[Show abstract][Hide abstract] ABSTRACT: Agave tequilana fructans are the source of fermentable sugars for the production of tequila. Fructans are processed by acid hydrolysis or by cooking in ovens at high temperature. Enzymatic hydrolysis is considered an alternative for the bioconversion of fructans. We previously described the isolation of Aspergillus niger CH-A-2010, an indigenous strain that produces extracellular inulinases. Here we evaluated the potential application of A. niger CH-A-2010 inulinases for the bioconversion of A. tequilana fructans, and its impact on the production of ethanol. Inulinases were analyzed by Western blotting and thin layer chromatography. Optimal pH and temperature conditions for inulinase activity were determined. The efficiency of A. niger CH-A-2010 inulinases was compared with commercial enzymes and with acid hydrolysis. The hydrolysates obtained were subsequently fermented by Saccharomyces cerevisiae to determine the efficiency of ethanol production. Results indicate that A. niger CH-A-2010 predominantly produces an exo-inulinase activity. Optimal inulinase activity occurred at pH 5.0 and 50 °C. Hydrolysis of raw agave juice by CH-A-2010 inulinases yielded 33.5 g/l reducing sugars, compared with 27.3 g/l by Fructozyme(®) (Novozymes Corp, Bagsværd, Denmark) and 29.4 g/l by acid hydrolysis. After fermentation of hydrolysates, we observed that the conversion efficiency of sugars into ethanol was 97.5 % of the theoretical ethanol yield for enzymatically degraded agave juice, compared to 83.8 % for acid-hydrolyzed juice. These observations indicate that fructans from raw Agave tequilana juice can be efficiently hydrolyzed by using A. niger CH-A-2010 inulinases, and that this procedure impacts positively on the production of ethanol.
[Show abstract][Hide abstract] ABSTRACT: Aspergillus sp. CH-Y-1043 grown on pectins with various degrees of esterification produces endo- and exo-pectinases at pH values as low as 2.5. Maximal production was attained at this pH, although fungal growth only approximated 50% of that obtained at higher pH values. Endopectinase was produced at pH 2.5–3.5 when the fungus was grown on low degree esterified pectin. With higher degree esterified pectin this enzyme was produced at all pH values analyzed. Exopectinase production was less affected by pH values. Still, maximal production was also reached at pH 2.5–3.5. Exopectinase was found to be associated to the cell and could be released after incubation at different pH values, whereas endo pectinase was not detected in the cellular fraction. Results confirmed by SDS–PAGE coupled with in situ activity assays in pectin–agarose gels allowed the identification of a protein band corresponding to endopectinase and a band with pectin esterase activity. Stability of Aspergillus sp. CH-Y-1043 pectinases at various pH values was also evaluated. Key words: Aspergillus sp. CH-Y-1043, extreme acidic pH pectinase production, in situ pectinase detection, cell-associated exopectinase.
Canadian Journal of Microbiology 02/2011; 37(12):912-917. · 1.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pectin lyases cleave the internal glycosidic bonds of pectin by β-elimination, producing non-saturated galacturonic oligomers. Genetic improvement of pectin lyase-overproducing strains is still necessary to improve industrial processes based on this enzyme. In the present study hybrids were obtained by protoplast fusion between mutant pectinolytic Aspergillus flavipes and Aspergillus niveus CH-Y-1043 strains. Prototrophic segregants showed different isoenzymatic profiles and produced increased levels of pectin lyase in cultures containing lemon peel as a sole carbon source. Hybrid HZ showed an increase of 450% and 1300% in pectin lyase production compared with that of A. niveus CH-Y-1043 and A. flavipes, respectively. Pectin lyase produced by the hybrid HZ was partially purified and used for the hydrolysis of orange peel. Pectin lyase was able to hydrolyze 56% of orange peel biomass. However, addition of 2 RFU and 20 U of endo- and exo-polygalacturonase, respectively, induced the hydrolysis of 92% of orange peel solids. In conclusion HZ is a pectin lyase-overproducing hybrid with potential applications in the pectin industry.
[Show abstract][Hide abstract] ABSTRACT: S. Solís, M.E. FLORES AND C. HUITRON. 1996. Protoplast release in pectinolytic strain mutants of Aspergillus sp. CH-Y-1043 (A13) and Aspergillus flavipes ATCC-16795 (F7) is described. Optimum yield of protoplasts A13 was obtained in a lapse of 1 h when commercially lytic enzymes of Trichoderma harzanium (2 mg ml−1) were added in 0.05 mol 1−1 citrate-phosphate buffer pH 5.0 containing 0.7 mol 1−1 KCl and 10 mg ml−1 BSA. Best results in F7 were obtained when the protoplasting system of A13 was supplemented with 10 mg ml−1Aureobasidium sp. lytic enzymes. Isolated protoplasts in A13 and F7 were capable of a high regeneration frequency of 87% and 53% when 0.7 mol 1−1 KCl and sorbitol were used as osmotic stabilizers. Endo-P, Exo-P and pectin lyase production were not modified during the process of regeneration.
