Influence du microbiote intestinal sur le métabolisme et la virulence des Escherichia coli entérohémorragiques

Les Escherichia coli entérohémorragiques (EHEC) représentent un problème majeur de santé publique dans les pays développés. Les EHEC sont régulièrement responsables de toxi-infections alimentaires graves chez l’humain et causent des colites hémorragiques et le symptôme hémolytique et urémique, mortel chez les enfants en bas âge. Les EHEC les plus virulents appartiennent au sérotype O157:H7 et le bovin constitue leur réservoir naturel. À ce jour il n’existe aucun traitement pour éviter l’apparition des symptômes liés à une infection à EHEC. Par conséquent, il est important d’augmenter nos connaissances sur les mécanismes employés par le pathogène pour réguler sa virulence et coloniser efficacement la niche intestinale. Dans un premier temps, l’adaptation de la souche EHEC O157:H7 EDL933 à l’activité métabolique du microbiote intestinal a été étudiée au niveau transcriptionnel. Pour se faire, EDL933 a été cultivée dans les contenus caecaux de rats axéniques (milieu GFC) et dans ceux provenant de rats colonisés par le microbiote intestinal humain (milieu HMC). Le HMC est un milieu cécal conditionné in vivo par le microbiote. Dans le HMC par rapport au GFC, EDL933 change drastiquement de profile métabolique en réponse à l’activité du microbiote et cela se traduit par une diminution de l’expression des voies de la glycolyse et une activation des voies de l’anaplérose (voies métaboliques dont le rôle est d’approvisionner le cycle TCA en intermédiaires métaboliques). Ces résultats, couplés avec une analyse métabolomique ciblée sur plusieurs composés, ont révélé la carence en nutriments rencontrée par le pathogène dans le HMC et les stratégies métaboliques utilisées pour s’adapter au microbiote intestinal. De plus, l’expression des gènes de virulence incluant les gènes du locus d’effacement des entérocytes (LEE) codant pour le système de sécrétion de type III sont réprimés dans le HMC par rapport au GFC indiquant la capacité du microbiote intestinal à réprimer la virulence des EHEC. L’influence de plusieurs composés intestinaux présents dans les contenus caecaux de rats sur l’expression des gènes de virulence d’EDL933 a ensuite été étudiée. Ces résultats ont démontré que deux composés, l’acide N-acétylneuraminique (Neu5Ac) et le N-acétylglucosamine (GlcNAc) répriment l’expression des gènes du LEE. La répression induite par ces composés s’effectue via NagC, le senseur du GlcNAc-6-P intracellulaire et le régulateur du catabolisme du GlcNAc et du galactose chez E. coli. NagC est un régulateur transcriptionnel inactivé en présence de GlcNAc-6-P qui dérive du catabolisme du Neu5Ac et du transport GlcNAc. Ce travail nous a permis d’identifier NagC comme un activateur des gènes du LEE et de mettre à jour un nouveau mécanisme qui permet la synchronisation de la virulence avec le métabolisme chez les EHEC O157:H7. La concentration du Neu5Ac et du GlcNAc est augmentée in vivo chez le rat par le symbiote humain Bacteroides thetaiotaomicron, indiquant la capacité de certaines espèces du microbiote intestinal à relâcher les composés répresseurs de la virulence des pathogènes. Ce travail a permis l’identification des adaptations métaboliques des EHEC O157:H7 en réponse au microbiote intestinal ainsi que la découverte d’un nouveau mécanisme de régulation de la virulence en réponse au métabolisme. Ces données peuvent contribuer à l’élaboration de nouvelles approches visant à limiter les infections à EHEC.

Extracellular beta-Glucosidase production by marine Aspergillus sydowii BTMFS 55

The beta-glucosidase enzyme purified from the marine fungus, Aspergillus sydowii BTMFS 55 showed a good yield of enzyme production under solid state fermentation. The statistical optimization of the media components revealed that moisture content, concentration of peptone and inoculum are the major parameters which supported the maximal enzyme production. The purified enzyme showed low pH activity and stability, glucose tolerance and activation by ethanol. It could produce ethanol from wheat bran and rice straw by simultaneous saccharification and fermentation with yeast.The glucosidase purified from Aspergillus sydowii BTMFS 55 shows great potential for several biotechnological applications such as the production of bio-ethanol from agricultural biomass and improvement in the aromatic character of wines and fruit juices through the hydrolysis of flavour glucosidic precursors. There is immense scope for the application of this marine fungus in the biofuel production besides in other industries provided further studies are pursued in exploiting this enzyme and the organism particularly scale up studies with respect to application. There is also ample scope for cloning of the gene encoding beta-glucosidase in domesticated hosts such as Pichia pastoris or S. cerevisiae that can produce ethanol directly from cellulosic biomass.

