Article

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

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Abstract

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 >0.3g/L) repressed the galactose consumption. Cellulase synthesis was growth-independent while lactase synthesis was growth-dependent, except at D0.1h−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.

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... Thus, selecting an inexpensive soluble inducer is an effective strategy to improve the enzyme production and decrease the costs. Interestingly, lactose, which is produced at around 1.2 million tons per annum worldwide as a byproduct of the cheese manufacturing and whey processing industries, can induce cellulase formation in some fungi, such as P. echinulatum and T. reesei (Lo et al. 2010;Sehnem et al. 2006). It is the only economic soluble carbon source for this purpose. ...
... This result indicated that lignocellulolytic enzymes synthesis by Bacillus BS-5 was regulated in ways different from the known Bacilli. Lactose is the only soluble and economically viable carbon source for cellulolytic enzyme production (Lo et al. 2010). The use of strain BS-5 with lactose as an inducer exhibited the economic and technical advantages of the enzyme production process. ...
... BS-5. In addition, the fermentation period of strain BS-5 was 32 h, which was 3-5 days shorter than those of the filamentous fungi such as Trichoderma and Penicillium (Liao et al. 2014;Mari et al. 2014;Lo et al. 2010;Sehnem et al. 2006). Consequently, the enzyme production cost can be significantly reduced. ...
Article
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In this study, with combined carboxymethyl cellulose agar plate, xylan agar plate and filter paper hydrolysis assay, a novel cellulase and xylanase-producing strain identified as Bacillus sp. was isolated. Using lactose as the only carbon source, a complete and balanced lignocellulolytic enzyme system containing at least endoglucanase (9.6 U/ml), exoglucanase (0.8 U/ml), Fpase (1.4 U/ml), xylanase (3.8 U/ml) and β-glucosidase (1.2 U/ml) was produced. Interestingly, a zymogram of the crude culture supernatant displayed a multifunctional lignocellulolytic enzyme system including at least four bonds with both endoglucanase activity and xylanase activity at 21.2, 23.8, 28.9 and 31.2 kDa, respectively, indicating that these enzymes might be bifunctional. More gratifyingly, according to the binding affinity analysis and scanning electron microscopy, the crude enzyme complex produced by strain BS-5 was capable of hydrolyzing not only pure insoluble polysaccharides, but also agricultural residues such as corn cob. At 5% substrate concentration and 20 FPU/g enzyme loading, the reducing sugar was 350.8 mg/g of alkali-pretreated corn cob after 72 h enzymatic hydrolysis. These results suggested that this strain could be a good candidate for the development of a more cost-effective and efficient lignocellulolytic enzyme cocktail for the saccharification of lignocellulosic biomass.
... Therefore most studies considered an uncoupled production model (Lo et al., 2010;Velkovska et al., 1997), which was assumed valid here for low growth rates between 0 and 0.03 h -1 (Eq. 3). ...
... During growth on lactose, a maximal yield Yx° of 0.6 g.g -1 was generally observed both at high growth rates (Lo et al. 2010), and at low growth rates (Pakula et al., 2005), so this value was assumed to be valid for this work. Bibliographic and personal data (submitted for publication) for cellulase production by T. reesei on lactose were used for the identification of the other parameters (Table 1). ...
Article
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A simple kinetic model of cellulase production by Trichoderma reesei on lactose was developed and parameters identified using bibliographic and personal data. Assuming a mean representation of industrial hyperproducer strains, this model was used to simulate and compare several cultivation strategies regarding three criteria: in addition to usual productivity and yield calculation, oxygen demand was studied and used as a constraint for protocol design. Results showed that none of the protocols maximizes both productivity and yield, requiring to find a compromise for these parameters. Moreover, substrate concentration in the feed was the main criterion for the choice of the protocol, while oxygen demand and biomass concentration were the main issues to reach high productivities. The model will be useful for an economic study of cultivation strategies.
... Increase in lactose concentration beyond that limit having negative impact on cellulase production. Being a byproduct of cheese manufacturing industry, lactose is considered inexpensive and economically feasible inducer for cellulase production and it was identified with reasonably good induction abilities for cellulase production by Trichoderma reesei [62]. However, induction ability of lactose alone for formation of cellulase is limited due its slower induction rate [63]. ...
Article
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A systematic evaluation of microorganism’s potential towards biosynthesis of cellulases from inexpensive lignocellulosic feedstock through appropriate kinetic modelling facilitates understanding, optimization and designing of an effective industrial cellulase enzyme production process. The present study aims to optimize a submerged fungal cultivation strategy for cellulase production from abundantly available newspaper wastes (NPW). A combined pretreatment strategy consisting diluted, 1% (v v⁻¹) H2SO4 followed by 2% (wv⁻¹) NaOH treatment was highly effective to convert newspaper waste to an effective cellulose-enriched inducer for the production of cellulase. In addition, the composition of the most influential nutrient components like peptone and lactose was optimized with the help of response surface methodology for enhanced cellulase production with maximum activity levels. Maximum cellulase production of 8.64 g L⁻¹ with 7.82 FPU mL⁻¹ total activity levels was achieved from optimized composition of pretreated NPW 3.29% (w v⁻¹), lactose 2.94% (w v⁻¹) and peptone 1.53% (w v⁻¹). To analyse intrinsic inhibition effect of the substrate concentration on cellulase production, modified Luedeking–Piret model simulated experiments were further conducted with 1.5% (w/v), 3.29% (w/v) and 4% (w/v) NPW concentrations. The developed kinetic model perfectly captured the trends of biomass production, substrate consumption and adsorption characteristic of cellulase enzyme on its activity during production. The rate constant for cellulase synthesis was evaluated to be increased to 0.040 IU g⁻¹ h ⁻¹ at 3.29% (w v⁻¹) of NPW concentration; however, it was further reduced to 0.024 IU g⁻¹ h ⁻¹ at higher NPW concentration of 4% (w v⁻¹).
... Lactose is a soluble inducer, but it is not directly absorbed by cells and is broken down into glucose and galactose after entry into the cell. The rate-limiting step in this process is the rate of decomposition of lactose [88]. Lactose is a by-product of the dairy production process and is inexpensive and economically viable. ...
Article
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Lignocellulosic biomass such as agricultural and forestry waste is the most abundant renewable organic carbon source on earth and can be used to produce source of clean energy such as ethanol. One of the disadvantages of the preparation of ethanol using lignocellulose as raw material is the high cost of production of cellulase. Fungi are capable of effectively degrading lignocellulose and secreting a large amount of cellulase, and have the advantages of ease of preparation, high yield, and full enzyme systems. Therefore, this paper reviews sources of lignocellulose and the biodegradation properties which limit the production of cellulase, proposes micro-organisms capable of degrading lignocellulose and explains the types of cellulase, and the mechanism of action, methods of fermentation optimization, and control are analyzed, and ways to increase the yield of cellulase are described. Finally, research on the effects of inducers on the production of cellulase by fungi is reviewed. The aims of this review are to provide a reference for the efficient production and industrial application of cellulase.
... Among all the above inducers lactose is inexpensive and has been exploited commercially for cellulase production, although the mechanism of induction is still a subject of research. It was assumed that lactose was hydrolysed extracellularly to give galactose and glucose and thus boosted T. reesei's growth [108] however, repressed cellulase production indicates that cellulase production is not growth related. Thus, for cellulase induction either the intracellular presence or the uptake and/or metabolism of lactose is essential. ...
