Fungal Cellulase Activity Research Articles

Production and Biochemical Characterization of Cellulase Enzyme by Trichoderma Strains from Harran Plain

In this research, Trichoderma spp. fungus, a major producer of cellulase, that was isolated from Harran plain had been investigated. Morphological, microscopic and genetic identification of the six fungi isolates were carried out and their cellulase production ability were determined. The ITS region of four of the isolates were displayed over 90% similarity with the DNA sequences of Trichoderma spp. currently deposited into the databases. The molecular size of fungal cellulase was found as to be 32 kDa by SDS-PAGE analysis. Optimum working conditions of cellulase had also been studied. The optimal temperature for cellulase enzyme activity was 40 ° C, optimum pH 5.0, best reacting time 60 min and ideal substrate concentration was determined as 2% CMC. The fungal cellulase activity was compared with the activity of the commercial cellulase enzyme. The fermentation produced enzyme by using Trichoderma spp. isolated from Harran plain displayed remarkable cellulase activity.

The Fusarium graminearum cerato-platanins loosen cellulose substrates enhancing fungal cellulase activity as expansin-like proteins

Cerato-platanin proteins (CPPs) are small non-catalytic, cysteine-rich hydrophobic proteins produced by filamentous fungi. The genome of Fusarium graminearum, the causal agent of Fusarium head blight disease of wheat and other cereal grains, contains two genes putatively encoding for CPPs. To better characterize their features, the two FgCPPs were heterologously expressed in Pichia pastoris. The recombinant FgCPPs reduced the viscosity of a cellulose soluble derivate (carboxymethyl cellulose, CMC). The same effect was not observed on other polysaccharide substrates such as chitin, 1,3-β-glucan, xylan and pectin. Indeed, differently from other fungal CPPs and similarly to expansins, FgCPPs are trapped by cellulose and not by chitin, thus suggesting that these proteins interact with cellulose. A double knock-out mutant deleted of both FgCPPs encoding genes produces much more cellulase activity than the corresponding wild type strain when grown on CMC, likely compensating the absence of FgCPPs. This result prompted us to investigate a possible synergistic effect of these proteins with fungal cellulases. The incubation of FgCPPs in the presence of a fungal cellulase (EC 3.2.1.4) determines an increased enzymatic activity on CMC, filter paper and wheat cell walls. The observation that FgCPPs act with a non-hydrolytic mechanism indicates that these proteins favor fungal cellulase activity in an expansin-like manner. Though the disruption of the FgCPP genes had no demonstrable impact on fungal virulence, our experimental data suggest their probable involvement in virulence, thus we refer to them as accessory virulence genes. Our results suggest also that the FgCPPs could be exploited for future biotechnological application in second-generation biofuels production on lignocellulosic biomasses rich in cellulose. Finally, we demonstrate that FgCPPs act as elicitors of defense responses on Arabidopsis leaves, increasing resistance to Botrytis cinerea infections.

Isolation and Screening of Microbial Isolates from Decomposing Palm Kernel Shaft for Cellulase Production

This study was designed to screen microorganisms from decomposing palm kernel shaft for cellulase palm oil processing sites in Akoko area of Ondo State, Nigeria. Isolation of microorganisms was carried out by serial dilution and pour plate methods andidentified using standard biochemical methods. The isolates were screened for cellulase production using standard assay methods. The microorganisms were grown in a mineral salt basal medium for maximum yield of cellulase production. The microorganisms isolated from the sample include Bacillus subtilis, Micrococcus varians, M. leteus, Cellulomonas blazotes, C. flavigina, Sarcina ventriculi, B. cereus, C. fimi, Aspergillus niger, Rhizopus stolonifer, Saccharomyces cerevisiae and Trichoderma viridae. The screened microorganisms exhibited varied cellulase activities. The diameters of zones of clearance of the isolates ranged from 1.45 to 1.83for bacteria and 0.00 to 2.06for fungi. The cellulase activity exhibited by bacteria ranged from 0.238 µmol/ml to 0.590 µmol/ml while fungal cellulase activity ranged from 0.452 µmol/ml to 0.775 µmol/ml. The high cellulase activity exhibited by fungi isolated from decomposing palm kernel shaft suggested that their predominance as a potential source of cellulase could be more promising in various industrial processes for the degradation of cellulose containing organic substances.