Biochemical and Biotechnological investigations on the water-fern Salvinia molesta Mitchell

The present work is focussed mainly on the utilization of this weed-biomass on a biochemical and biotechnological basis. Before designing scientific and systematic utilization of any given biomass, the detailed analysis of its chemical componets is essential. Hence, as the preliminary part of the experimental works, samples of Salvinia were analysed for its chemical constituents.Before designing scientific and systematic utilization of any given biomass, the detailed analysis of its chemical componets is essential .The composition of the substrate contributes much to the nutritive value of mushrooms. Hence, alterations in the nutritive value of mushrooms (in terms of total carbohydrates, proteins, lipids and minerals) in response to Salvinia as substrate were analyzed.Substrate after mushroom harvest (spent substrate) can be utilized for various purposes such as cattle feed, as a source of degradative enzymes, as a substrate for other mushrooms and as garden manure. But studies are limited with regard to the utilization of Pleurotus spent substrate as garden manure. So the value of spent substrate as an organic supplement and its multidimensional impacts on soil chemical status, soil microbial population dynamics and plant growth (Amhurium andreanum) were carried out.Major findings of this work have got much relevance in designing measures to utilize different types of plant biomass, especially aquatic weeds, with the aid of a powerful biological tool, the lignocellulolytic fungus, Pleurorus

Chitinase production by.marine fungi

In this thesis an attempt is made to explore the potential of marine fungi for the production of chitinolytic enzymes and to recognize the ability to hydrolyse native chitin through submerged as well as solid substrate fermentation culture conditions, using wheat bran and shellfish processing waste such as ‘prawn waste’ as solid substrates. Attempt was made to isolate a potential chitinase producing fungus from marine environment and to develop an ideal bioprocess for the production ofchitolytic enzymes.Present study indicate scope for utilization of B. bassiana for industrial production of chitinase using prawn waste as solid substrate employing solid substrate fermentation.

“Studies on glucose tolerant β-glucosidases from a novel Byssochlamys fulva and their applications in biomass to ethanol conversion”

Beta-glucosidases are critical enzymes in biomass hydrolysis process and is important in creating highly efficient enzyme cocktails for the bio-ethanol industry. Among the two strategies proposed for overcoming the glucose inhibition of commercial cellulases, one is to use heavy dose of BGL in the enzyme blends and the second is to do simultaneous saccharification and fermentation where glucose is converted to alcohol as soon as it is being generated. While the former needs extremely high quantities of enzyme, the latter is inefficient since the conditions for hydrolysis and fermentation are different. This makes the process technically challenging and also in this case, the alcohol generation is lesser, making its recovery difficult. A third option is to use glucose tolerant β-glucosidases which can work at elevated glucose concentrations. However, there are very few reports on such enzymes from microbial sources especially filamentous fungi which can be cultivated on cheap biomass as raw material. There has been very less number of studies directed at this, though there is every possibility that filamentous fungi that are efficient degraders of biomass may harbor such enzymes. The study therefore aimed at isolating a fungus capable of secreting glucose tolerant β- glucosidase enzyme. Production, characterization of β-glucosidases and application of BGL for bioethanol production were attempted.

Occurrence and Distribution of a Diatom-Diazotrophic Cyanobacteria Association during a Trichodesmium Bloom in the Southeastern Arabian Sea

Symbiotic diatom-diazotrophic cyanobacteria association (DDA) of Rhizosolenia hebetata and Rhizosolenia formosa with endosymbiotic cyanobacteria Richelia intracellularis was noticed and documented for the first time during a bloom of the cyanobacterium Trichodesmium erythraeum in the oligotrophic shelf waters along Kochi and Mangalore transects, southeastern Arabian Sea (SEAS), during spring intermonsoon (April 2012). Although the host is frequently seen, the symbiont is rarely reported in the Indian EEZ. The presence of nitrogen-fixing symbiotic association of Rhizosolenia-Richelia along with Trichodesmium erythraeum highlights the significance of DDAs on the nutrient and energy budgets of phytoplankton in the oligotrophic environments of the Arabian Sea during spring intermonsoon

