Simultaneous saccharification and fermentation of pretreated sugar cane leaves to ethanol

The simultaneous saccharification and fermentation (SSF) of pretreated sugar cane leaves to produce ethanol using a cellulolytic enzyme complex from Trichoderma reesei QM 9414 and Saccharomyces cerevisiae NRRL-Y-132 was optimized. Enzymic saccharification parameters were evaluated prior to SSF studies. A 92% conversion of 2·5% substrate (alkaline hydrogen peroxide pretreated) to sugars was achieved at 50°C and pH 4·5, using T. reesei cellulase (40 FPU/g substrate), in 48 h. The pretreated substrate was then subjected to an SSF process using the cellulase complex and S. cerevisiae cells. Optimization of the SSF system is described.     

Cellobiohydrolase I (Cel7A) from Trichoderma reesei has chitosanase activity

Cellobiohydrolase CBH I (Cel7A) from the filamentous fungus Trichoderma reesei (TrCBHI), which is a member of glycoside hydrolase family (GHF) 7, was expressed in Aspergillus oryzae. We found that the recombinant enzyme showed significant chitosanase activity, as well as cellulase activity, and acted in an endo-type manner on soluble polymeric substrate. Furthermore, another GHF7 CBH I from Aspergillus aculeatus (AaCBHI) expressed in A. oryzae also had chitosanase activity, while endoglucanase EG I (Cel7B) from T. reesei had no activity towards chitosan. To our knowledge, this is the first report of GHF7 enzymes possessing chitosanase activity.     

里氏木霉中纤维素酶的合成诱导及调控*

随着绿色化学的兴起,天然纤维素原料转化和利用的研究受到了高度重视和广泛应用。利用纤维素酶降解纤维素为燃料乙醇、生物柴油的生产铺设了道路。但纤维素酶的生产成本较高,限制了纤维素酶产业化应用。里氏木霉生产的纤维素酶组分丰富,是纤维素酶高产菌株,深入研究里氏木霉的纤维素酶诱导及表达调控机制,有助于提高其纤维素酶产率。近年来人们对里氏木霉的纤维素酶诱导过程和调控机制有了一定研究进展,综述了里氏木霉纤维素酶诱导和基因表达调控,首先介绍了纤维素、纤维二糖、槐糖、乳糖等几种诱导物及诱导物的转运蛋白,进一步综述了几种转录因子的调控作用,同时介绍了染色体调控、信号通路和光条件对纤维素酶诱导的影响。最后展望了未来里氏木霉纤维素酶诱导表达的研究方向,包括探明诱导物的本质及其具体过程、揭示转录因子之间的联系及转录调控网络、寻找信号转导关键功能蛋白及研究环境因素对纤维素酶的诱导作用等。     

Production of cellulase by freely suspended and immobilized cells of Trichoderma reesei

Trichoderma reesei (QM 9123) was immobilized within the open porous network of reticulated polyurethane foam matrices, and the growth pattern, glucose consumption and cellulase production were compared with those of freely suspended cells. It was found that the method of immobilization was simple and had no detrimental effect on cell activity. Various production media, to be used after the cultivation of T. reesei were tried. It was found that a nitrogen source-free production medium gave the highest enzyme titers of 1.5 × 10³ FPA U l⁻¹. Similar results were obtained with both freely suspended and immobilized cells.     

Cellulase finishing of woven, cotton fabrics in jet and winch machines

Some authors have reported that as the applied agitation rate increases, the apparent activity of the endoglucanases from Trichoderma reesei towards cotton cellulose increases more markedly than does the apparent activity of the cellobiohydrolases. This suggests that the quality of cellulase finishing effects on cellulosic textiles may be machine-type dependent. The present work using total crude, endoglucanase-rich and cellobiohydrolase-rich cellulases from T. reesei confirmed that the final properties of woven, cotton fabrics treated under realistic processing conditions in a jet machine, were measurably and perceivably different from those of the same fabrics, treated using the same processing conditions of temperature, time, pH, enzyme concentration and fabric to liquor ratio, but in a winch machine. The results are interpreted in terms of the effects of agitation rate on the adsorption–desorption behaviour of the T. reesei endoglucanases and cellobiohydrolases.     