The Stability Pattern of the Cellulase Enzyme Produced by Aspergillus flavus

In view of the fact that temperature plays key role in the activity of fungal cellulases, this experiment was designed to assess the stability pattern of the cellulase Enzyme produced by Aspergillus flavus derived from rotten wood. The characterization of the purified cellulase enzyme of Aspergillus flavus was carried out using Carboxymethyl cellulose, Filter paper and Cotton wool assays. The maximum and optimum temperatures were 90°C and 70°C, respectively. The enzyme retained above 50% of its original activity at 100°C for 1h and up to 80% at 80°C for 1h Enzyme decay study showed that Cellulase activity remained 100 % at 0 h for both CMC and filter paper. However, as incubation period increased, the activity decreased, but the enzyme still retained above 50 % activity in both substrates at 1h, over the range of 40-100°C. The study carried out showed that the purified Aspergillus flavus cellulases demonstrated useful properties among which include; high optimum temperature, increased thermal stability. They also offer additional advantage as they are more rigid than meso enzymes and moreover, are more resistant to thermal and chemical denaturation. Hence are remarkable tools for developing commercial biotechnologies and for studying protein stability.

Temporal changes in fungal communities from buckwheat seeds and their effects on seed germination and seedling secondary metabolism

Seed-associated fungal communities affect multiple parameters of seed quality at all stages of production, from seed development to post-harvest storage and germination. We therefore investigated the diversity and dynamics of fungal communities in the seeds of common buckwheat (Fagopyrum esculentum) and Tartary buckwheat (F. tataricum) from harvest to 1 y of storage. Fungal populations in seeds were relatively stable, comprised mainly of field fungi. Incidence of fungi was most likely determined by fungal interspecies direct interactions, as well as by their synthesis of volatile organic compounds. Most prominent antagonistic interactions were seen for two plant pathogens, Alternaria alternata on Botrytis cinerea. Detrimental effects of the fungi on seed germination and seedling development were related to fungal extracellular enzyme activity, and in particular to amylase, cellulase and, polyphenol oxidase. Polyphenol and tannin concentrations in buckwheat seedlings were related to fungal growth rate and intensity of fungal cellulase activity, respectively, which suggests that physical penetration of the fungi through the host tissues is probably the stimulus for the activation of plant defence reactions in these seedlings.

Enzymatic, kinetic and anti-microbial studies on Aspergillus terreus culture filtrate and Allium cepa seeds extract and their potent applications

Abstract Our search focused on the industrially important enzymes from unconventional sources. The microbial source ( Aspergillus terreus ) and plant source ( Allium cepa seeds extract) reveal the presence of economic critical industrial enzymes, mainly cellulase. Cellulase from fungal culture filtrate (FCF) and onion seeds extract (OSE) shows the optimum activities at temperature (60 °C and 55 °C), pH (5.5 and 6.0) and substrate concentration (15 and 12.5 mg/ml), respectively. The K m and V max for cellulase from FCF and OSE were 20 and 8 mg/ml and 10 and 2.78 U/ml, respectively. The calculated values of the half-life for cellulase from FCF and OSE at 55 and 70 °C were 577.5 and 414.97, 150.33 and 135.35 min, respectively. The fungal cellulase activity increased to four, three and two-fold in the presence of Ca 2+ , Ba + , Mg + and Mn 2+ , respectively. However, plant cellulase did not enhanced by all tested metal ions. The plant cellulase efficient in saccharification of potato shells and corn cobs after 48 h with 81.29% and 52.35% saccharification yield, respectively. While the fungal cellulase was more efficient in hydrolyzing of molokhia stems with saccharification yield 86.87%. The microbial and plant-derived cellulase showed good effect in bio-polishing of cotton fabric. The FCF exhibited a wide range of antimicrobial activity against gram positive, gram negative bacteria and fungi, where OSE displayed good antifungal activity against Alternaria solani and Alternaria alternata . Pretreatment of tomato fruits with OSE caused reduction in disease incidence DI of A. alternata and A. solani and delay in ripening of fruits.

