Direct ethanol production from cellulosic materials by Zymobacter palmae carrying Cellulomonas endoglucanase and Ruminococcus β-glucosidase genes

In order to reduce the cost of bioethanol production from lignocellulosic biomass, we conferred the ability to ferment cellulosic materials directly on Zymobacter palmae by co-expressing foreign endoglucanase and β-glucosidase genes. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, the six genes encoding the cellulolytic enzymes (CenA, CenB, CenD, CbhA, CbhB, and Cex) from Cellulomonas fimi were introduced and expressed in Z. palmae. Of these cellulolytic enzyme genes cloned, CenA degraded carboxymethylcellulose and phosphoric acid-swollen cellulose (PASC) efficiently. The extracellular CenA catalyzed the hydrolysis of barley β-glucan and PASC to liberate soluble cello-oligosaccharides, indicating that CenA is the most suitable enzyme for cellulose degradation among those cellulolytic enzymes expressed in Z. palmae. Furthermore, the cenA gene and β-glucosidase gene (bgl) from Ruminococcus albus were co-expressed in Z. palmae. Of the total endoglucanase and β-glucosidase activities, 57.1 and 18.1 % were localized in the culture medium of the strain. The genetically engineered strain completely saccharified and fermented 20 g/l barley β-glucan to ethanol within 84 h, producing 79.5 % of the theoretical yield. Thus, the production and secretion of CenA and BGL enabled Z. palmae to efficiently ferment a water-soluble cellulosic polysaccharide to ethanol.     

Response surface optimization for enhanced production of cellulases with improved functional characteristics by newly isolated Aspergillus niger HN-2

Fungi isolated from partially decayed wood log samples showing characteristic diversity for spore colour, colony morphology and arrangement of spores were assessed for cellulolytic enzyme production. Isolates showing a cellulolytic index of ≥2.0 were assayed for filter paper (FP) cellulase and β-glucosidase (BGL) production. Molecular characterization confirmed the identity of the selected cellulolytic isolate as a strain of Aspergillus niger (A. niger HN-2). Addition of 2 % (w/v) urea enhanced FP and BGL activity by about 20 and 60 %, respectively. Validation studies conducted at parameters (29 °C, pH 5.4, moisture content 72 % and 66 h) optimized through response surface methodology in a solid-state static tray fermentation resulted in FP, BGL, cellobiohydrolase I (CBHI), endoglucanase (EG), xylanase activity and protein content of 25.3 FPU/g ds, 750 IU/g ds, 13.2 IU/g ds, 190 IU/g ds, 2890 IU/g ds and 0.9 mg/ml, respectively. In comparison, A. niger N402 which is a model organism for growth and development studies, produced significantly lower FP, BGL, CBHI, EG, xylanase activity and protein content of 10.0 FPU/g ds, 100 IU/g ds, 2.3 IU/g ds, 50 IU/g ds, 500 IU/g ds and 0.75 mg/ml, respectively under the same process conditions as were used for A. niger HN-2. Process optimization led to nearly 1.8- and 2.2-fold increase in FP and BGL activity, respectively showing promise for cellulase production by A. niger HN-2 at a higher scale of operation. Zymogram analysis revealed two isoforms each for EG and cellobiohydrolase and three isoforms for BGL. Crude cellulase complex produced by A. niger HN-2 exhibited thermostability under acidic conditions showing potential for use in biofuel industry.     

A novel neutral,halophile <Emphasis Type="Italic">Stachybotrys microspora</Emphasis>-based endoglucanase active impact on β-glucan

The production of cellulases from Stachybotrys microspora strain (A19) has been improved by fed-batch fermentation on Avicel cellulose 10 mg/ml. An endoglucanase EG2 was purified to homogeneity. This cellulase has a molecular mass estimated to 50 kDa when analyzed by a denaturant gel electrophoresis. It exhibited an optimal activity at 50 °C, pH 7.0 and 0.85 M NaCl. Specifically, these results show the thermo-active, alkali-tolerant and halo-tolerant properties of EG2. In addition, this endoglucanase showed its highest activity on barley-β-glucan, compared to the CMC. Moreover, it was less active on Avicel cellulose. Furthermore, the EG2 activity was stimulated in the presence of EDTA, urea and β-mercaptoethanol whereas it was reduced in the presence of SDS. This cellulase was highly stable in the presence of organic solvents such as acetone and n-hexane. TLC showed that the main hydrolysis products from EG2 were cellobiose and glucose. This fungal endoglucanase could be potentially important in the conversion of grass-derived biomass into fermentable sugars.     