Depth wise variation of microbial load in the soils of midland region of Kerala: a function of important soil physicochemical characteristics and nutrients

Soil microorganisms play a main part in organic matter decomposition and are consequently necessary to soil ecosystem processes maintaining primary productivity of plants. In light of current concerns about the impact of cultivation and climate change on biodiversity and ecosystem performance, it is vital to expand a complete understanding of the microbial community ecology in our soils. In the present study we measured the depth wise profile of microbial load in relation with important soil physicochemical characteristics (soil temperature, soil pH, moisture content, organic carbon and available NPK) of the soil samples collected from Mahatma Gandhi University Campus, Kottayam (midland region of Kerala). Soil cores (30 cm deep) were taken and the cores were separated into three 10-cm depths to examine depth wise distribution. In the present study, bacterial load ranged from 141×105 to 271×105 CFU/g (10cm depth), from 80×105 to 131×105 CFU/g (20cm depth) and from 260×104 to 47×105 CFU/g (30cm depth). Fungal load varies from 124×103 to 27×104 CFU/g, from 61×103 to110×103 CFU/g and from 16×103 to 49×103 CFU/g at 10, 20 and 30 cm respectively. Actinomycetes count ranged from 129×103 to 60×104 CFU/g (10cm), from 70×103 to 31×104 CFU/g (20cm) and from 14×103 to 66×103 CFU/g (30cm). The study revealed that there was a significant difference in the depthwise distribution of microbial load and soil physico-chemical properties. Bacterial, fungal and actinomycetes load showed a decreasing trend with increasing depth at all the sites. Except pH all other physicochemical properties showed decreasing trend with increasing depth. The vertical profile of total microbial load was well matched with the depthwise profiles of soil nutrients and organic carbon that is microbial load was highest at the soil surface where organics and nutrients were highest

Spatial and Temporal Variation of Microbial Population in the Grassland Soils of Tropical Montane Forest: Influence of Soil Physico-Chemical Characteristics and Nutrients

Present study is focused on the spatiotemporal variation of the microbial population (bacteria, fungus and actinomycetes) in the grassland soils of tropical montane forest and its relation with important soil physico-chemical characteristics and nutrients. Different physico-chemical properties of the soil such as temperature, moisture content, organic carbon, available nitrogen, available phosphorous and available potassium have been studied. Results of the present study revealed that both microbial load and soil characteristics showed spatiotemporal variation. Microbial population of the grassland soils were characterized by high load of bacteria followed by fungus and actinomycetes. Microbial load was high during pre monsoon season, followed by post monsoon and monsoon. The microbial load varied with important soil physico-chemical properties and nutrients. Organic carbon content, available nitrogen and available phosphorous were positively correlated with bacterial load and the correlation is significant at 0.05 and 0.01 levels respectively. Available nitrogen and available phosphorous were positively correlated with fungus at 0.05 level significance. Moisture content was negatively correlated with actinomycetes at 0.01 level of significance. Organic carbon negatively correlated with actinomycetes load at 0.05 level of significance

Potential application of β-1, 3 glucanase from an environmental isolate of Pseudomonas aeruginosa MCCB 123 in fungal DNA extraction

Pseudomonas aeruginosa MCCB 123 was grown in a synthetic medium for β-1,3 glucanase production. From the culture filtrate, β-1,3 glucanase was purified with a molecular mass of 45 kDa. The enzyme was a metallozyme as its β-1,3 glucanase activity got inhibited by the metal chelator EDTA. Optimum pH and temperature for β-1,3 glucanase activity on laminarin was found to be 7 and 50 °C respectively. The MCCB 123 β-1,3 glucanase was found to have good lytic action on a wide range of fungal isolates, and hence its application in fungal DNA extraction was evaluated. β-1,3 glucanase purified from the culture supernatant of P. aeruginosa MCCB 123 could be used for the extraction of fungal DNA without the addition of any other reagents generally used. Optimum pH and temperature of enzyme for fungal DNA extraction was found to be 7 and 65 °C respectively. This is the first report on β-1,3 glucanase employed in fungal DNA extraction

Impact of process parameters on chitinase production by an alkalophilic marine Beau6eria bassiana in solid state fermentation

A chitinolytic fungus, Beau6eria bassiana was isolated from marine sediment and significant process parameters influencing chitinase production in solid state fermentation using wheat bran were optimised. The organism was strongly alkalophilic and produced maximum chitinase at pH 9·20. The NaCl and colloidal chitin requirements varied with the type of moistening medium used. Vegetative (mycelial) inoculum was more suitable than conidial inoculum for obtaining maximal enzyme yield. The addition of phosphate and yeast extract resulted in enhancement of chitinase yield. After optimisation, the maximum enzyme yield was 246·6 units g 1 initial dry substrate (U gIDS 1). This is the first report of the production of chitinase from a marine fungus.

