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.     

Comparative analysis of optimal endoglucanase production by Trichoderma reesei and intergeneric fusants of Trichoderma reesei Saccharomyces cerevisiae

Intergeneric fusants of Trichoderma reesei QM 9414/Saccharomyces cerevisiae NCIM 3288 developed in the authors' laboratory can convert cellulosic materials directly to ethanol in a single step process. The production of endoglucanase in this case is a key factor. The production profile of this enzyme by the intergeneric fusants is different from Trichoderma reesei QM 9414 (WT). The production of endoglucanase was studied seperately by Trichoderma reesei (WT) using optimal production medium which was designed as per the combined screening approach of Plackett-Burman followed by a central composite experimental plan and the intergeneric fusants using optimal production medium obtained by Box-Behnken optimization procedure. Dried grass was used as the cellulosic substance whose concentration was kept constant during the statistical optimization procedure. The concentration of dried grass was later varied keeping the other optimized medium constituents constant to find the final optimum medium composition for endoglucanase production.     

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.     

Induction and production of cellulases by l-sorbose in Trichoderma reesei

Summary Cellulase production in Trichoderma reesei mutants was induced by l-sorbose, known to be an inhibitor of -1,3-glucan synthesis. In the experiments the washed mycelia were used as resting cells. For CMCase induction over 24 h using T. reesei PC-3-7, the most effective pH, temperature and l-sorbose concentration were 2.8, 28° C and 0.3 mg/ml, respectively. Comparison with other cellulase inducers showed that the inductive level of CMCase by l-sorbose was similar to that by sophorose, known to be the most potent inducer of cellulases. Since the induction of CMCase was inhibited completely by 10 g of cycloheximide per ml, the induction process was considered to involve de novo synthesis. Although l-sorbose had the effective inducibility of CMCase, the assimilation rate of l-sorbose was very low in T. reesei PC-3-7.Production of Ethanol from Biomasses. Part III.Production of Ethanol from Biomasses. Part III.     

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.     

Secretome characteristics of pelletized Trichoderma reesei and cellulase production

Trichoderma reesei is an important cellulase producer and its secondary mycelial phase is responsible for cellulase expression and secretion in submerged fermentation. Little is known regarding the effects of fungal morphology on cellulase production by Trichoderma sp. In this study we aimed to extend the understanding of cellulase production by T. reesei, especially correlating cellulase productivity with pellet morphology and with its secretome characteristics. We found that T. reesei was more likely to form pellets in malt extract broth than in potato dextrose broth. CaCO(3) helped in formation of fine pellets in malt extract broth. 10(9) spores/ml resulted in formation of pellets with the size of 0.13 ± 0.047 mm. LC/MS spectrometry analysis indicated that the secretomes from pellet was different from that of mycelial mat under the same fermentation conditions. Optimization tests showed that lactose, xylose and Pluronic F68 are important for efficient production of cellulases with FPU activity in the pellets fermentation. This is the first report on the artificial formation of pellets by Trichoderma sp. as well as correlation between physiological characteristic of the pellets and cellulase production by T. reesei. The findings from this study can be used for improvement of cellulase productivity.     

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.     

Protein disulfide isomerase homolog TrPDI2 contributing to cellobiohydrolase production in Trichoderma reesei

The majority of the cysteine residues in the secreted proteins form disulfide bonds via protein disulfide isomerase (PDI)-mediated catalysis, stabilizing the enzyme activity. The role of PDI in cellulase production is speculative, as well as the possibility of PDI as a target for improving enzyme production efficiency of Trichoderma reesei, a widely used producer of enzyme for the production of lignocellulose-based biofuels and biochemicals. Here, we report that a PDI homolog, TrPDI2 in T. reesei exhibited a 36.94% and an 11.81% similarity to Aspergillus niger TIGA and T. reesei PDI1, respectively. The capability of TrPDI2 to recover the activity of reduced and denatured RNase by promoting refolding verified its protein disulfide isomerase activity. The overexpression of Trpdi2 increased the secretion and the activity of CBH1 at the early stage of cellulase induction. In addition, both the expression level and redox state of TrPDI2 responded to cellulase induction in T. reesei, providing sustainable oxidative power to ensure cellobiohydrolase maturation and production. The results suggest that TrPDI2 may contribute to cellobiohydrolase secretion by enhancing the capability of disulfide bond formation, which is essential for protein folding and maturation.     