Expression of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae

Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full‐length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum (TcEG1), and evaluated the activity of the transgenic product (rTcEG1). Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde‐3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β‐1,4‐endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments.     

Assessing the Feasibility of Biofuel Production from Lignocellulosic Banana Waste in Rural Agricultural Communities in Peru and Colombia

Banana cultivation is widespread in tropical and subtropical regions where many rural agricultural communities exist. The banana plant bears fruit once in its life cycle, leaving behind a significant amount of usable agricultural residue. Current practice leaves this residue to decompose in the field spreading diseases and polluting water supplies. We evaluated the technological feasibility of converting lignocellulosic banana residue to ethanol as a localized biofuel production strategy to improve the livelihoods of rural agricultural communities in Peru and Colombia. Liquid hot water (LHW) and steam explosion pretreatment followed by saccharification and fermentation using commercial cellulolytic enzymes and yeast strain were evaluated for three different lignocellulosic residues independently (pseudostems, leaves, and rachis). Stems and rachis, with higher glucan conversion, appeared more promising for biofuel production than leaves (up to 93 and 77 % glucose yields for rachis and pseudostems, respectively). Steam explosion pretreatment allowed higher glucan conversion for stems and leaves, while LHW was better suited for rachis. Pseudostem is the most abundant residue generated with 306,000 tons/year in Uraba Province (Colombia) and 15,000 tons/year in the Chira Valley (Peru) on a dry weight basis. Potential ethanol production in the Chira Valley was estimated in 4.8 and 76.8 ML year^?1 in Colombia, processing stems and rachis combined. This study indicated that there is potential for biofuel production using the lignocellulosic banana residue, which could be expanded to other banana growing communities around the world. Process improvements such as increasing solids loading, water recycling, and optimizing fermentation are still required.     

Improvement of Aspergillus oryzae NRRL 3484 by mutagenesis and optimization of culture conditions in solid-state fermentation for the hyper-production of extracellular cellulase

Spore suspensions of Aspergillus oryzae NRRL 3484 were subjected to mutagenesis using ultraviolet-irradiation followed by chemical treatments to improve the biosynthesis of cellulase. Ten mutant strains namely UEAC7, UEAR5, UNAC4, UNAC16, UNAR19, UNBC7, UNBR3, UNBR10, UNBR23 and UNBR25 were selected and their extracellular cellulase activities were assayed. Mutant UNAC4 gave the highest cellulase production [2,455 ± 28 U/g-dry substrate (ds) for filter paper-ase (FP-ase)] in a yield 4-fold exceeding that of the wild type strain (578 ± 5.0 U/g-ds for FP-ase). Rice straw (RS) was used as a sole carbon source for the enzyme production at a concentration of 10 % (w/v). Maximum cellulase production was achieved at initial medium pH 5.5, initial moisture content 77 % and an incubation temperature 28 °C on the fifth day of growth. NH₄Cl proved to be the suitable added nitrogen source for maximum enzyme production followed by peptone. These results clearly indicate the cost-effectiveness of solid state fermentation technology in the economic production of extracellular cellulase. The hyper-production of cellulase by mutant strain UNAC4 has potential for industrial processes that convert lignocellulosic material (e.g. RS) into products of commercial value such as glucose and biofuels.     

Biomass hydrolyzing enzymes from plant pathogen Xanthomonas axonopodis pv. punicae: optimizing production and characterization