L-Glutaminase production by marine Beau6eria sp. under solid state fermentation

Extracellular L-glutaminase production by Beau6eria sp., isolated from marine sediment, was observed during solid state fermentation using polystyrene as an inert support. Maximal enzyme production (49.89 U:ml) occurred at pH 9.0, 27°C, in a seawater based medium supplemented with L-glutamine (0.25% w:v) as substrate and D-glucose (0.5% w:v) as additional carbon source, after 96 h of incubation. Enzyme production was growth associated. Results indicate scope for production of salt tolerant L-glutaminase using this marine fungus

Lipase from marine Aspergillus awamori BTMFW032: Production, partial purification and application in oil effluent treatment

Marine fungus BTMFW032, isolated from seawater and identified as Aspergillus awamori, was observed to produce an extracellular lipase, which could reduce 92% fat and oil content in the effluent laden with oil. In this study, medium for lipase production under submerged fermentation was optimized statistically employing response surface method toward maximal enzyme production. Medium with soyabean meal- 0.77% (w/v); (NH4)2SO4-0.1 M; KH2PO4-0.05 M; rice bran oil-2% (v/v); CaCl2-0.05 M; PEG 6000-0.05% (w/v); NaCl-1% (w/v); inoculum-1% (v/v); pH 3.0; incubation temperature 35 8C and incubation period-five days were identified as optimal conditions for maximal lipase production. The time course experiment under optimized condition, after statistical modeling, indicated that enzyme production commenced after 36 hours of incubation and reached a maximum after 96 hours (495.0 U/ml), whereas maximal specific activity of enzyme was recorded at 108 hours (1164.63 U/mg protein). After optimization an overall 4.6- fold increase in lipase production was achieved. Partial purification by (NH4)2SO4 precipitation and ion exchange chromatography resulted in 33.7% final yield. The lipase was noted to have a molecular mass of 90 kDa and optimal activity at pH 7 and 40 8C. Results indicated the scope for potential application of this marine fungal lipase in bioremediation.

Propyl Gallate Synthesis Using Acidophilic Tannase and Simultaneous Production of Tannase and Gallic Acid by Marine Aspergillus awamori BTMFW032

Marine Aspergillus awamori BTMFW032, recently reported by us, produce acidophilic tannase as extracellular enzyme. Here, we report the application of this enzyme for synthesis of propyl gallate by direct transesterification of tannic acid and in tea cream solubilisation besides the simultaneous production of gallic acid along with tannase under submerged fermentation by this fungus. This acidophilic tannase enabled synthesis of propyl gallate by direct transesterification of tannic acid using propanol as organic reaction media under low water conditions. The identity of the product was confirmed with thin layer chromatography and Fourier transform infrared spectroscopy. It was noted that 699 U/ml of enzyme could give 60% solubilisation of tea cream within 1 h. Enzyme production medium was optimized adopting Box–Behnken design for simultaneous synthesis of tannase and gallic acid. Process variables including tannic acid, sodium chloride, ferrous sulphate, dipotassium hydrogen phosphate, incubation period and agitation were recognized as the critical factors that influenced tannase and gallic acid production. The model obtained predicted 4,824.61 U/ml of tannase and 136.206 μg/ml gallic acid after 48 h of incubation, whereas optimized medium supported 5,085 U/ml tannase and 372.6 μg/ml of gallic acid production after 36 and 84 h of incubation, respectively, with a 15-fold increase in both enzyme and gallic acid production. Results indicated scope for utilization of this acidophilic tannase for transesterification of tannic acid into propyl gallate, tea cream solubilisation and simultaneous production of gallic acid along with tannase

Continuous Production of Extracellular L-Glutaminase by Ca-Alginate-Immobilized Marine Beauveria bassiana BTMF S-10 in Packed-Bed Reactor