Dissuasive effect of an aqueous extract from Enterolobium cyclocarpum<br>(Jacq) Griseb on the drywood termite Incisitermes marginipennis<br>(Isoptera:Kalotermitidae) (Latreille)

Heartwood aqueous extracts from Enterolobium cyclocarpum (Jacq.) Griseb. exhibited chronic toxic lethal effect against Incisitermes marginipennis (Latreille) with more than 60% mortality at a concentration of 56.63 mg. Feeding rate correlated positively with the mortality of termites. The termite hindgut cellulolytic activity, the symbiotic microorganism population in termite hindgut and fungal cellulase activity were not inhibited by the aqueous extract. Two major chemical groups were detected in aqueous extract by gas chromatography; they were monoterpenes (46.5%) and phenols (18.7%). Fifty seven constituents, including, D-limonene, terpineol and eugenol were identified; these terpenes have repellent activity against insects. These results suggest that essential oils from E. cyclocarpum contain a slow acting toxin with a dissuasive effect on termites.

Application of the 2-Cyanoacetamide Method for SpectrophotometricAssay of Cellulase Enzyme Activity

Cellulose is the most abundant form of carbon on the planet. Breakdown of cellulose microfibrils in the plant cell wall is a means by which microbes gain ingress into their respective hosts. Cellulose degradation is also important for global carbon recycling and is the primary substrate for production of biofuels. In this study, we developed a cellulase assay method that rivals the commonly used dinitrosalicylic acid (DNS) assay. It was shown that the 2-cyanoacetamide method is capable of detecting D-glucose in a linear fashion, can function in various buffers at pH ranging from 4.0 to 8.0, and is as sensitive as the DNS test at detecting fungal cellulase activity using carboxymethylcellulose as a substrate. This method will be useful to others who desire to accurately and efficiently assay microbial cellulase activity without the use of phenol and other highly toxic and corrosive chemicals.

Thermophilic Bacillus coagulans Requires Less Cellulases for Simultaneous Saccharification and Fermentation of Cellulose to Products than Mesophilic Microbial Biocatalysts

Ethanol production from lignocellulosic biomass depends on simultaneous saccharification of cellulose to glucose by fungal cellulases and fermentation of glucose to ethanol by microbial biocatalysts (SSF). The cost of cellulase enzymes represents a significant challenge for the commercial conversion of lignocellulosic biomass into renewable chemicals such as ethanol and monomers for plastics. The cellulase concentration for optimum SSF of crystalline cellulose with fungal enzymes and a moderate thermophile, Bacillus coagulans, was determined to be about 7.5 FPU g(-1) cellulose. This is about three times lower than the amount of cellulase required for SSF with Saccharomyces cerevisiae, Zymomonas mobilis, or Lactococcus lactis subsp. lactis whose growth and fermentation temperature optimum is significantly lower than that of the fungal cellulase activity. In addition, B. coagulans also converted about 80% of the theoretical yield of products from 40 g/L of crystalline cellulose in about 48 h of SSF with 10 FPU g(-1) cellulose while yeast, during the same period, only produced about 50% of the highest yield produced at end of 7 days of SSF. These results show that a match in the temperature optima for cellulase activity and fermentation is essential for decreasing the cost of cellulase in cellulosic ethanol production.