[Show abstract][Hide abstract] ABSTRACT: Approximately 1 million tons of Agave tequilana plants are processed annually by the Mexican Tequila industry generating vast amounts of agricultural waste. The aim of this study was to investigate the potential use of Agave tequilana waste as substrate for the production of commercially important enzymes. Two strains of Aspergillus niger (CH-A-2010 and CH-A-2016), isolated from agave fields, were found to grow and propagate in submerged cultures using Agave tequilana waste as substrate. Isolates showed simultaneous extracellular inulinase, xylanase, pectinase, and cellulase activities. Aspergillus CH-A-2010 showed the highest production of inulinase activity (1.48 U/ml), whereas Aspergillus niger CH-A-2016 produced the highest xylanase (1.52 U/ml) and endo-pectinase (2.7U/ml) activities. In both cases production of enzyme activities was significantly higher on Agave tequilana waste than that observed on lemon peel and specific polymeric carbohydrates. Enzymatic hydrolysis of raw A. tequilana stems and leaves, by enzymes secreted by the isolates yielded maximum concentrations of reducing sugars of 28.2 g/l, and 9.9 g/l respectively. In conclusion, Agave tequilana waste can be utilized as substrate for the production of important biotechnological enzymes.
Journal of Environmental Biology 02/2008; 29(1):37-41. · 0.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the prevalence of mucosal autoantibodies to survivin in patients with human papillomavirus (HPV)-associated cervical cancer and precursor lesions.
Cervical mucus from 117 HPV-associated cervical cancer, 102 high-grade squamous intraepithelial lesion (HSIL), 107 low-grade SIL (LSIL), and 80 normal controls were tested by ELISA using either full length recombinant survivin or survivin-derived peptides. Survivin expression in cervical tissue biopsies was studied by Western Blotting.
Cervical mucus from 33 cervical cancer (28.2%), 17 HSIL (16.6%), and 8 LSIL (7.4%) patients reacted with recombinant survivin. The IgA-class antibody response was significantly higher than that observed in the normal controls. The level of mucosal anti-survivin response was associated to the level and intensity of survivin expression in the different lesions. Finally, reactivity against a survivin Nt-derived peptide was found more frequently than reactivity against a Ct-derived peptide.
IgA-class autoantibodies against survivin are present in a substantial proportion of cervical mucus from patients with HPV-associated cervical cancer, and precursor lesions. Mucosal anti-survivin response is positively associated with the level of survivin expression and the grade of cervical lesion.
[Show abstract][Hide abstract] ABSTRACT: Methylmalonyl-coenzyme A (CoA) mutase, methylmalonyl-CoA decarboxylase and NADP+-isocitrate dehydrogenase activity profiles were determined in Saccharopolyspora erythraea CA340, an erythromycin producer, grown in glucose or ammonium chloride. Results show that methylmalonyl-CoA mutase and isocitrate dehydrogenase activities were associated with growth, while methylmalonyl-CoA decarboxylase maximal activity was observed at stationary phase. Isocitrate dehydrogenase synthesis was stimulated by glucose and repressed by ammonium; glucose affected negatively the synthesis of methylmalonyl-CoA mutase, whereas methylmalonyl-CoA decarboxylase production was not affected by carbon or nitrogen sources. These results suggested that the negative effect of glucose or ammonium on erythromycin production could be due, in part, to lower pools of succinyl-CoA and methylmalonyl-CoA.
[Show abstract][Hide abstract] ABSTRACT: Debate continues over the physical characteristics and even the existence of arginase isoenzymes. This paper gives additional support for such multiplicity and reports differences in physical characteristics among the various forms.
After 2500–5000-fold purification of rat liver arginase, three molecular forms were separated on carboxymethyl-cellulose columns and were purified 2500–5000-fold, 800–1000-fold and 600–1000-fold, respectively. The molecular forms have also been identified by chromatography in the supernatant of tissue extracts. The isolation of these molecular forms by affinity chromatography, using Sepharose-lysine as a competitive inhibitor of arginase, shows only one main form, however.