... Among all the above inducers lactose is inexpensive and has been exploited commercially for cellulase production, although the mechanism of induction is still a subject of research. It was assumed that lactose was hydrolysed extracellularly to give galactose and glucose and thus boosted T. reesei's growth [108] however, repressed cellulase production indicates that cellulase production is not growth related. Thus, for cellulase induction either the intracellular presence or the uptake and/or metabolism of lactose is essential. ...
Article
Increasing population and industrialization caused increased demand for liquid fossil fuels which in turn increases the greenhouse gas emission. Bioethanol produced from lignocellulosic biomass via enzymatic route is a potential alternative to fossil fuels and is environmentally sustainable. Cellulases have been regarded as the limiting factor for bioethanol production from lignocellulosic biomass via enzymes. In the last few decades advances in bioprocesses led to reduction in the cost of cellulases by several folds, enabling bioethanol production to become cost-effective. This is the reason for existence of commercial plants for bioethanol production, however; still there are scope for further improvement in bioprocess for cellulase production and research is ongoing worldwide. Researchers face huge challenge while moving from flask and bioreactor research outcomes from a laboratory scale to the pilot scale production, which has been rarely discussed. This review will present those challenges and its probable solutions. Though commercial cellulases are available, it is highly required to have in-house cellulase production technology to be self-reliant. On-site and integrated cellulase production configuration is popular as it seems to be cost-effective. This review will address advances in bioprocesses and challenges for cellulase production which have surfaced in the last decade.
... Proteins production was described via a Monod-type kinetic model. Other similar models are available in the literature (Lo et al., 2010). Rate of cell (X) growth, rates of substrate (S), oxygen (O 2 ) consumption, and rate of products (P) formation can be expressed by the following equations: ...
Article
The application of Computational Fluid Dynamics to aerobic fermentations faces several issues, such as validation of multiphase models at high gas holdup and with complex liquids under turbulent condition. In this work, the Eulerian two-fluid model framework was adapted to simulate the enzyme production by the filamentous fungi T. reesei. Due to scarcity of data on turbulent mixing in complex fluids, every aspect of the numerical model (turbulence model, drag force law, rheology model, etc.) needed to be validated. First, the adequacy of the model was evaluated by the mean of comparison with new and previous experimental data in non-Newtonian aerated systems. Once coupled with an apparent oxygen and substrate uptake kinetics, the model was used to investigate the effect of scale-up on the enzyme productivity from biomass. Fully predictive results highlighted complex behaviors, such as: possible substrate heterogeneities, yield loss, and non-obvious interactions between mixing and oxygen transfer limitations.
... Cette valeur est d'environ 15 % chez T. reesei pour une pression légèrement supérieure (1,14 bar) à la pression atmosphérique (ce qui correspond à 0,0375 mmol.L -1 d'O 2 ) (Marten et al., 1996). Le (Lo et al., 2010) Le tableau (Tab. I.2.ii) établi par présente des données relatives à la production de cellulases pour trois souches de T. reesei. ...
Thesis
Le procédé de production d’éthanol à partir de biomasse lignocellulosique nécessite l’hydrolyse de cette dernière en sucres simples. Cette hydrolyse est le plus souvent réalisée par voie biologique grâce à des enzymes appelées cellulases. La production de ces enzymes représente cependant un verrou économique majeur au développement du procédé à grande échelle. Les cellulases sont généralement produites industriellement par le champignon filamenteux aérobie Trichoderma reesei, doté d’une forte capacité de sécrétion d’enzymes. Les cultures sont réalisées en bioréacteurs aérés et agités mécaniquement. Elles nécessitent de contrôler la concentration des substrats, ce qui requiert la maitrise de conditions hydrodynamiques et physicochimiques. En effet, le milieu de culture de T. reesei devient une suspension de cellules de champignons associées en filaments, de structure complexe, dont la viscosité augmente avec la concentration microbienne selon un comportement rhéofluidifiant. La viscosité est fonction de la morphologie du microorganisme qui peut, elle-même, varier avec les conditions de cultures. Cet accroissement de viscosité est un critère clef de l’extrapolation du procédé, car il affecte le transfert d’oxygène. Afin de maintenir une concentration en oxygène dissous suffisante, l’agitation et l’aération sont en général augmentées, entraînant un accroissement du cisaillement. Cet accroissement impacte en retour la morphologie du champignon, ralentit sa croissance puis diminue la production de cellulases. Ainsi, les conditions hydrodynamiques et rhéologiques engendrées au sein du bioréacteur sont complexes et variables dans le temps. L’interrelation entre conditions opératoires, morphologie, croissance du champignon et viscosité du moût de fermentation impose l’intégration de tous ces phénomènes pour l’optimisation du procédé, notamment à grande échelle. L’objectif de la thèse est de mettre en place une approche, visant à étudier au laboratoire la croissance de T. reesei et sa production d’enzymes, en reproduisant les contraintes hydrodynamiques associées aux conditions de fonctionnement des fermenteurs industriels. Pour ce faire, deux méthodologies originales ont été développées : une méthode de mesure de la viscosité du milieu, optimisée pour les champignons filamenteux, représentative des conditions rencontrées à grande échelle et qui s’appuie sur l’utilisation d’un rhéomètre rotatif équipé d’un rotor hélicoïdal ; une méthode d’analyse d’images associant un microscope motorisé et des algorithmes d’analyse d’images innovants, qui permet de générer des données sur la morphologie du champignon et d’identifier un critère morphologique pertinent basé sur le nombre de « trous » au sein d’un filament. Parallèlement à ces méthodes, différentes contraintes de cisaillement ont été mises en oeuvre en fermentation, afin de reproduire, à l’échelle du laboratoire, les conditions rencontrées à l’échelle industrielle. Ces outils ont été utilisés conjointement et validés lors de cultures non conventionnelles mimant les conditions industrielles en termes de cisaillement. Ils ont permis d’identifier un critère représentatif du cisaillement (EDCF) et d’établir, à partir de ce critère, des corrélations capables de prédire la viscosité du moût de fermentation, le taux de croissance maximum du microorganisme ainsi que certains paramètres morphologiques de la souche. De façon originale, ces corrélations déterminées à l’échelle du laboratoire ont été validées par des mesures effectuées à l’échelle industrielle. Au final, l’approche développée permet d’identifier au plus tôt les contraintes d’extrapolation à ne pas dépasser, afin d’orienter les choix technologiques des fermenteurs industriels impliquant des champignons filamenteux.
... Soluble oligosaccharides like cellobiose, lactose and sophorose (2-O-␤-glucopyranosyl-d-glucose) are considered as direct inducers of cellulase [5]. Though Lactose, an inexpensive soluble substrate, offers reasonably good induction for cellulase production by Trichoderma reesei, the extracellular lactose hydrolysis is the rate-limiting step for growth of fungus [6]. ...
Article
Nanocellulose prepared by controlled microbial hydrolysis (NCm) induced secretion of complete array of cellulase by the fungus, _Trichoderma reesei_. The nanocellulose prepared by conventional sulphuric acid hydrolysis (NCa) and high pressure homogenization process (NFC) were also evaluated in this study in addition to two commercial inducers namely, sophorose and cellobiose. Batch culture was grown on Mandel’s basal salt medium added with cellulose as the sole carbon source. Cell growth, substrate concentration and cellulase production were analysed over a period of 5 days. NCa, due to attachment of sulphate groups on surface, inhibited the growth of the fungus. NFC could not effectively induce secretion of cellulase as it tends to aggregate due to high aspect ratio ( > 1000). While sophorose induced an incomplete array of cellulase that too only up to 2 h, cellobiose induced complete array with 65% substrate utilization over a period of 5 days. NCm induced complete array of cellulase with 80% substrate utilized over a period of 5 days. This is attributed to higher crystallinity that helped in slower degradation and steady release of molecules needed for enzyme induction.
