A microfibril-generating factor from the cellulase of Trichoderma reesei

A combination of ionic strength reduction and diafiltration of Trichoderma reesei cellulate complex through a hollow fiber apparatus of 5000 molecular weight (MW) cutoff and subsequent passage of filtrate over a Spherogel-TSK 3000-SW column provided extracts that had the ability to generate microfibrils in filter paper and to disrupt filter paper and corn leaf tissue. Milligram quantities of material obtained from these extracts released small amounts of soluble carbohydrate from filter paper, required ferric iron for increased activity, and contained amino acids. Short fiber formation and disruption of filter paper during interaction with these extracts was enhanced by prior acid treatment and eliminated by prior base treatment. The amount of soluble carbohydrate hydrolyzed in 24 h from filter paper by whole cellulase complex was not changed by first disrupting the substrate with the extracts.     

A complete protein pattern of cellulase and hemicellulase genes in the filamentous fungus Trichoderma reesei

The complete protein pattern of cellulase and hemicellulase genes was studied through the Genome-wide analysis in Trichoderma reesei. The genome database revealed the presence of 39 ORFs encoding related proteins, including 32 enzymes with a catalysis domain related to cellulases and hemicellulases and 7 related proteins with a cellulose-binding module (CBM). Ten of these encoded yet undescribed enzymes, including six novel beta-glucosidases or xylosidases, two putative xylanases and two undescribed mannases. To better illustrate the relation of these 39 related proteins, four groups were created and analyzed by phylogenetic analysis: group A corresponding to xylanases, group B belonging to mannases and acting to degrade mannan; group C containing all known and putative cellulose-degrading proteins that have highly conserved CBMs; and group D containing beta-glucosidase and beta-xylosidase. Group D was the largest group, in which 8 beta-glucosidases appeared to be non-secreted proteins.     

Shear inactivation of cellulase of Trichoderma reesei

Inactivation of the cellulase of Trichoderma reesei (EC 3.2.1.4) by shear, is of sufficient magnitude to merit consideration in the design of equipment for the enzymatic hydrolysis of cellulose. The inac inactivation constant, k_d, is a function of the flow rate of the enzyme solution through a fine capillary tube. k_d increased slowly at low shear stress, and much more rapidly when the shear stress was greater than 15 dynes cm^?2.     

里氏木霉纤维素酶的分离纯化及酶学性质

通过(NH4)2SO4分级沉淀、HiPrep 26/10 Desalting凝胶色谱脱盐、Source 15 Q阴离子交换色谱技术,里氏木霉(Rut C-30)纤维素酶主要组分得以初步分开,再经过Source 15 S阳离子交换色谱、HiPrep Sephacryl S-100 HR凝胶过滤色谱、Superdex 75 PrepGrade凝胶过滤色谱进一步分离纯化,得到2个纯化的内切葡聚糖酶组分EGⅡ、EGⅠ和一个外切葡聚糖酶组分CBHⅠ;经过SDS-PAGE电泳鉴定为电泳纯,测得相对分子质量分别为5.22×104,5.62×104和6.90×104。EGⅡ的最适反应pH是5.6,最适反应温度为65℃;EGⅠ的最适反应pH是4.4,最适反应温度为55℃;以羧甲基纤维素(CMC)为底物时,EGⅠ、EGⅡ的米氏常数(Km)分别为2.20 mg/mL、3.38 mg/mL。CBHⅠ的最适反应pH是5.8,最适反应温度为60℃,以对硝基苯基-β-D-纤维二糖苷(PNPC)为底物时,米氏常数(Km)为0.12 mg/mL。     

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。     

Mathematical modeling and optimization of cellulase protein production using Trichoderma reesei RL-P37

The enzyme cellulase, a multienzyme complex made up of several proteins, catalyzes the conversion of cellulose to glucose in an enzymatic hydrolysis-based biomass-to-ethanol process. Production of cellulase enzyme proteins in large quantities using the fungus Trichoderma reesei requires understanding the dynamics of growth and enzyme production. The method of neural network parameter function modeling, which combines the approximation capabilities of neural networks with fundamental process knowledge, is utilized to develop a mathematical model of this dynamic system. In addition, kinetic models are also developed. Laboratory data from bench-scale fermentations involving growth and protein production by T. reesei on lactose and xylose are used to estimate the parameters in these models. The relative performances of the various models and the results of optimizing these models on two different performance measures are presented. An approximately 33% lower root-mean-squared error (RMSE) in protein predictions and about 40% lower total RMSE is obtained with the neural network-based model as opposed to kinetic models. Using the neural network-based model, the RMSE in predicting optimal conditions for two performance indices, is about 67% and 40% lower, respectively, when compared with the kinetic models. Thus, both model predictions and optimization results from the neural network-based model are found to be closer to the experimental data than the kinetic models developed in this work. It is shown that the neural network parameter function modeling method can be useful as a "macromodeling" technique to rapidly develop dynamic models of a process.     

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.     

Inhibition of Trichoderma reesei cellulase by sugars and solvents

Inhibition of Trichoderma reesei cellulase by sugars (glucose, delta-gluconolactone, and cellobiose) and solvents (ethanol, butanol, and acetone) was studied using cellulose azure. Glucose, cellobiose, ethanol, and butanol were noncompetitive inhibitors, delta-gluconolactone was a mixed inhibitor, and acetone was a noncompetitive activator. Converting cellobiose to glucose reduces the effective inhibitor binding constant by 6 times and converting cellobiose to ethanol reduces it by 16 times.     