Novel inducers derived from starch for cellulase production by Trichoderma reesei

Soluble inducers derived from starch were investigated for cellulase production by T. reesei. Various methods of starch treatment were compared. Acid-hydrolysed starch was found to be most effective. When 1·0 g starch was employed for cellulase production, the cellulase so produced after 6 days of fermentation, with supplementation of 1% (0·01 ml/g paper) β-glucosidase, saccharified more than 15 g shredded waste office paper in 9·34% suspension, resulting in 10 g fermentable sugars, 72% of which was glucose. The effectiveness of the starch-derived inducers was compared with that of lactose, pure cellulose, CMC, xylan and prehydrolysate of pine wood. The effects of calcium chloride and sorbose addition on cellulase production, and the kinetics of cellulase production by the starch-derived inducers are presented.     

Application of response surface methodology to evaluate the influence of temperature and initial pH on the production of β-1,3-glucanase and carboxymethylcellulase from Trichoderma harzianum

Response surface methodology was employed to study the effect of temperature and initial pH on the production of β-1,3-glucanase and carboxymethylcellulase from Trichoderma harzianum in both surface culture and submerged culture experiments. The efficiency of the enzymes in generating protoplasts from Trichoderma reesei mycelium was also studied. Regression analysis was performed on the data obtained. A temperature of 30°C and an initial pH of 4.7 were found to be optimal for β-1,3-glucanase production from T. harzianum in both surface culture and submerged culture processes.     

Unique mode of acetylation of oligosaccharides in aqueous two-phase system by Trichoderma reesei acetyl esterase

Trichoderma reesei RUT C-30 acetyl esterase, known to catalyze transacetylation reactions in water/vinyl acetate two-phase mixtures, was studied with respect to regioselectivity of acetylation of oligosaccharides in aqueous environment. Using series of oligosaccharides and their methyl glycosides, it was found that the enzyme catalyzes an efficient acetylation at O-3 position of the non-reducing terminal units of gluco-, xylo- and manno-oligosaccharides and a less efficient acetylation of O-2 position of the reducing end units of gluco- and xylo-oligosaccharides. The axial hydroxyl group at O-2 position of the reducing end mannose in mannooligosaccharides was not recognized by the enzyme and its acetylation was not observed. The structure of isolated transacetylation products was established by NMR, ESI-MS analysis and on the basis on their resistance towards action of glycosidases acting from the non-reducing end of oligosaccharides. The position of acetylation allowed deduce on some of the structural requirements of the enzyme for the acetyl group acceptors. T. reesei RUT C-30 acetyl esterase was also found to be capable of liberation of acetyl groups from terminal units of oligosaccharides, which speaks for its classification as an exo-acting acetyl esterase.     

增补UV-B辐射对菥蓂生理特性及次生代谢产物的影响

为了解药用植物菥蓂(Thlaspi arvense)在紫外线(UV-B)辐射增强下生长及响应规律,以2月龄菥蓂幼苗为研究对象,在自然光照基础上人工增补3.26μW·cm^-2(T1)和9.78μW·cm^-2(T2)2种不同辐射强度处理,以自然光照下生长的菥蓂幼苗为对照,研究菥蓂幼苗中光合指标和叶绿素荧光参数、光合色素(叶绿素a和b)含量、渗透调节物质(丙二醛、可溶性蛋白、可溶性糖和脯氨酸)含量、抗氧化酶(超氧化物歧化酶、过氧化物酶、过氧化氢酶和抗坏血酸过氧化氢酶)活性和总黄酮、总酚和黑芥子苷等次生代谢产物含量对不同UV-B辐射强度的响应。结果表明：2种辐射强度下净光合速率、气孔导度、蒸腾速率、胞间二氧化碳摩尔分数、最大荧光(F_m)、PSⅡ最大光化学效率(F_v/F_m)和PSⅡ潜在光化学效率(F_v/F_o)均随辐射强度增大而降低;叶绿素a和叶绿素b、可溶性蛋白、可溶性糖和脯氨酸等渗透调节物质、超氧化物歧化酶、过氧化物酶、过氧化氢酶和抗坏血酸过...     

