Cellulase and xylanase production by Trichoderma reesei Rut C-30

Summary When grown on cellulose or xylan, Trichoderma reesei (strain Rut C-30) produced extra-cellular enzymes which could hydrolyze both cellulose and xylan to their respective monosaccharides. At low O₂ saturation, -glucosidase activity is greatly reduced for cellulose-grown but not xylan-grown cells.     

Influence of pH on the production of xylanases by Trichoderma reesei Rut C-30

Trichoderma reesei Rut C-30 was cultivated in bioreactors at different pH on a medium with lactose as the main carbon source. Compared to an earlier study, in which T. reesei Rut C-30 was cultivated using polysaccharides (cellulose or xylan) as the main carbon sources, we now report a slightly lower pH value for maximal xylanase levels. The highest xylanase activity (IU/ml) on the lactose-based medium was observed at pH 6.0 compared to pH 7.0 on the polysaccharide-based media. When the pattern of different xylanases was analyzed by isoelectric focusing and activity zymogram, we observed that a low pH (4.0) favoured the production of xylanase I, whilst a high pH (6.0) favoured the production of xylanase III. Xylanase II was clearly produced at both pH values. The results at pH 4 and 6 correlate with the pH activity profiles of xylanase I, II and III. Hence, the different T. reesei xylanases were produced according to which enzyme is most active in that particular environment.     

Nutrient control for stationary phase cellulase production in Trichoderma reesei Rut C-30

This work describes the use of nutrient limitations with Trichoderma reesei Rut C-30 to obtain a prolonged stationary phase cellulase production. This period of non-growth may allow for dependable cellulase production, extended fermentation periods, and the possibility to use pellet morphology for easy product separation. Phosphorus limitation was successful in halting growth and had a corresponding specific cellulase production of 5 ± 2 FPU/g-h. Combined with the addition of Triton X-100 for fungal pellet formation and low shear conditions, a stationary phase cellulase production period in excess of 300 h was achieved, with a constant enzyme production rate of 7 ± 1 FPU/g-h. While nitrogen limitation was also effective as a growth limiter, it, however, also prevented cellulase production.     

The influence of culture conditions on mycelial structure and cellulase production by Trichoderma reesei Rut C-30

The morphology of Trichoderma reesei Rut C-30, during submerged cultivations in shake flask, was examined. The influence of the size inoculum and the composition of the fermentation medium on the morphology and cellulase production was studied. Different inoculum sizes were studied but the significative change in fungus morphology was observed for spores concentration between 10(5) and 10(7) spores/ml (i.e. 10(2) and 10(4) spores/ml in pre-culture medium). In the medium without Tween 80, at low inoculum size, the majority of the pellets were large and well individualized, in contrast, at higher inoculation densities small flocs were obtained, with higher production of soluble protein and higher filter paper activity. It was found that the average pellet size seems to be inversely proportional to the inoculum size. Medium composition, namely Tween 80, also influences the morphology of T. reesei Rut C-30 and enzyme production. The presence of Tween 80 in fermentation medium inhibited the pellet formation of this strain.     

Factors affecting foaming behavior in cellulase fermentation by Trichoderma reesei Rut C-30

Coupling fermentation with in situ foam fractionation may be beneficial to cellulase production in optimizing oligomer inducer generation, minimizing catabolite repression and reducing cellulase degradation by proteases. In this study, the potential factors that may affect the foaming behavior of broth from Trichoderma reesei Rut C-30 fermentation were examined. These factors included solid (both cell and cellulose) concentrations, cellulase activity and extracellular protein concentration. The loss of cellulase activity caused by the foaming process was minimal. The foamate generation was lower in the presence of higher solids (cell and/or cellulose) concentrations. Cellulase appeared to promote the broth foaming ability but its enrichment ratio was not high (lower than 1.2). The enrichment ratios for the individual component enzymes (beta-glucosidase, endo- and exo-glucanases) were found to be similarly low. None of the cellulase components were likely the primary foaming factors. The foam also carried out cells and cellulose solids. The hydrophobicity of cell surface, studied at various fermentation stages and in both media with and without cellulose, increased as the fermentation approached the stationary phase and then decreased gradually after entering the stationary phase.     