... Hence, rice straw grindings filtered by 80 mesh were selected for further experiments. In addition, lactose, which was found to be a good carbon source and inducer for cellulase production by some fungi [39][40][41], was tested in the presence of rice straw. However, no obvious inducer effect was observed in the fermentation of R. stolonifer TP-02 by addition of lactose. ...
Article
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Agricultural by-products, rice straw, wheat bran juice, and soybean residue, were used as substrates for cellulase production using Rhizopus stolonifer var. reflexus TP-02. The culture medium was optimized though uniform design experimentation during shaking flask fermentation, and the ideal formulation obtained for filter paper enzyme (FPase) production was 10 % bran diffusion juice, 1 % rice straw, 0.17 % urea, 0.17 % soybean residue, 0.11 % KH2PO4, and 0.027 % Tween 80, and the maximal FPase activity in the culture supernatant was 13.16 U/mL at an incubation time of 3 days. A kinetic model for cellulase production in batch fermentation was subsequently developed. The unstructured kinetic model considered three responses, namely biomass, cellulase, and sugar. Models for the production of three types of cellulase components (i.e., endoglucanases, cellobiohydrolases, and β-glucosidases) were established to adequately describe the cellulase production pattern. It was found that the models fitted the experimental data well under pH 5.0 and 6.0, but only the avicelase production model predicted the experimental data under pH-uncontrolled conditions.
... Both used cellulose as main carbon source. Lo et al. (2010) worked with T. reesei Rut C30 (NRRL 11460) for enzyme production using a soluble substrate (lactose) in batch and continuous fermentation process. Ma et al. (2013) studied the enzyme production from T. reesei Rut C30 (ATCC 56765) in batch and fed-batch process using cellulose as inducer. ...
Article
A mathematical model to describe the kinetics of enzyme production by the filamentous fungus Trichoderma harzianum P49P11 was developed using a low cost substrate as main carbon source (pretreated sugarcane bagasse). The model describes the cell growth, variation of substrate concentration and production of three kinds of enzymes (cellulases, beta-glucosidase and xylanase) in different sugarcane bagasse concentrations (5; 10; 20; 30; 40gL(-1)). The 10gL(-1) concentration was used to validate the model and the other to parameter estimation. The model for enzyme production has terms implicitly representing induction and repression. Substrate variation was represented by a simple degradation rate. The models seem to represent well the kinetics with a good fit for the majority of the assays. Validation results indicate that the models are adequate to represent the kinetics for a biotechnological process.
... T. reesei is able to grow on a wide range of carbon sources for enzyme production, including many monosaccharides, oligosaccharides, polyols, and amino acids [5]. The disaccharide lactose is commonly used in industrial enzyme production because it results in good growth and efficient induction of enzyme production [6]. In addition to lactose and other simple sugars, many complex materials have been used for enzyme production. ...
Article
The enzyme induction utility of soybean hulls (SBH), consisting in excess of 50 wt% non-starch polysaccharides (NSP) cellulose, hemicellulose, and pectin, was studied through cultivation of the carbohydrase-producing fungus Trichoderma reesei Rut C-30. Shake flask systems of T. reesei were grown in a medium consisting of defatted soybean flour as a nitrogen source and SBH, some of which were untreated and others pretreated by liquid hot water, alkaline, and supercritical carbon dioxide, as carbon source. Cellulase, xylanase, and polygalacturonase activities were measured for the systems, and the natural hull systems were found to yield optimum enzyme production. Controlled batch fermentation experiments were carried out to compare enzyme production resulting from media with Avicel® (FMC BioPolymer, Philadelphia, PA, USA) versus natural SBH with and without soybean flour as the nitrogen source. Soybean hull fermentations were also performed at several pH levels to observe the effects on enzyme production. Soybean hulls induced comparable levels of cellulase, and higher levels of xylanase and polygalacturonase, than Avicel®. With SBH, cellulase and xylanase production were enhanced at higher pH levels (6.0), and polygalacturonase was enhanced at lower pH levels (4.0–4.5). Enzyme production was largely unaffected by the presence of soybean flour as the nitrogen source.
... doi:10.1371/journal.pone.0088689.g007 inexpensive, soluble substrate, leads to reasonably good induction for cellulase production [43], [44]. The fungus does not directly take up lactose but instead hydrolyzes the compound to galactose and glucose. ...
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Profiling the transcriptome that underlies biomass degradation by the fungus Trichoderma harzianum allows the identification of gene sequences with potential application in enzymatic hydrolysis processing. In the present study, the transcriptome of T. harzianum IOC-3844 was analyzed using RNA-seq technology. The sequencing generated 14.7 Gbp for downstream analyses. De novo assembly resulted in 32,396 contigs, which were submitted for identification and classified according to their identities. This analysis allowed us to define a principal set of T. harzianum genes that are involved in the degradation of cellulose and hemicellulose and the accessory genes that are involved in the depolymerization of biomass. An additional analysis of expression levels identified a set of carbohydrate-active enzymes that are upregulated under different conditions. The present study provides valuable information for future studies on biomass degradation and contributes to a better understanding of the role of the genes that are involved in this process.
Thesis
Les cellulases industrielles sont principalement produites par un champignon filamenteux aérobie, Trichoderma reesei, en raison de sa grande capacité de sécrétion. L’extrapolation du procédé de production de cellulases est une condition préalable pour que les procédés de production d’éthanol à partir de biomasse lignocellulosique soient viables. Cette extrapolation est cependant difficile car la morphologie filamenteuse induit une augmentation de la viscosité du milieu, ce qui conduit à des hétérogénéités spatiales et temporelles au sein de la culture, notamment en termes de concentration en oxygène dissous, affectant négativement la production d’enzymes. L’objectif de cette thèse est d’étudier les effets du mélange de la culture, en termes de stress hydrodynamique et de gradients en oxygène dissous, sur la physiologie et la capacité de production de cellulases par T. reesei. Afin de reproduire à l’échelle du laboratoire les contraintes rencontrées à l’échelle industrielle, la méthodologie scale-down a été employée. L’impact du cisaillement sur la production de cellulases a été étudié en mode continu, en employant deux niveaux de stress hydrodynamique. L'effet des gradients d’oxygène a été étudié en cultures fed-batch à l'aide de trois approches scale-down : deux systèmes Bizone, constitués de deux bioréacteurs reliés, le plus grand étant aéré, l’autre non (ou l’inverse pour le système Bizone inversé) et un bioréacteur unique sur lequel une variation dynamique de la pO2 est appliquée. Les résultats montrent que les performances sont dégradées par l’anaérobiose. Toutefois, l’existence de petites bulles d’air, maintenues dans les milieux visqueux, évite que le milieu soit totalement anaérobie, ce qui limite l’effet délétère de l’absence d’oxygène. L’application de périodes d’hypoxie a également permis de faire émerger un variant non producteur, qui induit une baisse importante des performances du bioprocédé. Enfin, le développement de modèles originaux a confirmé l’importance de la prise en compte du hold-up lié aux petites bulles d’air pour bien représenter le comportement de T. reesei. L’ensemble de ces travaux permet enfin de démontrer une certaine « robustesse » du champignon face au cisaillement et aux hétérogénéités susceptibles d’apparaître à l’échelle industrielle.