外源水杨酸对高温胁迫下金莲花幼苗生理及电阻抗参数的影响

为解决金莲花幼苗在北方低海拔地区越夏过程中生长不良的问题，研究喷施水杨酸对高温胁迫下金莲花幼苗生理及电阻抗参数的影响，为提高金莲花的耐热性提供理论依据。以金莲花幼苗为试验材料，外源水杨酸（SA）设置4个浓度梯度（0.5、1.0、1.5、2.0 mmol·L^-1），喷施处理后置于人工气候箱内进行昼38℃/夜30℃的高温胁迫试验。结果表明：喷施0.5~1.0 mmol·L^-1水杨酸能有效降低金莲花幼苗的热害指数、相对电导率和丙二醛（MDA）含量，缓解叶绿素降解、显著提高可溶性糖含量、游离脯氨酸含量以及超氧化物歧化酶（SOD）活性，其中以1.0 mmol·L^-1的水杨酸（SA）处理效果最好，说明喷施适宜浓度的SA可以提高金莲花的耐热性，从而缓解高温伤害。相关性分析表明，热害指数与相对电导率呈极显著正相关（P<0.01），与叶绿素总量、可溶性糖含量胞外电阻率（r_e）呈显著负相关（P<0.05），胞内电阻率（r_i）与Pro含量呈显著正相关，弛豫时间（τ）与Pro含量和SOD活性呈极显著正相关，因此胞外电阻率、胞内电阻率和弛豫时间等电阻抗参数可作为金莲花对高温逆境反应的评价指标。     

中山杉及其父母本幼苗对干旱胁迫和复水的响应

本文研究了落羽杉和墨杉及其杂交后代中山杉302(落羽杉♀×墨杉♂)、中山杉407(墨杉♀×落羽杉♂)、回交代中山杉118(中山杉302♀×墨杉♂)在自然干旱胁迫和复水过程中,光合特征、抗氧化酶系统和形态特性等的响应.结果表明:随干旱时间的延长,所有植株的净光合速率逐渐降低、脯氨酸开始积累且抗氧化酶系统逐渐清除丙二醛的毒性.胁迫至第8天,落羽杉净光合速率的下降幅度最大,而中山杉118的水分利用效率最高、丙二醛含量最少;墨杉的超氧化物歧化酶活性和脯氨酸含量增长最大.复水2 d后,所有植物的参数均有不同程度的恢复,其中,中山杉118恢复速率最快,其净光合速率和脯氨酸含量分别恢复了74.4%和60.2%.复水9 d后,所有植株的测定指标基本恢复至或接近正常水平,其中,中山杉118的生物量未受影响且根冠比显著增加.植物的耐旱能力依次为墨杉>中山杉118>中山杉407>中山杉302>落羽杉.回交品种中山杉118的杂种优势明显,较大程度地遗传了墨杉的耐旱性,该结论可为耐旱中山杉品种的杂交选育和推广应用提供科学依据.     

东海带鱼的最适可捕规格

带鱼是中国东海的重要经济渔获种类,其可捕规格会随不同时期的捕捞强度而变化。为确定目前东海带鱼最适可捕规格,根据2016—2020年拖网、帆张网及延绳钓等主捕带鱼作业方式采集的带鱼样品和数据,利用FiSAT II软件对其生长、死亡参数进行估算,进而计算可捕规格。结果表明: 带鱼的拐点肛长、临界肛长、1龄鱼肛长分别为382.84、397.12、216.05 mm,50%带鱼性成熟肛长为230.38 mm,最小可捕肛长为219.23 mm。利用Beverton-Holt模型分析带鱼单位补充量渔获量可知,带鱼当前渔业参考点为:t_c=0.38 a,F=2.11,处于捕捞过度区域,并得出最适可捕肛长为364.64 mm。但过高的可捕规格会导致短期内带鱼产量急剧下降,渔民难以接受,因此,本研究综合考虑采用接近1龄鱼肛长与50%性成熟肛长作为最适可捕规格较为合适,即220 mm。     

Trichoderma reesei cellulases

Recent advances in the delineation of the biochemical mechanisms of cellulose hydrolysis, strain improvement, molecular cloning and process engineering are bringing T. reesei cellulases closer to being a commercially viable route to cellulose hydrolysis.     

