Streptomyces misionensis PESB-25 produces a thermoacidophilic endoglucanase using sugarcane bagasse and corn steep liquor as the sole organic substrates.
ABSTRACT: Streptomyces misionensis strain PESB-25 was screened and selected for its ability to secrete cellulases. Cells were grown in a liquid medium containing sugarcane bagasse (SCB) as carbon source and corn steep liquor (CSL) as nitrogen source, whose concentrations were optimized using response surface methodology (RSM). A peak of endoglucanase accumulation (1.01 U · mL(-1)) was observed in a medium with SCB 1.0% (w/v) and CSL 1.2% (w/v) within three days of cultivation. S. misionensis PESB-25 endoglucanase activity was thermoacidophilic with optimum pH and temperature range of 3.0 to 3.6 and 62° to 70 °C, respectively. In these conditions, values of 1.54 U mL(-1) of endoglucanase activity were observed. Moreover, Mn(2+) was demonstrated to have a hyperactivating effect on the enzyme. In the presence of MnSO4 (8 mM), the enzyme activity increased threefold, up to 4.34 U · mL(-1). Mn(2+) also improved endoglucanase stability as the catalyst retained almost full activity upon incubation at 50 °C for 4 h, while in the absence of Mn(2+), enzyme activity decreased by 50% in this same period. Three protein bands with endoglucanase activity and apparent molecular masses of 12, 48.5 and 119.5 kDa were detected by zymogram.
Project description:A novel gene (designated as cen219) encoding endoglucanase was isolated from a Bursaphelenchus xylophilus metagenomic library by functional screening. Sequence analysis revealed that cen219 encoded a protein of 367 amino acids. SDS-PAGE analysis of purified endoglucanase suggested that Cen219 was a monomeric enzyme with a molecular mass of 40 kDa. The optimum temperature and pH for endoglucanase activity of Cen219 was separately 50 °C and 6.0. It was stable from 30 to 50 °C, and from pH 4.0 to 7.0. The activity was significantly enhanced by Mn(2+) and dramatically reduced by detergent SDS and metals Fe(3+), Cu(2+) or Hg(2+). The enzyme hydrolyzed a wide range of ?-1, 3-, and ?-1, 4-linked polysaccharides, with varying activities. Activities towards microcrystalline cellulose and filter paper were relatively high, while the highest activity was towards oat gum. The Km and Vmax of Cen219 towards CMC was 17.37 mg/ml and 333.33 U/mg, respectively. The findings have an insight into understanding the molecular basis of host-parasite interactions in B. xylophilus species. The properties also make Cen219 an interesting enzyme for biotechnological application.
Project description:The effects of manganese (Mn) preconditioning, 96 h post-hatch followed by the replacement of inorganic Mn with different levels of organic Mn (5 to 21 D), on growth, tissue excreta Mn content, gene expression, and enzyme activity were evaluated. A total of 420 day-old male Cobb 500 broilers were divided into 2 groups. One group was fed a corn-soybean meal basal diet containing 17 mg of Mn/kg (preconditioning diet, MnPD); the second group was fed the non-preconditioning diet (NPCD), which was the MnPD supplemented with 60 mg of Mn/kg from manganese sulfate (MnSO4). On day 5, each group was divided into 5 subgroups and were randomly assigned to dietary treatments consisting of MnPD alone or MnPD supplemented with 12 or 60 mg Mn/kg Mn as MnSO4 or Mn proteinate (6 replicate cages of 6 birds). Broiler chicks that were fed the MnPD had lower (P ≤ 0.05) body weight gain (BWG) and G:F ratio when compared to those that were fed the NPCD for 4 D. Birds that were fed MnPD (1 to 4 D) and switched to MnPD supplemented with 60 mg/kg Mn (5 to 21 D) had lower (P ≤ 0.05) BWG compared to those that were fed NPCD (1 to 4 D) and switched to MnPD supplemented with 60 mg/kg Mn for 21 D. Excreta, tibia ash, liver, and heart Mn levels were increased (P ≤ 0.05) by supplemental Mn. The expression of jejunum divalent metal transporter-1 mRNA levels, as well as activities of plasma total super oxide dismutase and liver alanine transaminase, was not affected by MnPD or Mn source and levels. These results confirmed that feeding marginally deficient Mn diets to broiler chicks post-hatch does affect growth rate and tissue Mn concentration.