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Wheat chaff as an agricultural waste represents a cheap raw material for biotechnological processes. With its lignocellulosic composition, it is suitable for producing hydrolytic enzymes for second generation renewable fuel production technologies. The aim of this work was to optimize the process parameters (cultivation temperature 25–35°C, pH value 4–6 and cultivation time 3–7 days) of the cultivating fungi (Trichoderma reesei QM 9414) on a media based on wheat chaff by submerged and solid-state techniques, in order to enhance and compare the two types of simultaneous cellulase and xylanase production. Optimal conditions for the submerged fermentation were 29.65°C for temperature, pH 4.27 and 7 days of cultivation, while for the solid-state fermentation, the optimal conditions were 28.01°C, pH 6.00 and 7 days. The cellulolytic and xylanolytic activities of the obtained cultivation broth filtrates were 0.0535 and 0.1676 U mL-1 for the submerged fermentation, and 0.0407 and 0.1401 U mL-1 for the solid-state fermentation, respectively, and with a 26.77 and 13.39 % enhancement of enzyme activity for submerged fermentation, and a 22.96 and 42.66 % enhancement for solid-state fermentation, respectively, compared to the results obtained before optimization. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. TR-31002]
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A new strain Trichoderma koningii D-64 was isolated from Singapore soil samples. It produced cellulase, xylanase, and laccase on a variety of carbon sources. Enzyme activities of 3.8 ± 0.3, 40.3 ± 5.1, 6.6 ± 0.3 and 98.8 ± 10.3 U/mL were respectively obtained for FPase, CMCase, β-glucosidase and xylanase in a mixture of 1% cellulose and 2% wheat bran. About 70–95% saccharification efficiency of oil palm empty fruit bunch was obtained using T. koningii D-64 enzymes alone without the supplement of any other commercial enzymes. Strain T. koningii D-64 is therefore a potential cellulase producer for the efficient lignocellulosic biomass conversion to fermentable sugars.
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Acetic acid is one of the major potential inhibitors of cellular growth resulting from acid pretreatment of lignocellulosic biomass. To use lignocellulose hydrolysate in biorefineries it is necessary to assess their effect on the microorganisms likely to be used. An attractive option is to use a part of these hydrolysates for on-site cellulase production. Based on an industrial process that included a fast initial growth phase, the effect on Trichoderma reesei grown on glucose was assessed and modeled at different concentrations and pH. Acetic acid had a strong effect on T. reesei growth rate and yield, which correlated only with the concentration of the undissociated form in solution. The specific growth rate was accurately modeled by two different classic inhibition models as a function of pH and total acetic acid concentration. Co-consumption of glucose and acetic acid was observed, so that the culture medium was gradually detoxified by the cells. With or without glucose, acetic acid was mineralized into carbon dioxide at a similar specific rate, but no growth was observed without glucose. A 2.5-fold increase in the maintenance coefficient was observed, due to the need for glucose consumption to preserve cell integrity, which corresponded to a one-third decrease in the overall biomass yield. The resulting models can be used to simulate T. reesei growth on acetic acid-containing media and to choose the optimum pH for efficient growth on lignocellulosic biomass hydrolysates.
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The effects of varying initial concentrations of microcrystalline cellulose on cellulase production with Trichoderma reesei RUT-C30 as well as the effects of varying lactose and ammonium sulfate concentrations in the feed medium were studied simultaneously in parallel-operated shake flasks and, alternatively, in parallel-operated stirred-tank bioreactors on a 10-mL scale. Fifteen experiments were performed as triplicates in shake flasks as well as in stirred-tank bioreactors in parallel to identify the parameters of second-order polynomials for the estimation of the final filter paper activity of T. reesei RUT-C30 after a process time of 96 h. Even though parameter estimation was not possible based on the results of the shake flasks due to final enzyme activities at or below the detection limit (with the exception of one shake flask), the identification of the second-order polynomial was successful with the results of the parallel-operated stirred-tank bioreactors on a 10-mL scale. Reaction conditions with 53.3 g L(-1) microcrystalline cellulose in the initial medium, no lactose feeding and 3.3 g L(-1) day(-1) intermittent ammonium sulfate addition were estimated to be optimal. The final experimental validation of the optimum substrate supply on a L-scale resulted in the production of 4.88 filter paper units (FPU) mL(-1) with T. reesei RUT-C30 after 96 h. This is an improvement by a factor of 3.6 compared to the reference batch process (1.35 FPU mL(-1)).
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Basic features of regulation of expression of the genes encoding the cellulases of the filamentous fungus Trichoderma reesei QM9414, the genes cbh1 and cbh2 encoding cellobiohydrolases and the genes egl1, egl2 and egl5 encoding endoglucanases, were studied at the mRNA level. The cellulase genes were coordinately expressed under all conditions studied, with the steady-state mRNA levels of cbh1 being the highest. Solka floc cellulose and the disaccharide sophorose induced expression to almost the same level. Moderate expression was observed when cellobiose or lactose was used as the carbon source. It was found that glycerol and sorbitol do not promote expression but, unlike glucose, do not inhibit it either, because the addition of 1 to 2 mM sophorose to glycerol or sorbitol cultures provokes high cellulase expression levels. These carbon sources thus provide a useful means to study cellulase regulation without significantly affecting the growth of the fungus. RNA slot blot experiments showed that no expression could be observed on glucose-containing medium and that high glucose levels abolish the inducing effect of sophorose. The results clearly show that distinct and clear-cut mechanisms of induction and glucose repression regulate cellulase expression in an actively growing fungus. However, derepression of cellulase expression occurs without apparent addition of an inducer once glucose has been depleted from the medium. This expression seems not to arise simply from starvation, since the lack of carbon or nitrogen as such is not sufficient to trigger significant expression.
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For well over one hundred years, researchers around the world have pursued ways to make ethanol from biomass such as wood, grasses, and waste materials. To distinguish it from ethanol made from starch and sugars in traditional agricultural crops, we refer to ethanol made from biomass as "bioethanol." The effort to develop bioethanol technology gained significant momentum in the late 1970s as a result of the energy crises that occurred in that decade. This article briefly reviews the broader history of bioethanol technology development. With this as a background, we focus our attention on the strategic thinking behind the U.S. Department of Energy's Bioethanol Program, which envisions remarkable advances in cellulase enzyme research and as the basis for significant future process cost reductions.
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A low-foaming hydrophobin II deletant of the Trichoderma reesei strain Rut-C30 was used for production of cellulases by continuous cultivation on lactose medium in a laboratory fermenter. The control paradigm of the addition of new medium to the continuous process was based on the growth dynamics of the fungus. A decrease in the rate of base addition to the cultivation for pH-minimum control was used as an indicator of imminent exhaustion of carbon source for growth and enzyme induction. When the amount of base added per 5 min computation cycle decreased below a given value, new medium was added to the fermenter. When base addition for pH control thereafter increased above the criterion value, due to increased growth, the medium feed was discontinued or decreased. The medium feeding protocol employed was successful in locking the fungus in the stage of imminent, but not actual, exhaustion of carbon source. According to the results of a batch cultivation of the same strain on the same medium, this is the phase of maximal enzyme productivity. The medium addition protocol used in this work resulted in a very stable continuous process, in which cellulase productivity was maintained for several hundred hours at the maximum level observed in a batch cultivation for only about 10 h. Despite a major technical disturbance after about 420 h, the process was restored to stability. When the cultivation was terminated after 650 h, the level of enzyme production was still maximal, with no signs of instability of the process.