Chitinous Material in Trichoderma reesei

Trichoderma reesei was grown for 180h in batch culture in an 8 liter stirred fermenter using a glucose-rich medium. Concentrations of glucose, ammonia, cell dry weight, debris and lipid are presented for two runs. Cell dry weights reached 26.9g/L and 19.6g/L in these runs. The debris from solvent-extracted cells was chitin which accumulated to greater than 75% of the final cell dry weights.     

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.     

Cellulase production in continuous culture by Trichoderma reesei on xylose-based media

Trichoderma reesei (QM 9414) produced cellulase in continuous culture, on media containing xylose (1%) supplemented with sorbose (0.3%) to induce cellulase production. Maximum cell mass of 4.54 kg/m(3) occurred at pH 4.0 and a dilution rate of 0.0391 h(-1) where residual substrate was 0.43 kg/m(3), but no cellulase was produced. Maximum cellulase production of 0.69 FPU occurred at pH 3.5 and a dilution rate of 0.0110 h(-1), where cell mass production was 2.56 kg/m(3) and residual substrate was 0.15 kg/m(3). Monod kinetic constants, corrected for endogenous metabolism, were 0.091 h(-1), 0.469 kg/m(3), 0.00923 h(-1), and 0.470 kg cells/kg xylose at pH 3.5, for the maximum specific growth rate, Michaelis-Menten coefficient, endogenous metabolism coefficient, and yield coefficient, respectively. Specific growth rate fitted a maturation time model, which predicted decreasing maturation time with increasing pH.     

里氏木霉306产t-PA固态发酵条件的研究

对基因工程菌株里氏木霉(Trichodermareesei)306生物合成组织型纤溶酶原激活剂(t-PA)的固态发酵培养基成分和发酵条件进行了研究;分析了发酵过程中t-PA的生成、总糖的消耗和菌体生长的规律。在优化的固体发酵条件下,t-PA的最大酶活是924.63IU/g干重培养基,较初始条件下提高了5倍。通过浅盘进行了放大实验,最大酶活为983.64IU/g干重培养基,t-PA合成速率为:13.66IU/g干重培养基.h,较三角瓶固态发酵最高产酶提高了6.38%,产酶速率提高了24.07%。     

Process optimization for the production of diosgenin with Trichoderma reesei

Based on the response surface methodology, an effective microbial system for diosgenin production from enzymatic pretreated Dioscorea zingiberensis tubers with Trichoderma reesei was studied. The fermentation medium was optimized with central composite design (3⁵) depended on Plackett–Burmann design which identified significant impacts of peptone, K₂HPO₄ and Tween 80 on diosgenin yield. The effects of different fermentation conditions on diosgenin production were also studied. Four parameters, i.e. incubation period, temperature, initial pH and substrate concentration were optimized using 4⁵ central composite design. The highest diosgenin yield of 90.57% was achieved with 2.67% (w/v) of peptone, 0.29% (w/v) of K₂HPO₄, 0.73% (w/v) of Tween 80 and 9.77% (w/v) of substrate, under the condition of pH 5.8, temperature 30 °C. The idealized incubation time was 6.5 days. After optimization, the product yield increased by 33.70% as compared to 67.74 ± 1.54% of diosgenin yield in not optimized condition. Scale-up fermentation was carried out in a 5.0 l bioreactor, maximum diosgenin yield of 90.17 ± 3.12% was obtained at an aeration of 0.80 vvm and an agitation rate of 300 rpm. The proposed microbial system is clean and effective for diosgenin production and thus more environmentally acceptable than the traditional acid hydrolysis.     

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.     

Inheritance of endo-β-1,4-glucanase production in Trichoderma reesei

Abstract Mutants of Trichoderma reesei QM9414 were isolated and characterized with respect to their cellulolytic and auxotrophic markers. Fusion of protoplasts isolated from uninucleate conidia was used to obtain hybrids between different pairs of mutants. Induced haploidization of the hybrids allowed recovery of stable segregants, which were screened for endoglucanase production. Segregants of each cross displayed a range of titers divulging their quantitative nature and polygenic control. Biometrical analysis suggests the involvement of 4–7 operationally recognizable units of function (effective factors) in the overall endoglucanase phenotype. High titer segregants were obtained from most crosses.     

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。