Enhanced production of cellulase,hemicellulase, and β-glucosidase by Trichoderma reesei (Rut C-30)

The Production of cellulases and Hemicellulases was studied with Trichoderma reesei Rut C-30, This organism produced, together with high cellulase activities, considerable amounts of xylanases and β-glucosidase. Three cellulose concentration (1, 2.5, and 5.0%) were examined to determined the maximum levels of cellulase activity obtainable in submerged culture. Temperature and pH profiling was used to increase cell mass to maximum levels within two days and thereby enhancing fermentor productivity at higher substrate levels. The effect of temperature, pH, Tween-80 concentration, carbon sources, and substrate concentration on the ration of mycelial growth and extracellulose enzyme production are described.     

工业纤维素诱导里氏木霉RUT C-30产葡聚糖酶的条件

为了研究工业纤维素诱导里氏木霉RUT C-30产葡聚糖酶的最佳条件,根据单因素实验结果,以工业纤维素、(NH4)2SO4和生物素为实验因素,滤纸酶活为响应值,进行中心组合设计,建立一个二次多项式数学模型,进行响应面优化,寻找最优产酶结果.经过优化,选出工业纤维素、(NH4)2SO4和生物素的添加量分别为39.485 g/L、6.232 g/L和249.872 μg/L,最高的滤纸比酶活为6.298 U/mL,实验验证,滤纸比酶活为6.118 U/mL,与预测值相差了2.86%.     

Cellulase-poor xylanases produced by Trichoderma reesei RUT C-30 on hemicellulose substrates

Hemicellulose components from industrial viscose fibre production are characterized by a lower cellulose content than commercial xylan and the presence of a carboxylic acid fraction originating from the alkaline degradation of carbohydrates during the process. This substrate, after neutralization, can be used by Trichoderma reesei RUT C-30 for the production of cellulase-poor xylanases, useful for the pulp and paper industry. The yields of xylanase ranged up to almost 400 units/ml, with a ratio of carboxymethylcellulase/xylanase of less than 0.015. This crude xylanase enzyme mixture was shown to be superior to that obtained on beech-wood xylan when used for bleaching and, particularly, upgrading of hard-wood chemical pulp by selective removal of the xylan components. Biochemical studies indicate that the low cellulase production by T. reesei grown on these waste hemicelluloses is the result of a combination of at least three factors: (a) the comparatively low content of cellulose in these hemicellulosic wastes, (b) the inhibitory action of the carboxylic acid fraction present in the hemicellulosic wastes on growth and sporulation of T. reesei, and (c) the use of a mycelial inoculum that is unable to initiate the attack on the cellulose components within the carbon source. Correspondence to: G. Gamerith     

Promoting pellet growth of Trichoderma reesei Rut C30 by surfactants for easy separation and enhanced cellulase production

It is desirable to modify the normally filamentous Trichoderma reesei Rut C-30 to a pellet form, for easy biomass separation from the fermentation medium containing soluble products (e.g., cellulase). It was found in this study that this morphological modification could be successfully achieved by addition of the biosurfactant rhamnolipid (at ≥ 0.3g/L) and the synthetic Triton X-100 (at ≥ 0.1g/L) to the fermentation broth before the cells started to grow actively. Thirteen other surfactants tested were not as effective. Furthermore, the added rhamnolipid and Triton X-100 increased the maximum cellulase activity (Filter Paper Units) produced in the fungal fermentation; the increase was 68 ± 7.8% for rhamnolipid and 73 ± 12% for Triton X-100. At the concentrations required for pellet formation, rhamnolipid had negative effect on the cell growth: with increasing rhamnolipid concentrations, the growth rate decreased and the lag-phase duration increased linearly. Triton X-100 caused no significant differences in growth rate or lag phase.     