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Trichoderma reesei was cultivated in chemostat cultures on lactose-containing medium. The cultures were characterized for growth, consumption of the carbon source and protein production. Secreted proteins were produced most efficiently at low specific growth rates, 0.022-0.033 h(-1), the highest specific rate of total protein production being 4.1 mg g(-1) h(-1) at the specific growth rate 0.031 h(-1). At low specific growth rates, up to 29 % of the proteins produced were extracellular, in comparison to only 6-8 % at high specific growth rates, 0.045-0.066 h(-1). To analyse protein synthesis and secretion in more detail, metabolic labelling of proteins was applied to analyse production of the major secreted protein, cellobiohydrolase I (CBHI, Cel7A). Intracellular and extracellular labelled CBHI was quantified and analysed for pI isoforms in two-dimensional gels, and the synthesis and secretion rates of the molecule were determined. Both the specific rates of CBHI synthesis and secretion were highest at low specific growth rates, the optimum being at 0.031 h(-1). However, at low specific growth rates the secretion rate/synthesis rate ratio was significantly lower than that at high specific growth rates, indicating that at low growth rates the capacity of cells to transport the protein becomes limiting. In accordance with the high level of protein production and limitation in the secretory capacity, the transcript levels of the unfolded protein response (UPR) target genes pdi1 and bip1 as well as the gene encoding the UPR transcription factor hac1 were induced.
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Lactose is the only soluble and economically feasible carbon source for the production of cellulases or heterologous proteins regulated by cellulase expression signals by Hypocrea jecorina (Trichoderma reesei). We investigated the role of the major beta-galactosidase of H. jecorina in lactose metabolism and cellulase induction. A genomic copy of the bga1 gene was cloned, and this copy encodes a 1,023-amino-acid protein with a 20-amino-acid signal sequence. This protein has a molecular mass of 109.3 kDa, belongs to glycosyl hydrolase family 35, and is the major extracellular beta-galactosidase during growth on lactose. Its transcript was abundant during growth on l-arabinose and l-arabinitol but was much less common when the organism was grown on lactose, d-galactose, galactitol, d-xylose, and xylitol. Deltabga1 strains grow more slowly and accumulate less biomass on lactose, but the cellobiohydrolase I and II gene expression and the final cellulase yields were comparable to those of the parental strain. Overexpression of bga1 under the control of the pyruvate kinase promoter reduced the lag phase, increased growth on lactose, and limited transcription of cellobiohydrolases. We detected an additional extracellular beta-galactosidase activity that was not encoded by bga1 but no intracellular beta-galactosidase activity. In conclusion, cellulase production on lactose occurs when beta-galactosidase activity levels are low but decreases as the beta-galactosidase activities increase. The data indicate that bga1-encoded beta-galactosidase activity is a critical factor for cellulase production on lactose.
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Lactose (1,4-O-beta-d-galactopyranosyl-d-glucose) is a soluble and economic carbon source for the industrial production of cellulases or recombinant proteins by Hypocrea jecorina (anamorph Trichoderma reesei). The mechanism by which lactose induces cellulase formation is not understood. Recent data showed that the galactokinase step is essential for cellulase induction by lactose, but growth on d-galactose alone does not induce cellulases. Consequently, the hypothesis was tested that d-galactose may be an inducer only at a low growth rate, which is typically observed when growing on lactose. Carbon-limited chemostat cultivations of H. jecorina were therefore performed at different dilution rates with d-galactose, lactose, galactitol and d-glucose. Cellulase gene expression was monitored by using a strain carrying a fusion between the cbh2 (encoding cellobiohydrolase 2, Cel6A) promoter region and the Aspergillus niger glucose oxidase gene and by identification of the two major cellobiohydrolases Cel7A and Cel6A. The results show that d-galactose indeed induces cbh2 gene transcription and leads to Cel7A and Cel6A accumulation at a low (D=0.015 h(-1)) but not at higher dilution rates. At the same dilution rate, growth on d-glucose did not lead to cbh2 promoter activation or Cel6A formation but a basal level, lower than that observed on d-galactose, was detected for the carbon-catabolite-derepressible Cel7A. Lactose induced significantly higher cellulase levels at 0.015 h(-1) than d-galactose and induced cellulases even at growth rates up to 0.042 h(-1). Results of chemostats with an equimolar mixture of d-galactose and d-glucose essentially mimicked the behaviour on d-galactose alone, whereas an equimolar mixture of d-galactose and galactitol, the first intermediate of a recently described second pathway of d-galactose catabolism, led to cellulase induction at D=0.030 h(-1). It is concluded that d-galactose indeed induces cellulases at low growth rate and that the operation of the alternative pathway further increases this induction. However, under those conditions lactose is still a superior inducer for which the mechanism remains to be clarified.
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The ability of Hypocrea jecorina (Trichoderma reesei) to grow on lactose strongly depends on the formation of an extracellular glycoside hydrolase (GH) family 35 beta-galactosidase, encoded by the bga1 gene. Previous studies, using batch or transfer cultures of pregrown cells, had shown that bga1 is induced by lactose and d-galactose, but to a lesser extent by galactitol. To test whether the induction level is influenced by the different growth rates attainable on these carbon sources, bga1 expression was compared in carbon-limited chemostat cultivations at defined dilution (=specific growth) rates. The data showed that bga1 expression by lactose, d-galactose and galactitol positively correlated with the dilution rate, and that galactitol and d-galactose induced the highest activities of beta-galactosidase at comparable growth rates. To know more about the actual inducer for beta-galactosidase formation, its expression in H. jecorina strains impaired in the first steps of the two d-galactose-degrading pathways was compared. Induction by d-galactose and galactitol was still found in strains deleted in the galactokinase-encoding gene gal1, which is responsible for the first step of the Leloir pathway of d-galactose catabolism. However, in a strain deleted in the aldose/d-xylose reductase gene xyl1, which performs the reduction of d-galactose to galactitol in a recently identified second pathway, induction by d-galactose, but not by galactitol, was impaired. On the other hand, induction by d-galactose and galactitol was not affected in an l-arabinitol 4-dehydrogenase (lad1)-deleted strain which is impaired in the subsequent step of galactitol degradation. These results indicate that galactitol is the actual inducer of Bga1 formation during growth on d-galactose in H. jecorina.
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The 5' regulatory region of the cbh2 gene of Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for induction of cbh2 gene expression by sophorose and cellulose. The CAE consists of two motifs, a CCAAT box on the template strand and a GTAATA box on the coding strand, which cooperate during induction. Northern analyses of cbh2 gene expression has revealed an absolute dependence on induction, but no direct effect of Cre1-mediated carbon catabolite repression. Investigation of the chromatin structure in the wild-type strain showed that, under repressing conditions, there is a nucleosome free region (nfr) around the CAE, which is flanked by strictly positioned nucleosomes. Induction results in a loss of positioning of nucleosomes -1 and -2 downstream of the CAE, thus making the TATA box accessible. Simultaneous mutation of both motifs of the CAE, or of the CCAAT-box alone, also leads to shifting of nucleosome -1, which normally covers the TATA-box under repressing conditions, whereas mutation of the GTAATA element results in a narrowing of the nfr, indicating that the proteins that bind to both motifs in the CAE interact with chromatin, although in different ways. A cellulase-negative mutant strain, which has previously been shown to be altered in protein binding to the CAE, still displayed the induction-specific changes in nucleosome structure, indicating that none of the proteins that directly interact with CAE are affected, and that nucleosome rearrangement and induction of cbh2 expression are uncoupled. Interestingly, the carbon catabolite repressor Cre1 is essential for strict nucleosome positioning in the 5' regulatory sequences of cbh2 under all of the conditions tested, and induction can occur in a promoter that lacks positioned nucleosomes. These data suggest that Cre1, the Hap2/3/5 complex and the GTAATA-binding protein are all involved in nucleosome assembly on the cbh2 promoter, and that the latter two respond to inducing conditions by repositioning nucleosome -1.
