Influencia de la concentración de inóculo en la producción de celulasa y xilanasa por aspergillus niger / Influence of inoculum concentration on the cellulase and xylanase production by aspergillus niger

Existe un gran interés por el uso de enzimas lignocelulolíticas en varias industrias, y en la biodegradación de biomasa para la producción de biocombustibles y otras aplicaciones. Entre las fuentes microbianas de enzimas, Aspergillus niger es uno de los microorganismos más utilizados en la producción de enzimas industriales, debido a sus niveles altos de secreción de proteína y a su condición GRAS (generally regarded as safe). El objetivo del presente estudio fue evaluar la influencia de la concentración de inóculo en la morfología y producción de celulasas y xilanasas con A. niger en cultivo sumergido. Para ello, fueron inoculados matraces de 250 mL con 40 mL de medio con 3% (v/v) de una suspensión de 104 o 108 esporas por mililitro e incubados a 28 ºC y 175 rpm durante 120 horas. Se utilizaron 10 g*L-1 de lactosa como fuente de carbono. En cada caso se determinó la cantidad de biomasa, la proteína extracelular soluble, lactosa residual, actividad celulasa total y xilanasa cada 24 horas. Aunque no hubo un efecto notorio en la morfología de crecimiento, salvo en el color y el diámetro de pellets obtenidos, sí se afectó la µmax (0,06 y 0,03 h-1 para 104 y 108 esporas*mL-1, respectivamente) y la concentración máxima de biomasa. Además, mientras que las productividades volumétricas de celulasa (ΓFPA) (8,2 y 8,0 UI.*L-1*h-1 para 104 y 108 esporas*mL-1, respectivamente) fueron similares para ambos inóculos, la productividad de xilanasa (ΓXIL) fue mayor para el inóculo más concentrado (29,7 y 33,4 UI¨*L-1*h-1 para 104 y 108 esporas*mL-1, respectivamente). Los resultados indican que la productividad de celulasas y xilanasas está estrechamente relacionada con la concentración de inóculo.

There is a great interest for the use of lignocellulolytic enzymes in several industries and in biomass degradation for production of biofuels and other applications. Among the microbial sources of enzymes, Aspergillus niger is one of the most used microorganisms in the production of industrial enzymes due to its high levels of protein secretion and its GRAS (generally regarded as safe) condition. The aim of the present study was to evaluate the influence of A. niger inoculum concentration in the morphology and production of cellulases and xylanases in submerged cultures. For this, 250 mL flasks containing 40 mL culture medium were inoculated with a 3% (v/v) of either 104 or 108 spores per milliliter suspension and incubated at 28 º C and 175 rpm during 120 hours. Lactose (10 g*L-1) was used as the carbon source. In each case, the amount of biomass, the extracellular soluble protein, residual lactose, total celullase activity and xylanase activity were determined every 24 hours. Even thought there was not a notorious effect on the growth morphology, except in color and diameter of pellets; µmax was affected (0.06 and 0.03 h-1 for 104 and 108 spores*mL-1, respectively) as well as maximum biomass concentration. In addition, while the volumetric productivity of cellulase (8.2 and 8.0 UI*L-1*h-1 for 104 and 108 spores*mL-1, respectively) were similar for both inocula, the productivity of xylanase was greater for the more concentrated inoculum (29.7 and 33.4 UI*L-1*h-1 for 104 and 108 spores*mL-1, respectively).The results show that cellulase and xylanase productivities are closely related to the inoculum concentration.

Carbohydrate-binding module of a rice endo-beta-1,4-glycanase, OsCel9A, expressed in auxin-induced lateral root primordia, is post-translationally truncated.

