relationships were summarised with an equation of induction—repression model. Comparison of the chemostat results with those of batch fermentation provided some enhancement factors of a continuous fermentation: 1.23–9.82 regarding enzyme productivity and 0.53–2.88 regarding enzyme concentration. The production of β-fructosidase was much improved among the enzymes tested when continuous culture methods were used.     

Hydrothermal pre-treatment and enzymatic hydrolysis of sunflower stalks

The production of fermentable sugars by biochemical conversion of pretreated sunflower stalks is studied in this work. Liquid hot water pre-treatment, in a temperature range from 180 to 230 °C was used. After pre-treatment, solid residue and liquid fractions are separated by filtration. Pretreated solids are further submitted to enzymatic hydrolysis for glucose release using a commercial enzyme preparation. Enzymatic hydrolysis yields show that 90% glucose conversion can be obtained after 72 h enzyme action on pretreated sunflower stalks at 220 °C. Nevertheless, under such pre-treatment conditions most of the hemicellulosic sugars are lost due to solubilisation and degradation reactions. Taking into account all sugars that can be obtained from this agricultural residue, the best results in terms of potential ethanol production is found when pretreating sunflower stalks at 190 °C. The characterization of hydrolysate composition is also discussed as inhibitor compounds are also found in liquid fractions issued from pre-treatment.     

The quest for alternatives to microbial cellulase mix production: corn stover‐produced heterologous multi‐cellulases readily deconstruct lignocellulosic biomass into fermentable sugars

BACKGROUND: Production of cellulosic ethanol is still expensive compared with corn (maize) grain ethanol due to the high costs of bulk production of microbial cellulases. At least three cellulases including endo‐cellulase, exo‐cellulase and cellobiase are needed to convert cellulosic biomass into fermentable sugars. All these cellulases could be self‐produced within cells of transgenic bio‐energy crops. The production of heterologous Acidothermus cellulolyticus (E1) endo‐cellulase in endoplasmic reticulum and mitochondria of green tissues of transgenic corn plants was recently reported, and it was confirmed that the heterologous E1 converts cellulose into fermentable sugars. RESULTS: Biologically active A. cellulolyticus E1, Trichoderma reesei 1,4‐β‐cellobiohydrolases I (CBH I) exo‐cellulase and bovine rumen Butyrivibrio fibrisolvens cellobiase were expressed in corn plant endoplasmic reticulum (ER), apoplast (cell wall areas) and vacuole respectively. Results show that the ratio 1:4:1 (E1:CBH I:cellobiase) of crude heterologous cellulases is ideal for converting ammonia fiber explosion (AFEX) pretreated corn stover into fermentable sugars. CONCLUSIONS: Corn plants that express all three biologically active heterologous cellulases within their cellulosic biomass to facilitate conversion of pretreated corn stover into fermentable sugars is a step forward in the quest for alternatives to the present microbial cellulase mix production for cellulosic biofuels. Copyright © 2011 Society of Chemical Industry     

Enzymatic saccharification of dissolution pretreated waste cellulosic fabrics for bacterial cellulose production by Gluconacetobacter xylinus

BACKGROUND: Waste textiles, such as dyed cellulosic and/or polyester blended fabrics have the potential to serve as an alternative feedstock for the production of biological products via microbial fermentation. Dissolution pretreatment was employed to enhance the enzymatic saccharification of dyed and synthetic fiber blended cellulosic fabrics. The fermentable reducing sugars obtained from waste cellulosic fabrics were used to culture Gluconobacter xylinus for value‐added bacterial cellulose (BC) production. RESULTS: Concentrated phosphoric acid was the ultimate cellulose solvent for dissolution pretreatment since 5% w/w cellulosic fabric can be completed dissolved at 50 °C. After regeneration in water, the cellulosic precipitate was subjected to cellulase hydrolysis, resulting in at least 4‐fold enhancement of saccharification rate and reducing sugars yield. The colored saccharification products can be utilized by G. xylinus to produce BC, approximately 1.8 g L^?1 BC pellicle was obtained after 7 days static cultivation. CONCLUSION: Dyed and blended waste fabric can be pretreated effectively by dissolution to produce fermentable sugars by cellulase hydrolysis. Dissolution pretreatment can expose the dyed or polyester fiber covered digestible cellulosic fibers to cellulase and leads to a significant enhancement of saccharification yield. The colored saccharification products have no significant inhibiting effect on the fermentation activity of G. xylinus for BC production. Copyright © 2010 Society of Chemical Industry     

Mathematical model for ethanol production from mixed sugars by Pichia stipitis

A mathematical model for estimating the dynamic behavior of ethanol production from mixed sugars such as glucose and xylose is presented. This model was constructed by introducing the term for ethanol production into the diauxic growth model proposed previously by the authors. It was assumed that the first substrate, glucose, is metabolized by a constitutive enzyme and the second substrate, xylose, is utilized by an inducible enzyme. The synthesis of the inducible enzyme is controlled by the catabolite repression caused by glucose and with the induction caused by xylose as an inducer, and the xylose is then converted into cell mass and ethanol by the inducible enzyme. The parameters of the model were estimated from the experimental data in the medium containing glucose and xylose, singly or in combination in a batch culture. The application of the model was examined for experiments in both batch and continuous culture, with glucose and xylose as carbon sources. The calculated values, according to the model, corresponded satisfactorily with experimental data, such as cell growth, substrate consumption, and ethanol production, especially in the estimation of the lag times between the first log phase and the second log phase in cell growth and ethanol production. © 2001 Society of Chemical Industry     

Random exchanges of non-conserved amino acid residues among four parental termite cellulases by family shuffling improved thermostability

There have been two major problems preventing applications of termite cellulases; one was difficulty for their hetelologous overexpression, and another is their low thermostability. We previously achieved adaptation of termite cellulase genes to an overexpression system of Escherichia coli by family shuffling of four orthologous cDNAs (Biosci. Biotechnol. Biochem., 2005; 69: 1711-1720). Using the adapted mutant cDNAs as parental genes combined with native-form cDNAs, we performed further family shuffling and obtained mutant cDNAs, which gave enzymes with improved thermostability. The best-evolved clone (PA68) was improved by 10 degrees C in maximum stability (retaining 90% original activity for 30 min incubation) from the parental enzymes, and kept 54% of its original activity for 150 min at 50 degrees C, whereas the most thermostable enzyme amongst the parents (A18) retained 30% of its original activity. PA68 showed 889 (micromoles of reducing sugars/min/mg of protein) in V(max) and 560 (micromoles of reducing sugars/min/mg of protein) in the specific activity against carboxymethylcellulose, which corresponds to 9.8 and 13.1 times of those of one of the ancestral enzymes rRsEG. In summary, we improved thermostability of the termite cellulase and increased the V(max) value and specific activity by combining only cDNAs encoding enzymes adapted for normal temperatures.