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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 anti 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. (C) 1999 John Wiley & Sons, Inc.
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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.
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Growth and maintenance parameters μmax, Ks, m, and Ym for cellulase biosynthesis on lactose by T. reesei-C5 were estimated and compared with published data on other soluble substrates and mutant strains of T. reesei in continuous culture. Growth was favored at higher feed lactose but cellulase productivities did not increase proportionally, suggesting that a degree of inhibition and/or catabolic repression within the strain is possible. The estimated values of growth kinetics and maintenance parameters varied little but were within a reasonable range of published data on other soluble substrates and mutant strains of T. reesei in continuous cultures.
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Cellulase a multienzyme made up of several proteins finds extensive applications in food, fermentation and textile industries. Trichoderma reesei is an efficient producer of cellulase protein. The comparative study was made on various carbon sources on the production of cellulase using strains of T. reesei QM 9414, 97.177 and Tm3. Pretreatment of sugarcane bagasse and rice straw offers very digestible cellulose and potentially less inhibition. Cellulase production was enhanced by multiple carbon sources because of diauxic pattern of utilization of substrates. This is the first attempt of combining the synthetic substrate (xylose, lactose) with natural substrate (sugarcane bagasse, rice straw). The mixture of substrates produced the highest maximal enzyme activity on cellulose with xylose, cellulose with lactose, bagasse with xylose, bagasse with lactose, rice straw with xylose and rice straw with lactose. In addition Monod growth kinetics and Leudeking piret product formation kinetics were studied using T. reesei with optimized medium under optimized conditions of inoculum concentration, D.O. level, agitator speed, temperature and pH.
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Provides: (1) a glossary of terms used in biochemical engineering; (2) a list of key developments in the field; and (3) emphases placed in 15 topic areas in a course restructured on the basis of these developments. Topic areas include enzyme kinetics/applications, genetics and microbial control, transport phenomena, and others. (JN)
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By employing a two-stage continuous-culture system, some of the more important physiological parameters involved in cellulose biosynthesis have been evaluated with an ultimate objective of designing an optimally controlled cellulose process. The two-stage continuous-culture system was run for a period of 1350 hr with Trichoderma reesei strain MCG-77. The temperature and pH were controlled at 32°C and pH 4.5 for the first stage (growth) and 28°C and pH 3.5 for the second stage (enzyme production). Lactose was the only carbon source for the both stages. The ratio of specific uptake rate of carbon to that of nitrogen, Q(C)/Q(N), that supported good cell growth ranged from 11 to 15, and the ratio for maximum specific enzyme productivity ranged from 5 to 13. The maintenance coefficients determined for oxygen, MO, and for carbon source, MC, are 0.85 mmol O2/g biomass/hr and 0.14 mmol hexose/g biomass/hr, respectively. The yield constants determined are: YX/O = 32.3 g biomass/mol O2, YX/C = 1.1 g biomass/g C or YX/C = 0.44 g biomass/g hexose, YX/N = 12.5 g biomass/g nitrogen for the cell growth stage, and YX/N = 16.6 g biomass/g nitrogen for the enzyme production stage. Enzyme was produced only in the second stage. Volumetric and specific enzyme productivities obtained were 90 IU/liter/hr and 8 IU/g biomass/hr, respectively. The maximum specific enzyme productivity observed was 14.8 IU/g biomass/hr. The optimal dilution rate in the second stage that corresponded to the maximum enzyme productivity was 0.026 ∼ 0.028 hr−1, and the specific growth rate in the second stage that supported maximum specific enzyme productivity was equal to or slightly less than zero.
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Kinetic data are needed to develop basic understanding of fermentation processes and to permit rational design of continuous fermentation processes. The kinetics of the fermentation of glucose to lactic acid have been studied at six constant pH levels between 4·5 and 6·0 by measuring the instantaneous rates of bacterial growth and of lactic acid formation throughout each fermentation. It was found that the instantaneous rate of acid formation d P /d t , could be related to the instantaneous rate of bacterial growth d N /d t , and to the bacterial density N , throughout a fermentation at a given pH, by the expression where the constants α and β are determined by the pH of the fermentation.
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The present studies concern the production of cellulases, in batch and in continuous cultures, using a mutant strain T. reesei C-5 growing on soluble carbon substrate, lactose. The maximum specific growth rate (μmax) and saturation constant (Ks) in continuous culture at 2% feed lactose were estimated to be 0.066 h−1 and 1.17 g l−1, respectively. The corresponding values in batch culture on 4% lactose were estimated to be 0.067 h−1 and 1.36 g l−1, respectively, and the corresponding overall growth yield factor (Y0) was found to be 0.63 g cell g loctose−1 (in 60 h). The volumetric enzyme productivity (E), and specific enzyme productivity (v) in continuous culture with 2% feed lactose were found to be 51.52 IU l−1 h−1 and 4.58 IU mg cell−1 h−1, respectively, and the corresponding values with 4% feed lactose were estimated to be 56.02 IU l−1 h−1 and 2.68 IU l−1 h−1, respectively. From the pulse response of 0.2% glucose, partial catabolic repression was noticed. The addition of 0.2% cellulose produced an inducing effect for enzyme synthesis. It was noticed that T. reesei C-5 is a highly derepressed mutant strain capable of synthesizing active cellulases on soluble sugar lactose.
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The enzymatic hydrolysate of wastepaper was evaluated for its cellulase-inducing capability and production characteristics in continuous culture of Trichoderma reesei RUT C30. Under the study conditions, i.e., pH 5.0, temperature 25 °C, and typical medium C:N ratio, the apparent cell yield constant was found to be 0.76 (g of dry cell weight/g of reducing sugar), and the maximum specific cell growth rate was 0.26 h−1. The study on the effects of medium C:N ratio confirmed an important role of N sources in the cellulase synthesis. The cellulase production decreased significantly when the feed concentrations of N sources were reduced. An experiment at pH 7.5 with 4-fold N source concentrations also led to poorer cellulase production. When compared with cellulose, the wastepaper hydrolysate was found to have similar cellulase-inducing strength and to induce an apparently complete set of cellulase components. The hydrolysate was also concluded to be a better soluble inducer than sophorose. While comparable at a low dilution rate (0.012 h−1), the specific cellulase productivities of the hydrolysate-supported and the sophorose-induced systems exhibited opposite trends with increasing dilution rates. The specific productivity in sophorose-induced systems decreased with an increase in the dilution rate. On the other hand, with increasing dilution rate the specific productivity in the hydrolysate-supported systems increased from 2.2 FPU/g·h at D = 0.012 h−1 to 12.2 FPU/g·h at D = 0.122 h−1 before beginning to decline. The initial increasing trend was attributed to the higher concentrations of inducing oligomer intermediates at larger dilution rates.
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The secretion of multiple forms of cellulolytic enzymes by a Trichoderma reesei QM 9414 selectant exhibiting high protease activity (T. reesei QM 9414/A 30) was investigated using monoclonal, domain-specific antibodies against cellobiohydrolase (CBH) I, CBH II and -glucosidase, and a polyclonal antibody against endoglucanase I. The pattern of appearance of these proteins was followed during growth of the fungus on Avicel cellulose, using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting/immunostaining. Evidence was obtained that, at late cultivation stages, CBH I and II became partially modified to lower molecular weight components, whereas -glucosidase and endoglucanase I appeared to remain largely intact. Modification of CBH I appeared to commence from the carboxy-terminal AB region, whereas CBH II appeared to become modified both from the amino- (ABB') and the carboxy-terminal. Evidence for a protease activity that modifies the already truncated cellobiohydrolases in the culture filtrate was obtained. These results show that proteolysis at late culture stages may contribute to the multiplicity of cellulases found in T. reesei culture fluids. Initial proteolytic cleavage of CBH I and II may, however, involve an unusual protease not detectable by the azocasein method.
