Fermentative production and kinetics of cellulase protein on Trichoderma reesei using sugarcane bagasse and rice straw
TL;DR: This is the first attempt of combining the synthetic substrate (xylose, lactose) with natural substrate (sugarcane bagasse, rice straw) and the mixture of substrates produced the highest maximal enzyme activity on cellulose with xylose.
Abstract: 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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01 Jan 2012
TL;DR: In this article, thermophilic cellulolytic microorganisms were isolated and tested for their cellulytic activity and the enzyme production from potent isolates was optimized using cellulose basal broth medium.
Abstract: Cellulose, a major polysaccharide found in agricultural residues and industrial and municipal wastes. In the present study thermophilic cellulolytic microorganisms were isolated. The isolates were tested for their cellulytic activity. The enzyme production from potent isolates was optimised using cellulose basal broth medium. Activity of partially purified enzyme was determined. The most potent thermophilic cellulolytic isolates were identified as Bacillus subtilis. The crude cellulase enzyme concentrated at 80-85% ammonium sulfate produced highest zone of hydrolysis. The enzymatic degradation of cellulose waste has been suggested as a feasible alternative for the conversion of lignocelluloses substrate into fermentable sugars and application for biofuel production.
Dissertation•
01 Jan 2015
01 Jun 2008
TL;DR: TaqMan qRT PCR method was successful to demonstrate the most similarities of the Bacillus isolates in this study and they are closely related to “Bacillus subtilis group”.
Abstract: The basic goal of this study is the isolation of some Bacillus isolates from different sources for production of bioactive products such as detection of cellulose(s) and pectinase(s) and identification of these isolates using quantitative real-time PCR (qRT PCR) depending on 16S rRNA gene in each isolate. Nine, six and two isolates were obtained from municipal sewage water, composted rice straw and expired pharmaceutical drugs (Capozide), respectively. Out of nine isolates, only four Bacilli (SW2, SW3, SW6 and SW9) were selected and examined for cellulose(s) and pectinase(s) production, and revealed that only SW2 and SW9 exhibited maximum pectate lyase (PL) and polygalacturonase (PGase) viz. 465.55 and 222.53 (U/mg), respectively. Out of six Bacilli isolates (RS1, RS3 and RS5), only RS3 showed maximum productivity of carboxymethylcellulase (CMCase) viz. 657.21 (U/mg). On the other hand, only one Bacillus isolate out of two was selected ECC2 and exhibited the maximum Avicelase productivity viz. 533.32 (U/mg). TaqMan qRT PCR method was used to detect and quantify the 16S rRNA genes of Bacillus isolates tested. Forward primer Bsub5 F and the reverse primer Bsub3 R were used for the amplification of 16S rRNA of the “Bacillus subtilis group”, it is also successful to demonstrate the most similarities of the Bacillus isolates in this study and they are closely related to “Bacillus subtilis group”.
Cites background from "Fermentative production and kinetic..."
...44 cellooligosaccharides (Jorgensen et al., 2003; Muthuvelayudham and Viruthagiri, 2006)....
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...Enzymatic hydrolysis is an economic process in the conversion of cellulose to easily fermentable low cost sugars (Muthuvelayudham and Viruthagiri, 2006)....
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Dissertation•
01 Jan 2014
TL;DR: Employing in vivo and in vitro techniques the complex relationship between lignocellulose components and cellulase enzymes were investigated, as well as the effects of amino acids and nutrient limitations on cellulase activity.