Regulation of the Xylan-degrading Apparatus of Cellvibrio japonicus by a Novel Two-component System

The microbial degradation of lignocellulose biomass is not only an important biological process but is of increasing industrial significance in the bioenergy sector. The mechanism by which the plant cell wall, an insoluble composite structure, activates the extensive repertoire of microbial hydrolytic enzymes required to catalyze its degradation is poorly understood. Here we have used a transposon mutagenesis strategy to identify a genetic locus, consisting of two genes that modulate the expression of xylan side chain-degrading enzymes in the saprophytic bacterium Cellvibrio japonicus. Significantly, the locus encodes a two-component signaling system, designated AbfS (sensor histidine kinase) and AbfR (response regulator). The AbfR/S two-component system is required to activate the expression of the suite of enzymes that remove the numerous side chains from xylan, but not the xylanases that hydrolyze the beta1,4-linked xylose polymeric backbone of this polysaccharide. Studies on the recombinant sensor domain of AbfS (AbfS(SD)) showed that it bound to decorated xylans and arabinoxylo-oligosaccharides, but not to undecorated xylo-oligosaccharides or other plant structural polysaccharides/oligosaccharides. The crystal structure of AbfS(SD) was determined to a resolution of 2.6A(.) The overall fold of AbfS(SD) is that of a classical Per Arndt Sim domain with a central antiparallel four-stranded beta-sheet flanked by alpha-helices. Our data expand the number of molecules known to bind to the sensor domain of two-component histidine kinases to include complex carbohydrates. The biological rationale for a regulatory system that induces enzymes that remove the side chains of xylan, but not the hydrolases that cleave the backbone of the polysaccharide, is discussed.

Isolation and Characterization of Acid-Tolerant, Thermophilic Bacteria for Effective Fermentation of Biomass-Derived Sugars to Lactic Acid

Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals by the appropriate microbes. Due to the differences in the optimum conditions for the activity of the fungal cellulases that are required for depolymerization of cellulose to fermentable sugars and the growth and fermentation characteristics of the current industrial microbes, simultaneous saccharification and fermentation (SSF) of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity, leading to a higher-than-required cost of cellulase in SSF. We have isolated bacterial strains that grew and fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to l(+)-lactic acid at 50 degrees C and pH 5.0, conditions that are also optimal for fungal cellulase activity. Xylose was metabolized by these new isolates through the pentose-phosphate pathway. As expected for the metabolism of xylose by the pentose-phosphate pathway, [(13)C]lactate accounted for more than 90% of the total (13)C-labeled products from [(13)C]xylose. Based on fatty acid profile and 16S rRNA sequence, these isolates cluster with Bacillus coagulans, although the B. coagulans type strain, ATCC 7050, failed to utilize xylose as a carbon source. These new B. coagulans isolates have the potential to reduce the cost of SSF by minimizing the amount of fungal cellulases, a significant cost component in the use of biomass as a renewable resource, for the production of fuels and chemicals.

Simultaneous Saccharification and Co‐Fermentation of Crystalline Cellulose and Sugar Cane Bagasse Hemicellulose Hydrolysate to Lactate by a Thermotolerant Acidophilic Bacillus sp.

Polylactides produced from renewable feedstocks, such as corn starch, are being developed as alternatives to plastics derived from petroleum. In addition to corn, other less expensive biomass resources can be readily converted to component sugars (glucose, xylose, etc.) by enzyme and/or chemical treatment for fermentation to optically pure lactic acid to reduce the cost of lactic acid. Lactic acid bacteria used by the industry lack the ability to ferment pentoses (hemicellulose-derived xylose and arabinose), and their growth and fermentation optima also differ from the optimal conditions for the activity of fungal cellulases required for depolymerization of cellulose. To reduce the overall cost of simultaneous saccharification and fermentation (SSF) of cellulose, we have isolated bacterial biocatalysts that can grow and ferment all sugars in the biomass at conditions that are also optimal for fungal cellulases. SSF of Solka Floc cellulose by one such isolate, Bacillus sp. strain 36D1, yielded l(+)-lactic acid at an optical purity higher than 95% with cellulase (Spezyme CE; Genencor International) added at about 10 FPU/g cellulose, with a product yield of about 90% of the expected maximum. Volumetric productivity of SSF to lactic acid was optimal between culture pH values of 4.5 and 5.5 at 50 degrees C. At a constant pH of 5.0, volumetric productivity of lactic acid was maximal at 55 degrees C. Strain 36D1 also co-fermented cellulose-derived glucose and sugar cane bagasse hemicellulose-derived xylose simultaneously (SSCF). In a batch SSCF of 40% acid-treated hemicellulose hydrolysate (over-limed) and 20 g/L Solka Floc cellulose, strain 36D1 produced about 35 g/L lactic acid in about 144 h with 15 FPU of Spezyme CE/g cellulose. The maximum volumetric productivity of lactic acid in this SSCF was 6.7 mmol/L (h). Cellulose-derived lactic acid contributed to about 30% of this total lactic acid. These results show that Bacillus sp. strain 36D1 is well-suited for simultaneous saccharification and co-fermentation of all of the biomass-derived sugars to lactic acid.