Xanthomonas axonopodis pv. punicae strain—a potent plant pathogen that causes blight disease in pomegranate—was screened for cellulolytic and xylanolytic enzyme production. This strain produced endo-β-1,4-glucanase, filter paper lyase activity (FPA), β-glucosidase and xylanase activities. Enzyme production was optimized with respect to major nutrient sources like carbon and nitrogen. Carboxy methyl cellulose (CMC) was a better inducer for FPA, CMCase and xylanase production, while starch was found to be best for cellobiase. Soybean meal/yeast extract at 0.5 % were better nitrogen sources for both cellulolytic and xylanolytic enzyme production while cellobiase and xylanase production was higher with peptone. Surfactants had no significant effect on levels of extracellular cellulases and xylanases. A temperature of 28 °C and pH 6–8 were optimum for production of enzyme activities. Growth under optimized conditions resulted in increases in different enzyme activities of around 1.72- to 5-fold. Physico-chemical characterization of enzymes showed that they were active over broad range of pH 4–8 with an optimum at 8. Cellulolytic enzymes showed a temperature optimum at around 55 °C while xylanase had highest activity at 45 °C. Heat treatment of enzyme extract at 75 °C for 1 h showed that xylanase activity was more stable than cellulolytic activities. Xanthomonas enzyme extracts were able to act on biologically pretreated paddy straw to release reducing sugars, and the amount of reducing sugars increased with incubation time. Thus, the enzymes produced by X. axonopodis pv. punicae are more versatile and resilient with respect to their activity at different pH and temperature. These enzymes can be overproduced and find application in different industries including food, pulp and paper and biorefineries for conversion of lignocellulosic biomass.     

Efficient yeast cell-surface display of an endoglucanase of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and functional characterization of the whole-cell enzyme

The endoglucanase gene endo753 from Aspergillus flavus NRRL3357 strains was cloned, and the recombinant Endo753 was displayed on the cell surface of Saccharomyces cerevisiae EBY100 strain by the C-terminal fusion using Aga2p protein as anchor attachment tag. The results of indirect immunofluorescence and Western blot confirmed the expression and localization of Endo753 on the yeast cell surface. The hydrolytic activity test of the whole-cell enzyme revealed that Endo753 immobilized on the yeast cell surface had high endoglucanase activity. The functional characterization of the whole-cell enzyme was investigated, and the whole-cell enzyme displayed the maximum activity at pH 8 and 50 °C. The enzyme was stable in a pH range of 7.0–10.0. Furthermore, the whole-cell enzyme displayed high thermostability below 50 °C and moderate stability between 50 and 70 °C. These properties make endo753 a good candidate in bioethanol production from lignocellulosic materials after displaying on the yeast cell surface.     

Cellulases from Penicillium funiculosum: production, properties and application to cellulose hydrolysis

The objective of this work is to investigate the utilization of two abundant agricultural residues in Brazil for the production and application of cellulolytic enzymes. Different materials obtained after pretreatment of sugarcane bagasse, as well as pure synthetic substrates, were considered for cellulase production by Penicillium funiculosum. The best results for FPase (354 U L^?1) and β-glucosidase (1,835 U L^?1) production were observed when sugarcane bagasse partially delignified cellulignin (PDC) was used. The crude extract obtained from PDC fermentation was then partially characterized. Optimal temperatures for cellulase action ranged from 52 to 58°C and pH values of around 4.9 contributed to maximum enzyme activity. At 37°C, the cellulases were highly stable, losing less than 15% of their initial activity after 23 h of incubation. There was no detection of proteases in the P. funiculosum extract, but other hydrolases, such as endoxylanases, were identified (147 U L^?1). Finally, when compared to commercial preparations, the cellulolytic complex from P. funiculosum showed more well-balanced amounts of β-glucosidase, endo- and exoglucanase, resulting in the desired performance in the presence of a lignocellulosic material. Cellulases from this filamentous fungus had a higher glucose production rate (470 mg L^?1 h^?1) when incubated with corn cob than with Celluclast^®, GC 220^® and Spezyme^® (312, 454 and 400 mg L^?1 h^?1, respectively).     

Ethanol production from acid- and alkali-pretreated corncob by endoglucanase and β-glucosidase co-expressing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> subject to the expression of heterologous genes and nutrition added