L-Glutamine amidohydrolase (L-glutaminase, EC 3.5.1.2) is a therapeutically and industrially important enzyme. Because it is a potent antileukemic agent and a flavor-enhancing agent used in the food industry, many researchers have focused their attention on L-glutaminase. In this article, we report the continuous production of extracellular L-glutaminase by the marine fungus Beauveria bassiana BTMF S-10 in a packed-bed reactor. Parameters influencing bead production and performance under batch mode were optimized in the order-support (Na-alginate) concentration, concentration of CaCl2 for bead preparation, curing time of beads, spore inoculum concentration, activation time, initial pH of enzyme production medium, temperature of incubation, and retention time. Parameters optimized under batch mode for L-glutaminase production were incorporated into the continuous production studies. Beads with 12 × 108 spores/g of beads were activated in a solution of 1% glutamine in seawater for 15 h, and the activated beads were packed into a packed-bed reactor. Enzyme production medium (pH 9.0) was pumped through the bed, and the effluent was collected from the top of the column. The effect of flow rate of the medium, substrate concentration, aeration, and bed height on continuous production of L-glutaminase was studied. Production was monitored for 5 h in each case, and the volumetric productivity was calculated. Under the optimized conditions for continuous production, the reactor gave a volumetric productivity of 4.048 U/(mL·h), which indicates that continuous production of the enzyme by Ca-alginate-immobilizedspores is well suited for B. bassiana and results in a higher yield of enzyme within a shorter time. The results indicate the scope of utilizing immobilized B. bassiana for continuous commercial production of L-glutaminase

β-glucosidases from Aspergillus unguis NII 08123: Molecular characterization, properties and applications