Kinetic studies were done for two of the molecular forms isolated, specifically the activation energy (Ea) the energy of denaturatization (Ed), Km, pH and the effect of divalent cations were determined. Significant differences were found for the Ea, between the two molecular forms. The isolated isoenzymes are cationic at pH 5.5 and pH 8.8. However, they show different mobilities in electrophoresis. The molecular weight determination by gel filtration yields a value of 110000 to 115000 for both forms. The use of thin-layer immunochromatography plates, a combination of molecular weight and immunodiffusion technique, gave only one peak with the same molecular weight as that determined by gel filtration. The immunological studies showed that the isoenzymes have similar antigenic determinants.
[Show abstract][Hide abstract] ABSTRACT: Activation of the interleukin-2 receptor (IL-2R) induces signalling cascades promoting T cell proliferation. However, signal transduction pathways triggered in IL-2R-expressing solid tumours are unknown. This report shows that human papillomavirus (HPV)-associated cervical cancer cells express an IL-2R composed of beta and gamma chains (IL-2Rbetagamma), and that IL-2-mediated activation increases the phosphorylation of JAK3 and STAT5, stimulating cell proliferation. Interestingly, endogenous IL-2 is not produced by these cells, suggesting the activation of IL-2Rbetagamma by an alternative mechanism. Accordingly, we found that Stem Cell Factor (SCF)-activated c-Kit induces phosphorylation of the IL-2Rbeta chain in the absence of IL-2. Moreover, inhibition of IL-2Rbeta phosphorylation by blocking c-Kit tyrosine kinase activity abolishes both, IL-2 and SCF-mediated proliferation. Thus, these results demonstrate that IL-2 triggers a JAK3/STAT5 cascade in HPV-associated cervical cancer cells expressing IL-2Rbetagamma, and that this receptor can be alternatively activated by SCF-activated c-Kit in the absence of IL-2.
[Show abstract][Hide abstract] ABSTRACT: The taxonomic position of a thermophilic actinomycete strain isolated from soil was examined using a polyphasic approach. The strain, designated CH-M-1035T, was assigned to the genus Streptomyces on the basis of chemical and morphological criteria. It formed Rectiflexibiles aerial hyphae that carried long chains of rounded, smooth spores. The almost complete nucleotide sequence of the 16S rRNA gene of strain CH-M-1035T was determined and its comparison with the 16S rDNA sequences of previously studied streptomycetes confirmed the assignment of the novel strain to the genus Streptomyces. Strain CH-M-1035T clustered with species belonging to the Streptomyces thermodiastaticus clade in the 1 6S-rDNA-based phylogenetic tree. However, the phenotypic properties of strain CH-M-1035T differed from those of the recognized species within this clade. Therefore, it is proposed that strain CH-M-1035T be classified as a novel species within the genus Streptomyces, as Streptomyces mexicanus (type strain CH-M-1035T =DSM 41796T =BM-B-384T =NRRL B-24196T).
International Journal of Systematic and Evolutionary Microbiology 02/2003; 53(Pt 1):269-73. DOI:10.1099/ijs.0.002251-0 · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Streptomyces sp. DSM 41796 produced four major extracellular xylanases with Mr of 145, 120, 60 and 45 kDa. Those of 145 and 60 kDa formed a heterodimer. All xylanases, except that of 120 kDa, were induced by xylose, d-arabinose or sucrose, while commercial xylans induced the 60 kDa xylanase in a major proportion than others, and sugar-cane bagasse pith or lemon peel induced predominantly the 45 kDa xylanase.
[Show abstract][Hide abstract] ABSTRACT: Extracellular xylanase activity and cell-bound β-xylosidase production by a selected strain of Streptomyces sp. CH-M-1035 was characterized during growth on three xylans, sugar cane bagasse pith and lemon peel as sole carbon source. The cell-bound β-xylosidase and extracellular endoxylanase had pH optima of 6·0 and 5·0, and temperature optima of 50°C and 60°C, respectively. The highest level of β-xylosidase activity was obtained when Streptomyces sp. CH-M-1035 was grown on larchwood xylan, whereas the maximal endoxylanase production was found on lemon peel. Reducing sugars accumulated in the culture media when Streptomyces sp. CH-M-1035 was grown on xylans, but not on agroindustrial residues.
[Show abstract][Hide abstract] ABSTRACT: Protoplast fusion induced by polyethyleneglycol and Ca2+ was carried out between two auxotrophic mutants of Aspergillus sp. CH-Y-1043. The hybrids obtained showed significant differences in endopectinase activity and morphology compared to the prototrophic strain. Strains grown on lemon peel showed production improvement with respect to the parental strain. Since H15 hybrid showed up to 90% higher endopectinase production than the wild type CH-Y-1043, kinetics of enzyme production in Fernbach flasks and Fermentor (14L) by H15 were determined.