Roles of extracellular lactose hydrolysis in cellulase production by Trichoderma reesei Rut C30 using lactose as inducing substrate

Lactose, an inexpensive, soluble substrate, offers reasonably good induction for cellulase production by Trichoderma reesei. The fungus does not uptake lactose directly. Lactose is hydrolyzed to extracellular glucose and galactose for subsequent ingestion. The roles of this extracellular hydrolysis step were investigated in this study. Batch and continuous cultures were grown on the following substrates: lactose, lactose–glycerol mixtures, glucose, galactose, and glucose–galactose mixtures. Cell growth, substrate consumption, lactose hydrolysis, and lactase and cellulase production were followed and modeled. Cells grew much faster on glucose than on galactose, but with comparable cell yields. Glucose (at >0.3 g/L) repressed the galactose consumption. Cellulase synthesis was growth-independent while lactase synthesis was growth-dependent, except at D < ∼0.065 h⁻¹ where a basal level lactase production was observed. For cellulase production the optimal D was 0.055–0.065 h⁻¹ where the enzyme activity and productivity were both near maxima. The model suggested that lactase synthesis was subject to weak galactose repression. As the galactose concentration increased at high D (>0.1 h⁻¹), lactase synthesis became repressed. The insufficient lactase synthesis limited the lactose hydrolysis rate. Extracellular lactose hydrolysis was concluded to be the rate-limiting step for growth of T. reesei Rut C30 on lactose.     

Lactase production in continuous culture by Trichoderma reesei Rut-C30

Summary Trichoderma reesei Rut-C30 was found to produce extracellular lactase when grown on lactose medium. Maximum enzyme levels in continuous culture were observed at dilution rates (D) between 0.02 and 0.027 hr^-1. The enzyme productivity reached 27.3 U/L hr at D = 0.027 hr^-1. Lactase synthesis appears to be inducible and subject to catabolite repression. Optimal growth temperature and pH for enzyme production were 28°C and pH 5. Maximum enzyme activity was observed at 63°C and pH 4.6. The apparent K_m, based on the nitrophenyl-galactopyranoside assay was estimated as 0.4 mM. The enzyme is suitable for lactose hydrolysis in acid whey.     

Cellulase production by Trichoderma reesei

Summary A model is proposed for the enzyme production by Trichoderma reesei (QM 9414), which assumes control of the active enzyme transport through the cell membrane as a key parameter for the enzyme activity change in the culture filtrate. In a stirred tank reactor, continuous cultivation of the fungus was carried out in the dilution rate range of D=0.01–0.032 h^–1. After changing the dilution rate it took 3–4 weeks to attain a steady state in enzyme activity. Reducing sugars, dissolved protein, enzyme activity (filter-paper and glucosidase activities), cellulose and nitrogen content of the sediment, the elementary analysis of the cell and the composition of the outlet gas were all determined during cultivation. At a dilution rate of D=0.025 h^–1 all of these properties change due to derepression (for D<0.025 h^–1) or repression (for D>0.025 h^–1) of the enzymes which are responsible for the active transport of cellulases from the cell into the medium. The cellulase excretion causes a decrease of the yield coefficient of growth and a reduction of the nitrogen content of the cells.In a two-stage system the time to attain a steady state increases to 4–6 weeks. At low dilution rates the enzyme activity is only slightly higher in the second stage than in the first. At high dilution rates, at which the enzyme is not excreted into the medium in the first stage, enzyme activity can be increased considerably in the second stage.     

Cellulase production by Trichoderma reesei (RUT-C30) in fed-batch culture

Summary Fed-batch cultures of Trichoderma reesei RUT-C30 attained quasi-steady state conditions, in respect of biomass concentration and enzyme production rate, commensurate with a specific cell maintenance coefficient of 0.029 g cellulose.g biomass.^–1h^–1 and specific cellulase production rate of between 9.6 and 11.9 IU (filter paper activity).g biomass.^–1h^–1. A maximum enzyme yield of 57 IU.m1^–1 at an overall productivity of 201 IU.L.^–1h^–1 resulted from a cellulose feed rate of 1.0g.L.^–1h^–1.     