Lignocellulosic biomass is probably the best alternative resource for biofuel production and it is composed mainly of cellulose, hemicelluloses and lignin. Cellulose is the most abundant among the three and conversion of cellulose to glucose is catalyzed by the enzyme cellulase. Cellulases are groups of enzymes act synergistically upon cellulose to produce glucose and comprise of endoglucanase, cellobiohydrolase and β-glucosidase. β -glucosidase assumes great importance due to the fact that it is the rate limiting enzyme. Endoglucanases (EG) produces nicks in the cellulose polymer exposing reducing and non reducing ends, cellobiohydrolases (CBH) acts upon the reducing or non reducing ends to liberate cellobiose units, and β - glucosidases (BGL) cleaves the cellobiose to liberate glucose completing the hydrolysis. . β -glucosidases undergo feedback inhibition by their own product- β glucose, and cellobiose which is their substrate. Few filamentous fungi produce glucose tolerant β - glucosidases which can overcome this inhibition by tolerating the product concentration to a particular threshold. The present study had targeted a filamentous fungus producing glucose tolerant β - glucosidase which was identified by morphological as well as molecular method. The fungus showed 99% similarity to Aspergillus unguis strain which comes under the Aspergillus nidulans group where most of the glucose tolerant β -glucosidase belongs. The culture was designated the strain number NII 08123 and was deposited in the NII culture collection at CSIR-NIIST. β -glucosidase multiplicity is a common occurrence in fungal world and in A.unguis this was demonstrated using zymogram analysis. A total 5 extracellular isoforms were detected in fungus and the expression levels of these five isoforms varied based on the carbon source available in the medium. Three of these 5 isoforms were expressed in higher levels as identified by the increased fluorescence (due to larger amounts of MUG breakdown by enzyme action) and was speculated to contribute significantly to the total _- β glucosidase activity. These isoforms were named as BGL 1, BGL3 and BGL 5. Among the three, BGL5 was demonstrated to be the glucose tolerant β -glucosidase and this was a low molecular weight protein. Major fraction was a high molecular weight protein but with lesser tolerance to glucose. BGL 3 was between the two in both activity and glucose tolerance.121 Glucose tolerant .β -glucosidase was purified and characterized and kinetic analysis showed that the glucose inhibition constant (Ki) of the protein is 800mM and Km and Vmax of the enzyme was found to be 4.854 mM and 2.946 mol min-1mg protein-1respectively. The optimumtemperature was 60°C and pH 6.0. The molecular weight of the purified protein was ~10kDa in both SDS as well as Native PAGE indicating that the glucose tolerant BGL is a monomeric protein.The major β -glucosidase, BGL1 had a pH and temperature optima of 5.0 and 60 °C respectively. The apparent molecular weight of the Native protein is 240kDa. The Vmax and Km was 78.8 mol min-1mg protein-1 and 0.326mM respectively. Degenerate primers were designed for glycosyl hydrolase families 1, 3 and 5 and the BGL genes were amplified from genomic DNA of Aspergillus unguis. The sequence analyses performed on the amplicons results confirmed the presence of all the three genes. Amplicon with a size of ~500bp was sequenced and which matched to a GH1 –BGL from Aspergillus oryzae. GH3 degenerate primers producing amplicons were sequenced and the sequences matched to β - glucosidase of GH3 family from Aspergillus nidulans and Aspergillus acculateus. GH5 degenerate primers also gave amplification and sequencing results indicated the presence of GH5 family BGL gene in the Aspergillus unguis genomic DNA.From the partial gene sequencing results, specific as well as degenerate primers were designed for TAIL PCR. Sequencing results of the 1.0 Kb amplicon matched Aspergillus nidulans β -glucosidase gene which belongs to the GH1 family. The sequence mainly covered the N-Terminal region of the matching peptide. All the three BGL proteins ie. BGL1, BGL3 and BGL5 were purified by chromatography an electro elution from Native PAGE gels and were subjected to MALDI-TOF mass spectrometric analysis. The results showed that BGL1 peptide mass matched to . β -glucosidase-I of Aspergillus flavus which is a 92kDa protein with 69% protein coverage. The glucose tolerant β -glucosidase BGL5 mass matched to the catalytic C-terminal domain of β -glucosidase-F from Emericella nidulans, but the protein coverage was very low compared to the size of the Emericella nidulans protein. While comparing the size of BGL5 from Aspergillus unguis, the protein sequence coverage is more than 80%. BGL F is a glycosyl hydrolase family 3 protein.The properties of BGL5 seem to be very unique, in that it is a GH3 β -glucosidase with a very low molecular weight of ~10kDa and at the same time having catalytic activity and glucose 122 tolerance which is as yet un-described in GH β -glucosidases. The occurrence of a fully functional 10kDA protein with glucose tolerant BGL activity has tremendous implications both from the points of understanding the structure function relationships as well as for applications of BGL enzymes. BGL-3 showed similarity to BGL1 of Aspergillus aculateus which was another GH3 β -glucosidase. It may be noted that though PCR could detect GH1, GH3 and GH5 β-glucosidases in the fungus, the major isoforms BGL1 BGL3 and BGL5 were all GH3 family enzymes. This would imply that β-glucosidases belonging to other families may also co-exist in the fungus and the other minor isoforms detected in zymograms may account for them. In biomass hydrolysis, GT-BGL containing BGL enzyme was supplemented to cellulase and the performances of blends were compared with a cocktail where commercial β- glucosidase was supplemented to the biomass hydrolyzing enzyme preparation. The cocktail supplemented with A unguis BGL preparation yielded 555mg/g sugar in 12h compared to the commercial enzyme preparation which gave only 333mg/g in the same period and the maximum sugar yield of 858 mg/g was attained in 36h by the cocktail containing A. unguis BGL. While the commercial enzyme achieved almost similar sugar yield in 24h, there was rapid drop in sugar concentration after that, indicating probably the conversion of glucose back to di-or oligosaccharides by the transglycosylation activity of the BGl in that preparation. Compared this, the A.unguis enzyme containing preparation supported peak yields for longer duration (upto 48h) which is important for biomass conversion to other products since the hydrolysate has to undergo certain unit operations before it goes into the next stage ie – fermentation in any bioprocesses for production of either fuels or chemicals.. Most importantly the Aspergillus unguis BGL preparation yields approximately 1.6 fold increase in the sugar release compared to the commercial BGL within 12h of time interval and 2.25 fold increase in the sugar release compared to the control ie. Cellulase without BGL supplementation. The current study therefore leads to the identification of a potent new isolate producing glucose tolerant β - glucosidase. The organism identified as Aspergillus unguis comes under the Aspergillus nidulans group where most of the GT-BGL producers belong and the detailed studies showed that the glucose tolerant β -glucosidase was a very low molecular weight protein which probably belongs to the glycosyl hydrolase family 3. Inhibition kinetic studies helped to understand the Ki and it is the second highest among the nidulans group of Aspergilli. This has promoted us for a detailed study regarding the mechanism of glucose tolerance. The proteomic 123 analyses clearly indicate the presence of GH3 catalytic domain in the protein. Since the size of the protein is very low and still its active and showed glucose tolerance it is speculated that this could be an entirely new protein or the modification of the existing β -glucosidase with only the catalytic domain present in it. Hydrolysis experiments also qualify this BGL, a suitable candidate for the enzyme cocktail development for biomass hydrolysis