[Show abstract][Hide abstract] ABSTRACT: The specificity of induction of β-xylosidase and endoxylanase from Streptomyces sp. CH-M-1035 was investigated using mono-, di- and poly-saccharides, among other compounds. This microorganism was able to grow on all carbon sources except cellulose microcrystalline and methyl-β-D-xylopyranoside. β-Xylosidase was induced by larchwood xylan, birchwood xylan, oat spelts xylan and D-xylose. Basal levels of activity were obtained in sucrose, lactose, maltose and pectin. Endoxylanase activity was induced in presence of xylans, D-xylose, sucrose and D-arabinose. Both enzymes, β-xylosidase and endoxylanase, were repressed by the addition of glucose, glycerol and succinic acid to the culture medium containing 1% birchwood xylan. On the other hand, addition of methyl-β-D-xylopyranoside in the same conditions induced β-xylosidase but not endoxylanase synthesis. An unexpected stimulatory effect was observed on β-xylosidase biosynthesis when pyruvic acid was added to the culture medium independently of the xylan used.
[Show abstract][Hide abstract] ABSTRACT: Filter paper, carboxymethylcellulase, β-glucosidase, and xylanase activities were determined and compared to cellulases originating
fromAureobasidium sp. (Ab),Penicillium sp. (Pe), andAspergillus terreus (At). The formation of total reducing sugar was measured as a function of time for the hydrolysis of the native sugar cane
bagasse pith using culture filtrates with the same quantity of extracellular protein content from Ab, Pe, At, or from the
following mixtures Ab:Pe/ Ab:At and Pe:At at different volumetric ratios. The saccharification progress indicated a synergistic
effect for the mixture of all the enzymes, the highest being in the 40:60 relation of Pe and At, respectively. The synergistic
action has been assigned to a better balance of endo- and exoglucanases in this system and essentially to the addition of
β-glucosidase and xylanases from At to Pe cellulases.
[Show abstract][Hide abstract] ABSTRACT: Aspergillus sp. CH-Y-1043 synthesizes pectin lyase when grown on citrus pectin at 37 C. Production is favoured by increased esterification degree of the pectin used as carbon source. This enzyme displays higher activity at pH values of 8.5–8.8 and temperatures of 40–45 C. The optimal substrate for the enzyme was highly esterified pectin and no enzymatic activity was registered on polygalacturonic acid. The activity is stimulated by, though not dependent on, divalent cations (Ca2+, Mg2+, Mn2+, Ba2+ and Co2+) and inhibited by Zn2+, and it is not sensitive to the addition of EDTA. The enzyme is very stable when exposed to pH variations: at 4 C it preserves more than 95% of its activity at pHs ranging from 2.0 to 10.0, and at 30 C stability is preserved at pHs ranging from 4.0 to 8.0. At a constant pH of 5.0, the enzyme conserves its stability at temperatures ranging from 4 to 50 C and at pH 8.0 sensitivity to temperature increased. The results on the endo-exo nature of the enzyme suggest that this is an exo-pectin lyase.
[Show abstract][Hide abstract] ABSTRACT: Glucose and glycerol at concentrations of 2 % negatively affected amylase synthesis in plate and submerged Streptomyces kanamyceticus cultures. This microorganism was insensitive to growth inhibition by glucose analogs and deregulated mutants were identified by a clearing zone around colonies grown on starch and glycerol or glucose, and selected. Three kinds of mutants were obtained: one insensitive to glucose (Mutant 41), another insensitive to glycerol repression (Mutant E) and the last (Mutant 29) an amylase-hyperproducing mutant, albeit regulated by glucose or glycerol like the wild type. The levels of glucokinase, an enzyme involved in catabolite regulation of Enterobacteria, were determined and results showed no differences between the parental strain and the mutants.
[Show abstract][Hide abstract] ABSTRACT: Intact conidia of Aspergillus sp. were able to degrade pectin 'in vitro' even when protein synthesis was inhibited, thus indicating the presence of cell bound pectinases. At least an exo-pectinase was found and this enzyme was also present in the mycelium of Aspergillus sp. Its presence was not dependent on the carbon source used for growth, suggesting its constitutive nature. This exo-pectinase could be released from conidia or mycelium by incubation at different pH values and the amount of enzyme released could be increased by treatments with chemical agents and hydrolytic enzymes.
[Show abstract][Hide abstract] ABSTRACT: With the aim of obtaining hyperproducing strains of pectinases,Aspergillus sp. CH-Y-1043 was mutated with NTG and mutants resistant to glycerol catabolic repression were selected. Among the mutants obtained, CH-SS/ M63 produced an endo-PG activity 400% higher than the wild type, using lemon peel as the sole carbon source.