Cellulolytic activity of Actinomycetes isolated from termites (Termitidae) gut

Abstract Cellulolytic actinomycetes were isolated from the hindgut of four different termites: Macrotermes, Armitermes, Odontotermes and Microcerotermes spp.
The isolated actinomycetes ( Streptomyces sp. and Micromonospora sp.) were grown on cellulosic substrates and their extracellular cellulase (C_l, C_x and cellobiase) activity evaluated; using filter paper as a substrate for C_l, carboxymethylcellulose (CMC) for C_x and d -cellobiose for cellobiase, all strains were shown to degrade soluble and insoluble cellulose; optimum pH for growth was 6.2–6.7 at 28°C; three strains could grow at 48°C on cellulosic substrates.
Some strains exhibited high cellulase activity, constant for 5–7 days, but inhibition by glucose was a common feature for almost all isolates.     

Mutation of fungal endoglucanases into glycosynthases and characterization of their acceptor substrate specificity

Humicola insolens mutant Cel7B E197A is a powerful endo-glycosynthase displaying an acceptor substrate specificity restricted to β-d-glucosyl, β-d-xylosyl, β-d-mannosyl and β-d-glucosaminyl in +1 subsite. Our aim was to extend this substrate specificity to β-d-N-acetylglucosaminyl, in order to get access to a wider array of oligosaccharidic structures obtained through glycosynthase assisted synthesis. In a first approach a trisaccharide bearing a β-d-N-acetylglucosaminyl residue was docked at the +1 subsite of H. insolens Cel7B, indicating that the mutation of only one residue, His209, could lead to the expected wider acceptor specificity. Three H. insolens Cel7B glycosynthase mutants (H209A, H209G and H209A/A211T) were produced and expressed in Aspergillus oryzae. In parallel, sequence alignment investigations showed that several cellulases from family GH7 display an alanine residue instead of histidine at position 209. Amongst them, Trichoderma reesei Cel7B, an endoglucanase sharing the highest degree of sequence identity with Humicola Cel7B, was found to naturally accept a β-d-N-acetylglucosaminyl residue at +1 subsite. The T. reesei Cel7B mutant nucleophile E196A was produced and expressed in Saccharomyces cerevisiae, and its activity as glycosynthase, together with the H. insolens glycosynthase mutants, was evaluated toward various glycosidic acceptors.     

Fungal antagonists of the plant pathogen Rhizoctonia solani: selection, control efficacy and influence on the indigenous microbial community

A broad spectrum of fungal antagonists was evaluated as potential biocontrol agents (BCAs) against the soil-borne pathogen Rhizoctonia solani using a new combination of in vitro and in vivo assays. The in vitro characterisation of diverse parameters including the ability to parasitise mycelium and to inhibit the germination of Rhizoctonia sclerotia at different temperatures resulted in the selection of six potential fungal antagonists. These were genotypically characterised by their BOX-PCR fingerprints, and identified as Trichoderma reesei and T. viride by partial 18S rDNA sequencing. When potato sprouts were treated with Trichoderma, all isolates significantly reduced the incidence of Rhizoctonia symptoms. Evaluated under growth chamber conditions, the selected Trichoderma isolates either partly or completely controlled the dry mass loss of lettuce caused by R. solani. Furthermore, the antagonistic Trichoderma strains were active under field conditions. To analyse the effect of Trichoderma treatment on indigenous root-associated microbial communities, we performed a DNA-dependent SSCP (Single-Strand Conformation Polymorphism) analysis of 16S rDNA/ITS sequences. In this first assessment study for Trichoderma it was shown that the pathogen and the vegetation time had much more influence on the composition of the microbiota than the BCA treatment. After evaluation of all results, three Trichoderma strains originally isolated from Rhizoctonia sclerotia were selected as promising BCAs.     