Abstract: Lignocellulose degradation is being explored as part of the process of bioethanol production from waste products, such as wood-flour. Identified as a natural lignin degrader and its ability to produce cellulase enzymes, Streptomyces viridosporus was deemed worthy of investigation for lignocellulose degradation. It has the ability to degrade lignin, hemicellulose and cellulose components of lignocellulose substrates by secreting a large range of lignocellulose degrading enzymes, including peroxidases, esterases, endoglucanases and xylanases. Experiments in other laboratories had show that this Streptomyces species is capable of degrading both hard- and soft- wood substrates, as well as grass lignin lignocellulose. Using a continuous (chemostat) culture and employing carboxymethylcellulose (CMC) as the main carbon source, five dilution rates were investigated (0. 05h-1, 0. 07b-1, 0. 09h-1, 0. 11h-1 and 0. 13h-1) and the effect of mycelium taken from steady state cultures, on the lignocellulose degradation of wood-flour by S. viridosporus analysed. Employing in vivo and in vitro techniques the complex relationship between lignocellulose components and cellulase enzymes were investigated, as well as the effects of amino acids and nutrient limitations on cellulase activity. In particular, a critical sequence of enzyme activity and precursor liberation for successful degradation to glucose was identified. We have obtained conditions whereby S. viridosporus can successfully degrade lignocellulose, without using all the liberated sugars as a carbon source. Liquid cultures of S. viridosporus were able to liberate glucose from wood-flour (mesh size 40). This finding indicated considerable potential for using this species in lignocellulose degradation for commercial exploitation (as a precursor to bioethanol production). However, S. viridosporus liquid cultures have a pre-disposition to form pellets (discrete islands of hyphal growth) which can inhibit the effectiveness of some hyphal species in biotechnological processes (such as antibiotic biosynthesis). The pelleting of cultures was reduced by the selection of a non- pelleting strain via a prolonged continuous culture.
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TL;DR: The conversion of both cellulose and hemicellulose for the production of fuel ethanol is being studied intensively, with a view to developing a technically and economically viable bioprocess.
492 citations
"Fermentative production and kinetic..." refers background in this paper
...…growth rate (day)-1; µ, specific growth rate (day)–1; Ks, Monod constant; P, product concentration (g/l); α and β, Leudeking Piret constants; and X, substrate concentration (g/l). a number of applications including animal feed, pharmaceutical and textile industries (Aristidou and Penttilä, 2000)....
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...a number of applications including animal feed, pharmaceutical and textile industries (Aristidou and Penttilä, 2000)....
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TL;DR: 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, showing that distinct and clear-cut mechanisms of induction and glucose repression regulate cellulase expression in an actively growing fungus.
Abstract: 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.
483 citations
"Fermentative production and kinetic..." refers background in this paper
...Hypercelluloytic mutant strains secrete large amounts of cellulases (Ilmen et al., 1997)....
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TL;DR: This mini-review summarises the considerable amount of data accumulated over the past three decades regarding promoters of genes encoding hydrolytic enzymes, inducer molecules, and models for the recognition of the insoluble substrates cellulose and xylan.
Abstract: The genus Trichoderma comprises a group of filamentous ascomycetes that are now widely used in industrial applications because of their ability to produce extracellular hydrolases in large amounts. In addition, strong inducible promoters together with high secretory capacity have made Trichoderma an attractive host for heterologous protein production. Several promoters of genes encoding hydrolytic enzymes have been investigated in detail regarding their cis-acting elements and trans-acting factors. Potent inducer molecules, for both xylanolytic and cellulolytic enzyme systems, have been identified and characterized. Furthermore, models for the recognition of the insoluble substrates cellulose and xylan have been developed based on a large set of experiments. This mini-review summarises the considerable amount of data accumulated over the past three decades.
207 citations
"Fermentative production and kinetic..." refers background in this paper
...The genus Trichoderma, filamentous ascomycetes are widely used in industrial applications because of high secretory capacity and inducible promoting characteristics (Mach and Zeilinger, 2003)....
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TL;DR: The growth and enzyme production by Trichoderma reesei Rut C-30 using different lignocellulosic materials as carbon source were investigated and it was found that endoglucanase and endoxylanse activities were produced throughout the cultivations, whereas α-arabinosidase was induced late during the cultivation.
157 citations
"Fermentative production and kinetic..." refers background in this paper
...Biosynthesis of cellulase was also made on lignocellulo-sic materials such as sugar beet pulp and alkaline extracted sugar beet pulp and cellulose (Olsson et al., 2003)....
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...The general trend is that more cellulose in the mixture results in higher levels of endoglucanase (Olsson et al., 2003)....
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TL;DR: The filamentous fungus Penicillium brasilianum IBT 20888 was cultivated on a mixture of 30 g l−1 cellulose and 10 g xylan for 111 h and the resulting culture filtrate was used for protein purification and revealed similarity in molecular mass, pI and hydrolytic properties suggested that these two enzymes were identical, but the smaller was lacking the cellulose-binding domain or an essential part of it.
134 citations