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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 28C and pH 5. Maximum enzyme activity was observed at 63C and pH 4.6. The apparent Km, based on the nitrophenyl-galactopyranoside assay was estimated as 0.4 mM. The enzyme is suitable for lactose hydrolysis in acid whey.
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Extracellular cellulase and amylase productions byTrichoderma sp. were examined in continuous cultures using either cellobiose or glucose as the sole carbon source. Straightforward enhancements of these hydrolase potencies were observed when the dilution rate was decreased from nearly 0.10 to 0.02 h–1. This decrease in dilution rate reflected that of the specific uptake rate of carbon source. In the specific production rate of hydrolases, (8)"e = aYG (v - m)v - b(8)''\varepsilon = aY_G (v - m)v^{ - b} where a, m, YG=empirical coefficient and/or constant, b=4.4 for cellulase and 3.1 for amylase.Obviously, a controlled addition of carbon source to the culture medium so as to restrain the specific uptake rate of carbon source would enhance the hydrolase production. The simultaneous rate equations constituted by growth, carbon-source uptake, respiration and hydrolase production by this strain are a basis which may lead ultimately to an optimal design of the hydrolase-production process.
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The interaction between catalytically active and inactive Trichoderma reesei cellulase components and cotton fibers has been examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Cellobiohydrolase I (CBH I), the major component, was rendered catalytically inactive by its treatment with ammonium hexachloropalladate; however, the inactive enzyme still had the ability to bind to the cotton fiber. SEM and AFM provided evidence suggesting that the catalytic activity of CBH I was required for fiber disruption, AFM allowing resolution of cotton fibers to the microfibril level. However, at high magnification slightly elongated holes were observed throughout the surface of the microfibrillar surface of cotton fibers treated with inactivated CBH I. No disruption of cotton fibers was observed by a palladium-inactivated CBH II/EG II mixture.
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In the present work the genetically modified Trichoderma reesei strain QM9414 was used to produce full-length Cel7B (endoglucanase I, EGI) under the control of the constitutive Aspergillus nidulans gpdA promoter in the presence of glucose. However, the full-length Cel7B enzyme was found to be truncated to lower molecular weight components in the culture broth. Truncation of recombinant proteins produced in fungi may be due to protease activity. In order to identify major sectreted proteases, protease activity was assessed in culture filtrate of the T. reesei QM9414 recombinant. Zymogram analysis revealed the presence of proteolytic activity corresponding to one protein, which was subsequently purified by a combination of ion exchange and size exclusion chromatography. The protein has a molecular mass of 25 kDa, and an isoelectric point of 7.3. By matching tryptic peptide fragments analyzed by tandem mass spectrometry to fungal proteins available in databases as well as to expressed sequence tag (EST) sequences, and comparing the coded amino acids to full-length amino acid sequences, the purified protein was found to be homologous to several trypsin-like fungal serine proteases, with the highest homology to the protease P27 from Trichoderma harzianum. The purified protein was further characterized using benzoyl-arginyl-p-nitroanilide (BApNA) as substrate. It was found to have maximum activity at pH 8 and 50 °C, with a km-value of 0.3 mM.
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A strategy based on plate screening tests was designed for the selection of mutant strains of the fungus Trichoderma reesei suitable for cellulase (EC 3.2.1.4) production on an industrial scale. Six mutant generations were successively isolated, each of them fulfilling all of the three criteria: (1) improved productivity compared to the previous one, (2) high stability, (3) ability to be further improved. The mutant ultimately selected, CL 847, exhibited a four-fold increase in cellulase productivity in cellulose media as compared to the starting strain QM 9414, resistance to catabolite repression, increased β-d-glucosidase (EC 3.2.1.21) specific activity and it was partially constitutive. High cellulase concentrations were obtained in simple media with lactose or glucose as sole carbon source. The strain is currently used in an industrial fermentation plant.
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The production and properties of a protease of Trichoderma reesei QM 9414, which is secreted during growth on cellulose, was investigated: formation of protease activity was mainly induced by the presence of organic nitrogen in the medium, and only little affected by changes in the pH between 3 and 5. Variations in the carbon source were without effect. High levels of protease in the extracellular culture fluid correlated with the appearance of proteolytic cellulase degradation products. The major protease was partially purified and shown to resemble a pepstatin-insensitive, N-chlorosuccinimide sensitive aspartate protease.
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Amorphous cellulose induced the synthesis of endoglucanase in Trichoderma reesei to a greater extent than did cellobiose, lactose, sucrose or other commercial celluloses. In contrast, none of these carbohydrates was able to induce significant levels of endoglucanase activity in Aspergillus niger or Pseudomonas pickettii. Both endoglucanase and β-glucosidase activities were found in cell wall, cell-free extracts and extramycelial fractions of Trichoderma reesei cultures grown on amorphous cellulose. When carboxymethyl cellulose was used as substrate, Km and Vmax values of 1.32% (w/v) and 405.5 μmol glucose ml−1 h−1 for carboxymethyl cellulase were obtained Additionally, the activity was maximum over a pH range of 4.5–5.5, declining sharply beyond 5.5. The optimum temperature was between 50 and 70°C, with maximum activity at 60°C. The half-life of the enzyme appeared to be 9.4 h at 55°C and 4.3 h at 60°C.
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An acid hydrolysate was prepared by a procedure chosen for retaining more oligosaccharides to improve the cellulase-inducing capability when used as substrate in the fungal fermentation for cellulase production. The effect was evaluated with continuous culture of Trichoderma reesei Rut C30 at the dilution rates of 0.03-0.08 h(-1). The specific cellulase production rates were found to be relatively constant at 8.9+/-0.3 (FPU/g dry cells-h), except for the lower rate, i.e., 7.2 (FPU/g-h), at the lowest dilution rate investigated (0.03 h(-1)). The former value was slightly higher than the rate obtained with a lactose-based medium, i.e., 8.2 (FPU/g-h). The maximum specific cell growth rate supported by the hydrolysate-based medium was 0.096 (h(-1)) and the apparent cell yield increased from 0.44 to 0.57 (g dry cells)/(g consumed reducing sugars) with increasing dilution rates. The best-fit maximum/ideal cell yield (without endogenous metabolism) was 0.68 (g/g), the endogenous substrate consumption rate was 0.023 (g reducing sugars)/(g dry cells-h), and the specific cell death rate was 0.016 h(-1).
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Continuous culture studies have been carried out growing Trichoderma viride QM 9123 in a 10 liter stirred fermentor on a medium containing commercial glucose as the carbon source. Experiments were carried out at 30 degrees C and at three controlled pH values of 2.5, 3.0, and 4.0 over a range of dilution rates from 0.01 to 0.11 hr-1. Steady-state values of cell, glucose, and cellulase concentration oxygen tension, and outlet gas oxygen partial pressure were recorded. Values of maximum specific growth rate, endogenous metabolism coefficient, Michaelis-Menten coefficient, yield and maintenance coefficient for glucose were derived and correlated the effect of the hydrogen ion concentration. Specific oxygen uptake rates were correlated with specific growth rates and absorption coefficients were shown to be a function of dilution rate independent of pH. Some data on cellulase biosynthesis were examined and correlated in terms of a maturation time model.