液态发酵条件下拟康宁木霉8985产纤维素酶能力初探

液态发酵条件下,以微晶纤维素为唯一碳源,比较了拟康宁木霉Trichoderma koningiopsis 8985和里氏木霉T.reesei QM9414产纤维素酶的能力。8985发酵12 h开始产生纤维素酶,36 h时酶活达到产酶峰值的50%,此时QM9414尚未诱导产酶。测定8985发酵84 h时上清液中滤纸纤维素酶、羧甲基纤维素酶、β-葡萄糖苷酶和木聚糖酶的酶活分别为1.06、3.62、1.80和6.67 IU/mL,分别是QM9414上述酶活的1.72、1.70、6.35和1.12倍。8985滤纸纤维素酶酶活的最适反应条件为pH 4.5,反应温度50℃,在Fe³⁺(≤4 mmol/L)和Cu²⁺(0–10 mmol/L)存在条件下酶活稳定。     

Production of cellulase using a mutant strain of Trichoderma reesei growing on lactose in batch culture

The production of cellulases in batch culture was studied using a mutant strain of Trichoderma reesei C-5 growing on lactose. Growth kinetic parameters on 2% lactose were studied and the comparative results for growth and enzyme productivities at two different lactose levels are discussed. The cellulase synthesis rate depended on the lactose concentration in the medium. Although growth was favoured at a higher lactose level, the volumetric enzyme productivity did not increase in proportion and the specific enzyme productivity decreased to a certain extent, indicating that partial catabolic inhibition at higher lactose concentrations may be possible. However, it was noted that the mutant strain was highly depressed and capable of synthesising active cellulases on lactose.     

Modelling of microbial growth for sequential utilization in a multisubstrate environment

Microbial growth in multisubstrate environments is posed as a problem of multivariable constraint optimization. The optimization aims at maximizing the instantaneous growth rate of cells. The model developed for microbial growth using this hypothesis involves simple representation of complex cell structure as an optimization function which regulates the interplay of cellular machinery. The model parameters are estimated using single substrate growth data. Model simulation fits very well with earlier published experimental data of bacterial growth of Klensiella oxytoca on a variety of sugar mixtures involving glucose, fructose, lactose, and xylose. Moreover, the model is also able to predict the diauxic growth of Saccharomyces cerevisiae on glucose and galactose. One of the interesting outcomes of the above representation is the ability to prove analytically that the growth on the mixture of two sugars will be diauxic if one of the substrates has a very low K_s value and a high μ_m value.     

The intracellular galactoglycome in Trichoderma reesei during growth on lactose

Lactose (1,4-0-β-d-galactopyranosyl-d-glucose) is used as a soluble carbon source for the production of cellulases and hemicellulases for—among other purposes—use in biofuel and biorefinery industries. The mechanism how lactose induces cellulase formation in T. reesei is enigmatic, however. Previous results from our laboratory raised the hypothesis that intermediates from the two galactose catabolic pathway may give rise to the accumulation of intracellular oligogalactosides that could act as inducer. Here we have therefore used high-performance anion-exchange chromatography–mass spectrometry to study the intracellular galactoglycome of T. reesei during growth on lactose, in T. reesei mutants impaired in galactose catabolism, and in strains with different cellulase productivities. Lactose, allo-lactose, and lactulose were detected in the highest amounts in all strains, and two trisaccharides (Gal-β-1,6-Gal-β-1,4-Glc/Fru and Gal-β-1,4-Gal-β-1,4-Glc/Fru) also accumulated to significant levels. Glucose and galactose, as well as four further oligosaccharides (Gal-β-1,3/1,4/1,6-Gal; Gal-β-1,2-Glc) were only detected in minor amounts. In addition, one unknown disaccharide (Hex-β-1,1-Hex) and four trisaccharides were also detected. The accumulation of the unknown hexose disaccharide was shown to correlate with cellulase formation in the improved mutant strains as well as the galactose pathway mutants, and Gal-β-1,4-Gal-β-1,4-Glc/Fru and two other unknown hexose trisaccharides correlated with cellulase production only in the pathway mutants, suggesting that these compounds could be involved in cellulase induction by lactose. The nature of these oligosaccharides, however, suggests their formation by transglycosylation rather than by glycosyltransferases. Based on our results, the obligate nature of both galactose catabolic pathways for this induction must have another biochemical basis than providing substrates for inducer formation.