In vitro expression of Penicillium janthinellum cellobiohydrolase I gene in a coupled transcription–translation system

An enzymatically-active fungal cellobiohydrolase I (CBH I) was first synthesized in a coupled reticulocyte lysate system lacking of glycosylation modification by the template DNACbh1 in the presence of T7 RNA polymerase. The synthesized CBH I had the expected size (57 kDa) and catalyzed the substrate of p-nitrophenyl-β-d-cellobioside (pNPC), and had no activity against carboxymethyl cellulose (CMC-Na). The Km and Vmax values of the CBH I for pNPC were 0.82 mmol and 0.067 μmol min−1 per μg enzyme, respectively. The results indicated that glycosylation may not be necessary for enzymatic activity of fungal cellulases.

The enumeration and characterization of bacteria and fungi associated with marine wood-boring isopods, and the ability of these microorganisms to digest cellulose and wood

Bacteria and fungi associated with the marine wood-boring isopods Limnoria lignorum and Sphaeroma serratum, and with their wood burrows and seawater were investigated. Plate counts using nutrient agar and Czapex Dox agar media in natural and in artificial seawater were used. The diversity and number of bacteria associated with the two isopods were much higher than those associated with wood or seawater, whereas the opposite was true for fungi. The hydrolytic activity of the predominant genera on carboxymethylcellulose and on different types of wood was investigated. The magnitude of fungal cellulase activity exceeded that of bacteria. Activities of bacterial extracellulases exceeded those of intracellulases, while the opposite was observed for fungi. Cellulolytic activity of the predominant isolates biodegraded labkh, peach, white oak and mahogany in decreasing order of activity. Evidence provided by the cellulolytic activities, detected in woodcontaining cultures, suggests that the predominant micro-organisms isolated may play an important role in the woodboring process. Specimens used in the present study were collected from Port Fouad at Port Said Harbour, Egypt.

Termitomyces: A New Source of Potent Cellulases

The association of certain species of termites with fungi is a very complex phenomenon. Previously, it was demonstrated the importance of a basidiomycete fungus belonging to the genus Termitomyces as a source of potent cellulases. In this paper, the saccharifying activities of Termitomyces conidiophores against various oligo- and polysaccharides were compared. In addition, partial purification of the cellulase complex of conidiophores was achieved by using a Sephadex column G-IOO. The fungal cellulase activity was also compared with five commercial cellulases namely: A. niger cellulase (from Calbiochem comp.), Drisellase-NIO (Kyomo Hakko Comp.), Trichoderma-SPI22 (The Novo Comp.), Trichoderma-4 (Natick, US Army) and Onozuka SSPI500 (Kink Yakult Comp.). Cellulase( s) activities were measured against cotton wool (CJ, filter paper (FP), carboxymethylcellulose (CMC) and cellobios (Bglucosidase). Results showed that the activity of Termitomyces cellulase was far more superior than others when the substrates were filter paper and CMC. However, the cellulolytic activity on cotton wool was intermediate.