Low-cost technologies to overcome the recalcitrance of cellulose are the key to widespread utilization of lignocellulosic biomass for ethanol production. Efficient enzymatic hydrolysis of cellulose requires the synergism of various cellulases, and the ratios of each cellulase are required to be regulated to achieve the maximum hydrolysis. On the other hand, engineering of cellulolytic Saccharomyces cerevisiae strains is a promising strategy for lignocellulosic ethanol production. The expression of cellulase-encoding genes in yeast would affect the synergism of cellulases and thus the fermentation ability of strains with exogenous enzyme addition. However, such researches are rarely reported. In this study, ten endoglucanase and β-glucosidase co-expressing S. cerevisiae strains were constructed and evaluated by enzyme assay and fermentation performance measurement. The results showed that: (1) maximum ethanol titers of recombinant strains exhibited high variability in YPSC medium (20 g/l peptone, 10 g/l yeast extract, 100 g/l acid- and alkali-pretreated corncob) within 10 days. However, they had relatively little difference in USC medium (100 g/l acid- and alkali-pretreated corncob, 0.33 g/l urea, pH 5.0). (2) Strains 17# and 19#, with ratio (CMCase to β-glucosidase) of 7.04 ± 0.61 and 7.40 ± 0.71 respectively, had the highest fermentation performance in YPSC. However, strains 11# and 3# with the highest titers in USC medium had a higher ratio of CMCase to β-glucosidase, and CMCase activities. These results indicated that nutrition, enzyme activities and the ratio of heterologous enzymes had notable influence on the fermentation ability of cellulase-expressing yeast.     

First Thermostable Endo-β-1,4-Glucanase from Newly Isolated Xanthomonas sp. EC102

A novel gene encoding thermostable endoglucanase was identified in Xanthomonas sp. EC102 from soil. The gene had 1,458 base pairs of open reading frame, which encode a 52-kDa protein of 486 amino acid residues. Sequence of the amino acid residues was similar with the endoglucanase from Xanthomonas campestris pv. campestris ATCC33913 (GenBank Accession No. NP_638867.1) (94 % identity). The endoglucanase was overexpressed in Escherichia coli BL21 and purified. Temperature for the highest enzymatic activity was 70 °C and pH optima was pH 5.5. The specific activity of the endoglucanase toward carboxymethylcellulose (CMC) was approximately 2 μmol min^?1 mg^?1, V _max for CMC was 1.44 μmol mg^?1 min^?1, and K _m values was 25.6 mg mL^?1. The EC102 endoglucanase was stable at temperatures up to 60 °C, and it was activated by 0.1 mM of Mn²⁺ and Co²⁺. This is the first report about thermostable endoglucanase from Xanthomonas sp.     

A metagenomic approach for the identification and cloning of an endoglucanase from rice straw compost

Over the years, culturable cellulase-producing microorganisms have been isolated from a variety of sources and genes of cellulolytic enzymes have been cloned. Then again, the “great plate count anomaly” phenomenon necessitates a culture-independent metagenomic approach for the isolation of cellulolytic genes from microorganisms in their natural environment. We have constructed a metagenomic library derived from rice straw composts. Of 2739 clones screened, a lambda clone carrying a 12 kb genomic fragment was able to yield a clear zone on an agar plate containing carboxymethyl cellulose (CMC). A 4.7 kb subclone, generated by restriction enzyme digestion, was found to harbor a GH12 cellulase gene, RSC-EG1, encoding 464 amino acids along with two other ORFs. The identified cellulolytic gene showed more than 70% similarity on the amino acid level with cellulase from Micromonospora aurantiaca and Thermobispora sp. Interestingly, RSC-EG1 contains a stretch of approximately 86 amino acids not present in either of these close relatives. Our results demonstrated that RSC-EG1, stable over a wide temperature and pH range, is a novel endoglucanase, and provided the first example of metagenomics approach to isolate cellulolytic gene from rice straw composts.     

Expression and surface display of Cellulomonas endoglucanase in the ethanologenic bacterium Zymobacter palmae

In order to reduce the cost of bioethanol production from lignocellulosic biomass, we developed a tool for cell surface display of cellulolytic enzymes on the ethanologenic bacterium Zymobacter palmae. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, to express and display heterologous cellulolytic enzymes on the Z. palmae cell surface, we utilized the cell-surface display motif of the Pseudomonas ice nucleation protein Ina. The gene encoding Ina from Pseudomonas syringae IFO3310 was cloned, and its product was comprised of three functional domains: an N-terminal domain, a central domain with repeated amino acid residues, and a C-terminal domain. The N-terminal domain of Ina was shown to function as the anchoring motif for a green fluorescence protein fusion protein in Escherichia coli. To express a heterologous cellulolytic enzyme extracellularly in Z. palmae, we fused the N-terminal coding sequence of Ina to the coding sequence of an N-terminal-truncated Cellulomonas endoglucanase. Z. palmae cells carrying the fusion endoglucanase gene were shown to degrade carboxymethyl cellulose. Although a portion of the expressed fusion endoglucanase was released from Z. palmae cells into the culture broth, we confirmed the display of the protein on the cell surface by immunofluorescence microscopy. The results indicate that the N-terminal anchoring motif of Ina from P. syringae enabled the translocation and display of the heterologous cellulase on the cell surface of Z. palmae.     