Enhancement in cellulase production by Trichoderma reesei Rut-C30 due to citric acid

Summary This report discusses enhancement in cellulase production by Trichoderma reesei Rut-C30. A combination of 75 mM citrate buffer and low operating pH values; e.g., 3.5, resulted in about 100% higher enzyme production in shake flasks. A similar extent of improvement was seen at lower citrate buffer concentrations such as 5 and 10 mM. It is suggested that the undissociated form of citric acid is responsible for this improvement; no such acid moiety has been previously reported to enhance cellulase production.     

Enhanced cellulase production in fed-batch culture of Trichoderma reesei C30

The use of a fed-batch cultivation of the fungus Trichoderma reesei (C30) allows cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] production to occur under optimum conditions, and results in extremely high enzyme titres and productivities. Enzyme levels of 26 U ml^?1 at productivities >130 U l^?1 h^?1 have been achieved. These results are compared with the values obtained in two-stage continuous cultivation of the organism at optimum pH and temperature.     

Xylanase production by Trichoderma harzianum E58

Summary Growth of Trichoderma harzianum E58 on hemicellulose-rich media, both in batch and fermentor cultures, resulted in independent profiles for the production of xylanase and endoglucanase enzymes. Dramatic differences in the ratio of xylanase to endoglucanase activities were observed among cultures grown on cellulose-rich Solka Floc and xylan. These results indicated that the induction of xylanases and cellulases was likely to be under separate regulatory control. The specific activity and amount of xylanases produced were found to be dependent on the concentration of xylan in the growth media. Growth on oat spelts xylan or the hemicellulose-rich, water-soluble fraction from steam-treated aspenwood (SEA-WS) greatly enhanced the production of xylanases and xylosidase in the culture filtrates. Constitutive levels of xylanase and endoglucanase enzymes were detected during growth of the fungus on glucose.Offprint requests to: D. J. Senior     

Enzyme production by recombinant Trichoderma reesei strains.

The production of both homologous and heterologous proteins with the cellulolytic filamentous fungus Trichoderma reesei is described. Biotechnically important improvements in the production of cellulolytic enzymes have been obtained by genetic engineering methodology to construct strains secreting novel mixtures of cellulases. These improvements have been achieved by gene inactivation and promoter changes. The strong and highly inducible promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has also been used for the production of eukaryotic heterologous proteins in Trichoderma. The expression and secretion of active calf chymosin is described in detail.     

人工锌指蛋白介导调控的里氏木霉纤维素酶生产

里氏木霉Trichoderma reesei Rut-C30是目前研究最广泛的纤维素酶生产菌,选育高产纤维素酶的里氏木霉菌株有助于提高木质纤维素资源生物炼制的经济性。利用人工锌指蛋白文库转化T.reeseiRut-C30,筛选获得了两株高产纤维素酶的突变株T. reesei M1和M2,与出发菌株比较,突变株M1和M2滤纸酶活分别提高100%和53%,且M1突变株外泌蛋白量提高69%,M2内切纤维素酶活提高64%。实时定量PCR分析结果表明,与对照菌株相比,突变株M1和M2中主要纤维素酶基因转录均上调,但不同酶基因在两株菌中有不同的变化特征。此外,纤维素酶抑制转录因子基因ace1在两株突变株中都转录下调,而纤维素酶正调控转录因子基因xyr1仅在M1突变株中上调。以上结果表明,不同人工锌指蛋白对纤维素酶活性的影响具有多样性。对这些突变体中人工锌指蛋白靶基因进行深入分析,为进一步深入探究里氏木霉纤维素酶合成调控的机理,以及利用代谢工程选育更高效的产酶菌株提供了基础。