Production of cellulase by Trichoderma reesei from dairy manure

Cellulase production by the fungi Trichoderma reesei was studied using dairy manure as a substrate. Data showed that T. reesei RUT-C30 had higher cellulase production than T. reesei QM 9414 and that a homogenized manure, treated by a blender to reduce fiber size, led to higher cellulase production. The cellulase production was further optimized by growing T. reesei RUT-C30 on homogenized manure. The effects of manure concentration, pH, and temperature on cellulase production were investigated with optimal parameter values determined to be 10 g/l manure (dry basis), 25.5 degrees C, and pH 5.7, respectively. Elimination of CaCl2, MgSO4, nitrogen sources (NH4+ and urea) and trace elements (Fe2+, Zn2+, Co2+ and Mn2+) from the original salt solution had no negative influence on the cellulase production, while phosphate elimination did reduce cellulase production. Based on above results, the final medium composition was simplified with manure additives being KH2PO4, tween-80 and CoCl2 only. Using this medium composition and a reaction time of 6-8 days, a maximum cellulase production activity of 1.74 IU/ml of filter paper activity, 12.22 IU/ml of CMCase activity, and 0.0978 IU/ml of beta-glucosidase was obtained. This filter paper activity is the highest ever reported in cellulase production from agricultural wastes.     

Adsorption of Trichoderma reesei CBHI cellulase on silanized silica

Adsorption kinetics and surfactant-mediated elution of Trichoderma reesei CBHI cellulase were recorded in situ, at hydrophobic, silanized silica. Experiments were performed at different solution concentrations, ranging from 0.001 to 0.98 mg/mL. Adsorbed enzyme was partially elutable upon rinsing, with the amount of adsorbed mass remaining being highest at intermediate concentrations. In addition, the resistance to elution with buffer was generally lower at higher concentrations, and the resistance to elution with surfactant was generally lower at intermediate concentrations. These observations are tentatively explained with reference to a mechanism allowing for adsorption of associated monomers of CBHI as well as free monomers.     

A comparison between the cellulase systems of Trichoderma harzianum E58 and Trichoderma reesei C30

Summary Nearly all of the filter paper, endoglucanase and -glucosidase activities of T. harzianum E58 were located extracellularly, with low amounts of these activities detected in the cell extracts and relatively little associated with the cell wall. Most of the filter paper and endoglucanase activities of T. reesei C30 were detected extracellularly. The half lives of the different cellulase activities were assayed at various temperatures over a period of time. When the pH of the filtrate was adjusted to 4.8, the cellulase activities were considerably enhanced, with the average half-life at 50°C extended to 25 hrs. When various lignocellulosic substrates were hydrolyzed by T. harzianum E58 cellulases approximately 90% of the reducing sugars were present as glucose while 50–60% of the reducing sugars were detected as glucose when T. reesei C30 cellulases were used.     

Reversibility and competition in the adsorption of Trichoderma reesei cellulase components

Adsorption reversibility and competition between fractionated components of the Trichoderma reesei cellulase system were studied. Specific endoglucanase (EGI), nonspecific endoglucanases (EGII, EGIII), and cellobio-hydrolase (CBHI) were previously grouped according to their hydrolytic function. At 5 degrees C, direct evidence of exchange between adsorbed and free enzyme was obtained for each component using [(3)H] and [(14)C] radiolabeled tracers. No release of bound enzymes was detected upon dilution of the free enzyme solution. In simultaneous adsorption of enzyme pairs, CBHI was shown to predominate adsorption. Endoglucanase EGI was preferentially adsorbed over EGII and EGIII. Sequential adsorption studies have shown that interaction between enzyme components largely determines the degree of their adsorption. Evidence suggests that both common and distinct adsorption sites exist and that their occupation depends on which components are involved. Predominance in adsorption by any one of the enzyme components is decreased at 50 degrees C. Light microscopy and monitoring of sugar production during cellulose hydrolysis provided evidence that reduction in the ionic strength decreases the adsorption predominance of CBHI and enhances the synergism between the cellulase components.     

Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains

Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and -octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but — unlike in extracellular culture fluids from Trichoderma — CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.     

Temperature sensitive mutant of Trichoderma reesei defective in secretion of cellulase

Summary Temperature sensitive mutants of Trichoderma reesei derived from hypersecretory strain RL-P37 were isolated and characterized. Compared to the parent strain, one mutant (LU-ts 1) grew well in the mycelial phase at both permissive (25°C) and non-permissive (37°C) temperatures. However, the secretion of overall protein and active cellulases was significantly reduced in the mutant at the higher temperature. No accumulation of active cellulases or intracellular proteins was observed in the mycelia of LU-ts 1 at 37°C. The inhibitory effects of temperature on cellulase secretion in LU-ts 1 were reversible. Isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses confirmed that the secretion of the major cellulases was greatly reduced in LU-ts 1 at 37°C. Molecular characterization of the various temperature sensitive secretion mutants of T. reesei should help elucidate the crucial aspects of the secretory pathway of this cellulolytic fungus.     

Effects of gamma-ray irradiation on cellulase secretion of Trichoderma reesei

Trichoderma reesei was irradiated with gamma rays to investigate the effects of different dosages on cellulase production. Doses above 0.7 kGy induced cell lysis. Cell growth began to be obstructed at 2.0 kGy. As a result, the cells irradiated at 2.0 kGy secreted 1.8 times as much cellulase as the untreated cells.