Regulation of cellulase production in two thermophilic fungi Melanocarpus sp. MTCC 3922 and Scytalidium thermophilum MTCC 4520

This paper reports regulation of cellulase production in two thermophilic fungi, Melanocarpus sp. MTCC 3922 and Scytalidium thermophilum MTCC 4520. The expression of endoglucanase (EG), avicel adsorbable endoglucanase (AAEG) and β-glucosidase in both fungi was inducible. Of the different carbon sources tested, rice straw induced maximal levels of cellulase in both fungi. While, the addition of fructose (1%, w/v) to the carboxymethylcellulose (CMC) medium resulted in two-fold increase in endoglucanase production in Melanocarpus sp., however, the addition of ethanol (1%, v/v) resulted in eight-fold-increased expression of endoglucanase in S. thermophilum. The expression profiles of different components of cellulase complex were shown to be co-regulated in S. thermophilum but independently regulated in Melanocarpus sp.     

Determination of endoglucanase activity of paper decaying fungi from an old library at the ancient Medina of Fez

Paper from an ancient library of the cultural city of Fez (Morocco) is exposed to rapid deterioration by variety of microorganisms, especially cellulolytic fungi. For this, ten isolates fungi previously isolated from historical biodeteriorated paper were screened for their ability to produce endoglucanase (CMCase), amylase, polygalacturonase and ligninase enzymes. The CMCase activity of cellulolytic strains was essayed in liquid media at 25°C for 10 days. Influence of temperature and pH were assessed for the production of CMCase by all the fungus isolated from decaying paper. The research findings from the present study demonstrate that all the tested isolates had cellulase, amylase, pectinase and ligninase activities. It was found that Mucor racemosus PF15, Aspergillus niger, and Aspergillus oryzae exhibited the maximum endoglucanase activity in liquid medium (0.256, 0.236, and 0.216 UI/mL in descending order) for six days. Temperature profiling revealed optimum endoglucanase activity at 25 and 30°C. Maximum activity was observed at pH 5 and pH 6.     

Industrial yeast strain engineered to ferment ethanol from lignocellulosic biomass

In this study an industrial Saccharomyces cerevisiae yeast strain capable of fermenting ethanol from pretreated lignocellulosic material was engineered. Genes encoding cellulases (endoglucanase, exoglucanase and β-glucosidase) were integrated into the chromosomal ribosomal DNA and delta regions of a derivative of the K1-V1116 wine yeast strain. The engineered cellulolytic yeast produces ethanol in one step through simultaneous saccharification and fermentation of pretreated biomass without the addition of exogenously produced enzymes. When ethanol fermentation was performed with 10% dry weight of pretreated corn stover, the recombinant strain fermented 63% of the cellulose in 96 h and the ethanol titer reached 2.6% v/v. These results demonstrate that cellulolytic S. cerevisiae strains can be used as a platform for developing an economical advanced biofuel process.     

Improvement of cellulose degradation by cloning of endo-β-1, 3-1, 4 glucanase (bgls) gene from Bacillus subtilis BTN7A strain