Project description:This study assessed the in vitro effects of the bioaccessible food components released during the simulated human digestion of a coffee fibre-containing biscuit (CFB) on ?-glucosidase activity, antioxidant capacity and satiety hormones. Digest of CFB presented a significantly (p < 0.05) lower amount of sugar (68.6 mg/g) and a higher antioxidant capacity (15.1 mg chlorogenic acid eq./g) than that of a sucrose-containing biscuit (SCB). The CFB significantly reduced (p < 0.05) ?-glucosidase activity (IC50 = 3.3 mg/mL) compared to the SCB (IC50 = 6.2 mg/mL). Serotonin and glucagon-like peptide-1 (GLP-1) release by differentiated Caco-2 and HuTu-80 cells, respectively, was stimulated by the CFB (355% at a concentration of 0.5 mg/mL and 278% at a concentration of 0.05 mg/mL) to the same order of magnitude as those of the SCB. To summarize, the CFB was demonstrated to reduce monosaccharide bioaccessibility, to inhibit a diabetes-related digestive enzyme, and to improve the release of satiety hormones.
Project description:A thermophilic Thermobifida fusca strain UPMC 901, harboring highly thermostable cellulolytic activity, was successfully isolated from oil palm empty fruit bunch compost. Its endoglucanase had the highest activity at 24?hours of incubation in carboxymethyl-cellulose (CMC) and filter paper. A maximum endoglucanase activity of 0.9?U/mL was achieved at pH 5 and 60?°C using CMC as a carbon source. The endoglucanase properties were further characterized using crude enzyme preparations from the culture supernatant. Thermal stability indicated that the endoglucanase activity was highly stable at 70?°C for 24?hours. Furthermore, the activity was found to be completely maintained without any loss at 50?°C and 60?°C for 144?hours, making it the most stable than other endoglucanases reported in the literature. The high stability of the endoglucanase at an elevated temperature for a prolonged period of time makes it a suitable candidate for the biorefinery application.
Project description:This study was conducted to isolate the cellulolytic microorganism from the rumen of Holstein steers and characterize endoglucanase gene (Cel5A) from the isolated microorganism.To isolate anaerobic microbes having endoglucanase, rumen fluid was obtained from Holstein steers fed roughage diet. The isolated anaerobic bacteria had 98% similarity with Eubacterium cellulosolvens (E. cellulosolvens) Ce2 (Accession number: AB163733). The Cel5A from isolated E. cellulolsovens sp. was cloned using the published genome sequence and expressed through the Escherichia coli BL21.The maximum activity of recombinant Cel5A (rCel5A) was observed at 50°C and pH 4.0. The enzyme was constant at the temperature range of 20°C to 40°C but also, at the pH range of 3 to 9. The metal ions including Ca2+, K+, Ni2+, Mg2+, and Fe2+ increased the endoglucanase activity but the addition of Mn2+, Cu2+, and Zn2+ decreased. The Km and Vmax value of rCel5A were 14.05 mg/mL and 45.66 μmol/min/mg. Turnover number, Kcat and catalytic efficiency, Kcat/Km values of rCel5A was 96.69 (s-1) and 6.88 (mL/mg/s), respectively.Our results indicated that rCel5A of E. cellulosolvens isolated from Holstein steers had a broad pH range with high stability under various conditions, which might be one of the beneficial characteristics of this enzyme for possible industrial application.
Project description:One factor at a time approach was employed for the optimization of media components, physical factors and additional additives for lignin peroxidase (LiP) production under submerged fermentation by the endophytic fungus Endomelanconiopsis sp. The optimized conditions derived were: carbon source (dextrose 10 g/L), nitrogen source (ammonium tartarate 0.027 g/L), veratryl alcohol (1 mM), MnSO4 (0.5 g/L), inoculum concentration (3 mycelial plugs), pH (5), temperature (30 °C) and Tween 80 (1.5%). A 40 fold increase of enzyme activity was observed after the single parameter optimization. The lignin peroxidase (LiP) production to the level of 345.26 ± 0.52 IU/mL indicates that the fungus has the commercial potential for LiP.
Project description:The endoglucanase isolated from culture filtrates of Streptomyces lividans IAF74 was shown to have an Mr of 46,000 and a pI of 3.3. The specific enzyme activity of 539 IU/mg, determined by the reducing assay method on carboxymethyl cellulose, is among the highest reported in the literature. The cellulase showed typical endo-type activity when reacting on oligocellodextrins. Optimal enzyme activity was obtained at 50 degrees C and pH 5.5. The kinetic constants for this endoglucanase, determined with carboxymethyl cellulose as the substrate, were a Vmax of 24.9 IU/mg of enzyme and a Km of 4.2 mg/ml. Activity was found against neither methylumbelliferyl- nor p-nitrophenyl-cellobiopyranoside nor with xylan. The DNA sequence contains one possible reading frame validated by the N terminus of the mature purified protein. However, neither ATG nor GTG starting codons were identified near the ribosome-binding site. A putative TTG codon was found as a good candidate for the start codon. Comparison of the primary amino acid sequence of the endoglucanase of S. lividans revealed that the N terminus contains a bacterial cellulose-binding domain. The catalytic domain at the C terminus showed similarity to endoglucanases from a Bacillus sp. Thus, the endoglucanase CelA belongs to family A of cellulases as described before (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991.