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A filter paper assay method and unit value is described for the measurement of enzyme saccharification action. The method is simple, reproducible, and quantitative and predicts enzyme action under practical saccharification conditions. (JSR)
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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.
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Lactose is the only soluble carbon source which can be used economically for the production of cellulases or heterologous proteins under cellulase expression signals by Hypocrea jecorina (=Trichoderma reesei). Towards an understanding of lactose metabolism and its role in cellulase formation, we have cloned and characterized the gal1 (galactokinase) gene of H. jecorina, which catalyses the first step in d-galactose catabolism. It exhibits a calculated Mr of 57 kDa, and shows moderate identity (about 40%) to its putative homologues of Saccharomyces cerevisiae and Kluyveromyces lactis. Gal1 is a member of the GHMP family, shows conservation of a Gly/Ser rich region involved in ATP binding and of amino acids (Arg 51, Glu 57, Asp 60, Asp 214, Tyr 270) responsible for galactose binding. A single transcript was formed constitutively during the rapid growth phase on all carbon sources investigated and accumulated to about twice this level during growth on d-galactose, l-arabinose and their corresponding polyols. Deletion of gal1 reduces growth on d-galactose but does only slightly affect growth on lactose. This is the result of the operation of a second pathway for d-galactose catabolism, which involves galactitol as an intermediate, and whose transient concentration is strongly enhanced in the delta-gal1 strain. In this pathway, galactitol is catabolised by the lad1-encoded l-arabinitol-4-dehydrogenase, because a gal1/lad1 double delta-mutant failed to grow on d-galactose. In the delta-gal1 strain, induction of the Leloir pathway gene gal7 (encoding galactose-1-phosphate uridylyltransferase) by d-galactose, but not by l-arabinose, is impaired. Induction of cellulase gene expression by lactose is also impaired in a gal1 deleted strain, whereas their induction by sophorose (the putative cellulose-derived inducer) was shown to be normal, thus demonstrating that galactokinase is a key enzyme for cellulase induction during growth on lactose, and that induction by lactose and sophorose involves different mechanisms.
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An inducible mycelial beta-glucosidase from Scytalidum thermophilum was characterized. The enzyme exhibited a pI of 6.5, a carbohydrate content of 15%, and an apparent molecular mass of about 40 kDa. Optima of temperature and pH were 60 degrees C and 6.5, respectively. The enzyme was stable up to 1 h at 50 degrees C and exhibited a half-life of 20 min at 55 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-d-glucopyranoside, p-nitrophenyl-beta-d-xylopyranoside, o-nitrophenyl-beta-d-galactopyranoside, p-nitrophenyl-alpha-arabinopyranoside, cellobiose, laminaribiose and lactose. Kinetic studies indicated that the same enzyme hydrolyzed these substrates. Beta-Glucosidase was activated by glucose or xylose at concentration varying from 50 to 200 mM. The apparent affinity constants (K0.5) for glucose and xylose were 36.69 and 43.24 mM, respectively. The stimulatory effect of glucose and xylose on the S. thermophilum beta-glucosidase is a novel characteristic which distinguish this enzyme from all other beta-glucosidases so far described.
Article
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.
Article
The extracellular bga1-encoded beta-galactosidase of Hypocrea jecorina (Trichoderma reesei) was overexpressed under the pyruvat kinase (pki1) promoter region and purified to apparent homogeneity. The monomeric enzyme is a glycoprotein with a molecular mass of 118.8 +/- 0.5 kDa (MALDI-MS) and an isoelectric point of 6.6. Bga1 is active with several disaccharides, e.g. lactose, lactulose and galactobiose, as well as with aryl- and alkyl-beta-D-galactosides. Based on the catalytic efficiencies, lactitol and lactobionic acid are the poorest substrates and o-nitrophenyl-beta-D-galactoside and lactulose are the best. The pH optimum for the hydrolysis of galactosides is approximately 5.0, and the optimum temperature was found to be 60 degrees C. Bga1 is also capable of releasing D-galactose from beta-galactans and is thus actually a galacto-beta-D-galactanase. beta-Galactosidase is inhibited by its reaction product D-galactose and the enzyme also shows a significant transferase activity which results in the formation of galacto-oligosaccharides.
Article
The Hypocrea jecorina D-xylose reductase encoding gene xyl1 shows low basal transcript levels, and is induced by D-xylose, L-arabinose and L-arabinitol and, to a lesser extent, by lactose, D-galactose, galactitol and xylitol. The recombinantly expressed XYL1 catalyzes the NADPH-dependent reduction of the pentoses D-xylose and L-arabinose and the hexose D-galactose. Deletion of xyl1 slightly reduces growth on all carbon sources, but a significant decrease is found on D-xylose, L-arabinose and D-galactose. Similar to pentose degradation, XYL1 reduces D-galactose to galactitol in a recently identified second D-galactose pathway. Strains impaired in both D-galactose pathways are almost unable to grow on D-galactose. Deltaxyl1 strains show reduced growth on lactose and are impaired in beta-galactosidase expression and induction of the major cellobiohydrolase gene cbh1. A strain deleted in the cellulase regulator XYR1 is even more severely impaired in growth and beta-galactosidase expression on lactose, and does not produce any cbh1 transcript at all. In this strain, only a low basal level of xyl1 transcription is found on lactose. Galactitol, but not D-galactose is able to induce xyl1 transcription in a XYR1-independent manner. Our results show that the role of the H. jecorina XYL1 is not restricted to D-xylose catabolism and demonstrates its importance for induction of cellulases and beta-galactosidases.
Article
Distillers dried grain with solubles (DDGS) is the major coproduct produced at a dry grind ethanol facility. Currently, it is sold primarily as a ruminant animal feed. DDGS is low cost and relatively high in protein and fiber contents. In this study, DDGS was investigated as carbon source for extracellular hydrolytic enzyme production. Two filamentous fungi, noted for their high cellulolytic and hemicellulolytic enzyme titers, were grown on DDGS: Trichoderma reesei Rut C-30 and Asper gillus niger NRRL 2001. DDGS was either used as delivered from the plant (untreated) or after being pretreated with hot water. Both microorganisms secreted a broad range of enzymes when grown on DDGS. Higher xylanase titers were obtained when cultured on hot water DDGS compared with growth on untreated DDGS. Maximum xylanase titers were produced in 4 d for A. niger and 8 d for T. reesei in shake flask cultures. Larger amounts of enzymes were produced in bioreactors (5 L) either equipped with Rushton (for T. reesei) or updraft marine impellers (A. niger). Initial production titers were lower for bioreactor than for flask cultures, especially for T. reesei cultures. Improvement of enzyme titers were obtained using fed-batch feeding schemes.
A guide to Microsoft Excel 2002 for scientists and engineers
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Liengme BV. A guide to Microsoft Excel 2002 for scientists and engineers. third edition New York, NY: Butterworth Heinemann; 2002.
A kinetic study of the lactic acid fermentation. Batch process at controlled pH
  • Luedeking
Luedeking R, Piret EL. A kinetic study of the lactic acid fermentation. Batch process at controlled pH. Journal of Biochemical and Microbiological Technology and Engineering 1959;1:393-412.
-Glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose
  • Ff Zanoelo
  • Mt Polizeli
  • Hf Terenzi
  • Ja Jorge
Zanoelo FF, Polizeli MT, Terenzi HF, Jorge JA.-Glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose. FEMS Microbiology Letters 2004;240:137-43.
Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei
  • IImen