The aim of this study is to construct a new recombinant strain able to degrade cellulose efficiently. The endo-β-1, 3-1, 4 glucanase (bgls) gene was cloned from Bacillus subtilis BTN7A strain by using PCR technique. The specific primers of bgls gene were deduced. Optimization of PCR mixture and program were identified. The nucleotide sequence of bgls was placed in the public domain (GenBank accession number KM009051.1). The obtained bgls DNA was cloned with pGEM®-T Easy Vector. The recombinant plasmid designated as Bgls-NRC-1 was transformed into E. coli DH5α. The successful cloning of the bgls gene was tested either by PCR or by evaluating its expression in its new bacterial host. The bgls gene was expressed efficiently in E. coli and the enzyme activity of the transformant was compared to the enzyme activity of the donor bacterial strain. The new constructs produce much higher enzyme yields than the donor bacterial strain, they produce about 29% and about 57% higher cellulase specific activity at 37?°C and 55?°C respectively. Optimization of cellulolytic activity of the new recombinant strain were described. The effect of minimal medium supplemented with CMC or cellulose, or complete medium (LB) on bgls expression were tested, the order of cellulase activity production was CMC27.2?>?cellulose 21.9?>?LB 19.8?U/mg protein, respectively at 24?h. CMC was proved to be the best medium for cellulase production. Results also showed that double the initial inoculum resulted in more cellulase activities in all media.     

Hydrolytic enzyme(s) production in Micrococcus roseus growing on different cellulosic substrates

Micrococcus roseus (G12) isolated from higher termite Odontotermes obesus gut exhibited cellulose digesting properties. A lignocellulosic substrate, rice husk induced endoglucanase, β-glucosidase, β-xylanase and β-xylosidase production. Besides rice husk, CMC also induced endoglucanase production. β-Glucosidase activity was quite pronounced when rice husk was supplemented with CMC or cellobiose. Both β-xylanase and β-xylosidase activities could be induced by xylan as well as xylobiose, whereas CMC induced partial activity. Endoglucanase and β-xylanase enzymes were secreted into the culture medium, whereas β-glucosidase and β-xylosidase activities were intracellular in nature. Enzyme production was subject to end product inhibition. The extracellular enzyme(s) possessed the potential to saccharify rice husk, xylan and CMC to reducing sugars.     

Extracellular endoglucanase activity from Paenibacillus polymyxa BEb-40: production,optimization and enzymatic characterization

Endoglucanase activity produced by Paenibacillus polymyxa BEb-40 was studied. In submerged culture with minimal medium supplemented with carboxymethylcellulose (CMC), this microorganism produced up to 0.37 U/mL endoglucanase activity with high specific activity (14.3 U/mg_{total protein}). Detection of endoglucanase activity through zymography revealed at least 14 isoenzymes with molecular weights between 38 and 220 kDa. This high variety of secreted endoglucanases has not been described previously in Paenibacillus genus. The optimum conditions, determined by response surface methodology, were 48 °C and pH 3.4, which allowed an increase of 33.7 % in the relative endoglucanase activity obtained with respect to the standard conditions. Nevertheless, high levels of hydrolysis of at least 70 % of the maximum activity could be obtained at wide ranges of pH (2–9) and temperature (40–60 °C). Under optimal conditions, high levels of CMC hydrolysis were reached, of about 40 %, after only 12 h of reaction with substrate/total protein ratios between 19 and 76. Kinetic analysis revealed that endoglucanase activity followed a mixed inhibition model (K _m = 8.4 mM, K _ic = 0.03 mM, K _iu = 0.35 mM, V _max = 33.3 U/mg_{total protein}). These results allow to consider P. polymyxa BEb-40 as a promising microorganism for the production of endoglucanases, with possibilities of application in the breakdown of lignocellulosic biomass. The high specific activity at wide ranges of pH and temperature can allow its use in a wide variety of processes, under both acidic and alkaline conditions, as well as in mesophilic and thermophilic temperatures, further reducing the amount of enzymes used.     

Characterization of Cellulolytic Activities of Environmental Bacterial Consortia from an Argentinian Native Forest

Cellulolytic activities of three bacterial consortia derived from a forest soil sample from Chaco region, Argentina, were characterized. The phylogenetic analysis of consortia revealed two main highly supported groups including Achromobacter and Pseudomonas genera. All three consortia presented cellulolytic activity. The carboxymethylcellulase (CMCase) and total cellulase activities were studied both quantitatively and qualitatively and optimal enzymatic conditions were characterized and compared among the three consortia. Thermal and pH stability were analyzed. Based on its cellulolytic activity, one consortium was selected for further characterization by zymography. We detected a specific protein of 55 kDa with CMCase activity. In this study, we have shown that these consortia encode for cellulolytic enzymes. These enzymes could be useful for lignocellulosic biomass degradation into simple components and for different industrial applications.