Project description:In an attempt to screen out cellulase producing bacteria from herbivorous animal fecal matter it was possible to isolate a potent bacterium from cow dung. The bacterium was identified as Bacillus sp. using 16S rDNA based molecular phylogenetic approach. The effect of different agricultural wastes, paper wastes and carboxymethyl cellulose on endoglucanase production was tested and was found to produce maximally at 8% carboxymethyl cellulose. The endoglucanase was precipitated by ammonium sulfate saturation and purified by DEAE- Sepharose column. The purification was achieved 8.5 fold from the crude extract with a yield of 68.1%. The molecular weight of the protein was determined to be 97 kDa by SDS-PAGE. The enzymatic activity was moderately reduced by detergents (SDS, Tween-80), metal ions (MnCl2, ZnCl2) and EDTA. The endoglucanase was stable between pH 5.0 - 9.0 and temperature between 20-70°C with optimal activity at pH 7.0 and temperature 50°C. The apparent Km value of the enzyme for the substrate carboxymethyl cellulose was recorded to be 0.25 mg/ml. The endoglucanase was stable in the presence of commercial detergents such as Ariel, Surf Excel and Tide, indicated might be of potential applications in detergent industry. The enzyme from this strain could also be applied in bioconversion of lignocellulosic biomass into fermentable sugars.
Project description:The zygomycete fungus Lichtheimia ramosa H71D, isolated from sugarcane bagasse compost, was identified by applying phylogenetic analysis based on the DNA sequence of the Internal Transcribed Spacer (ITS), and subsequent secondary structure analysis of ITS2. L. ramosa H71D was able to grow over a wide range of temperatures (25-45 °C), manifesting optimal growth at 37 °C. A 64 kDa xylanase (named LrXynA) was purified from the culture supernatant of L. ramosa H71D grown on 2% carboxymethylcellulose (CMC), as the only carbon source. LrXynA displayed optimal activity at pH 6 and temperature of 65 °C. The enzyme retained more than 50% of its maximal activity over a broad range of pH values (4.5-7.5). Enzyme half-life (t½) times at 55, 65 and 75 °C were 80, 25, and 8 min, respectively. LrXynA showed higher affinity (k M of 2.87 mg/mL) and catalytic efficiency (k cat /k M of 0.651 mg s/mL) towards Beechwood xylan in comparison to other substrates such as Birchwood xylan, Oat-spelt xylan, CMC, Avicel and Solka floc. The predominant final products from LrXynA-mediated hydrolysis of Beechwood xylan were xylobiose and xylotriose, suggesting that the enzyme is an endo-?-1,4 xylanase. Scanning electron microscopy (SEM) imaging of sugar cane bagasse (SCB) treated with LrXynA, alone or in combination with commercial cellulases, showed a positive effect on the hydrolysis of SCB. To our knowledge, this is the first report focusing on the biochemical and functional characterization of an endo-?-1,4 xylanase from the thermotolerant and fast-growing fungus Lichtheimia ramosa.
Project description:Aspergillus nidulans is poorly exploited as a source of enzymes for lignocellulosic residues degradation for biotechnological purposes. This work describes the A. nidulans Endoglucanase A heterologous expression in Pichia pastoris, the purification and biochemical characterization of the recombinant enzyme. Active recombinant endoglucanase A (rEG A) was efficiently secreted as a 35?kDa protein which was purified through a two-step chromatography procedure. The highest enzyme activity was detected at 50°C/pH 4. rEG A retained 100% of activity when incubated at 45 and 55°C for 72?h. Purified rEG A kinetic parameters towards CMC were determined as K m = 27.5 ± 4.33?mg/mL, V max = 1.185 ± 0.11?mmol/min, and 55.8?IU (international units)/mg specific activity. Recombinant P. pastoris supernatant presented hydrolytic activity towards lignocellulosic residues such as banana stalk, sugarcane bagasse, soybean residues, and corn straw. These data indicate that rEG A is suitable for plant biomass conversion into products of commercial importance, such as second-generation fuel ethanol.