We report the cloning of a glycoside hydrolase family (GHF) 9 gene of rice (Oryza sativa L. cv. Sasanishiki), OsCel9A, corresponding to the auxin-induced 51 kDa endo-1,4-beta-glucanase (EGase). This enzyme reveals a broad substrate specificity with respect to sugar backbones (glucose and xylose) in beta-1,4-glycans of type II cell wall. OsCel9A encodes a 640 amino acid polypeptide and is an ortholog of TomCel8, a tomato EGase containing a carbohydrate-binding module (CBM) 2 sequence at its C-terminus. The expression of four rice EGase genes including OsCel9A showed different patterns of organ specificity and responses to auxin. OsCel9A was preferentially expressed during the initiation of lateral roots or subcultured root calli, but was hardly expressed during auxin-induced coleoptile elongation or in seed calli, in contrast to OsCel9D, a KORRIGAN (KOR) homolog. In situ localization of OsCel9A transcripts demonstrated that its expression was specifically up-regulated in lateral root primordia (LRP). Northern blotting analysis showed the presence of a single product of OsCel9A. In contrast, both mass spectrometric analyses of peptide fragments from purified 51 kDa EGase proteins and immunogel blot analysis of EGase proteins in root extracts using two antibodies against internal peptide sequences of OsCel9A revealed that the entire CBM2 region was post-translationally truncated from the 67 kDa nascent protein to generate 51 kDa EGase isoforms. Analyses of auxin concentration and time course dependence of accumulation of two EGase isoforms suggested that the translation and post-translational CBM2 truncation of the OsCel9A gene may participate in lateral root development.

Use of recombinant cellulose-binding domains of Trichoderma reesei cellulase as a selective immunocytochemical marker for cellulose in protozoa.

Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in Acanthamoeba, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent beta-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. We have used a recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from Trichoderma reesei cellulases linked together in combination with monoclonal anticellulase antibodies and anti-mouse immunoglobulin fluorescein conjugate to specifically stain cellulose in the cysts of Acanthamoeba strains for fluorescence microscopy imaging. Staining was observed in ruptured cysts and frozen sections of cysts but not in intact mature cysts. No staining reaction was observed with the chitin-containing cyst walls of Giardia intestinalis, Entamoeba dispar, or Pneumocystis carinii. Thus, the recombinant CBD can be used as a marker to distinguish between cellulose and chitin. Thirteen of 25 environmental or clinical isolates of amoebae reacted in the CBD binding assay. All 13 isolates were identified as Acanthamoeba spp. Five isolates of Hartmannella and seven isolates of Naegleria tested negative in the CBD binding assay. Whether cyst wall cellulose really is a unique property of Acanthamoeba spp. among free-living amoebae, as suggested by our findings, remains to be shown in more extensive studies.

In planta localization of a beta-1,4-endoglucanase secreted by Heterodera glycines.

Polyclonal sera specific to beta-1,4-endoglucanases (cellulases) synthesized in the subventral esophageal gland cells of the soybean cyst nematode, Heterodera glycines, were used to provide the first identification of a nematode esophageal gland protein that is secreted into host plant tissue. Sera generated to proteins encoded by Hg-eng-1 and Hg-eng-2 (endoglucanases) did not cross-react with soybean root proteins on Western blots (immunoblots) or in immunofluorescence microscopy of noninoculated (control) soybean root sections. In cross sections of soybean roots at 24 h after inoculation of roots with second-stage juveniles of H. glycines, HG-ENG-1 was localized within the nematode's subventral gland cells and was not detected in root tissue. HG-ENG-2 was localized within the subventral gland cells and was secreted from the juvenile's stylet into root cortical tissue at 24 h after inoculation of roots with second-stage juveniles of H. glycines. HG-ENG-2 was localized along the juvenile's migratory path through the root cortex.

Electron and fluorescence microscopy of extracellular glucan and aryl-alcohol oxidase during wheat-straw degradation by Pleurotus eryngii.

The ligninolytic fungus Pleurotus eryngii grown in liquid medium secreted extracellular polysaccharide (87% glucose) and the H2O2-producing enzyme aryl-alcohol oxidase (AAO). The production of both was stimulated by wheat-straw. Polyclonal antibodies against purified AAO were obtained, and a complex of glucanase and colloidal gold was prepared. With these tools, the localization of AAO and extracellular glucan in mycelium from liquid medium and straw degraded under solid-state fermentation conditions was investigated by transmission electron microscopy (TEM) and fluorescence microscopy. These studies revealed that P. eryngii produces a hyphal sheath consisting of a thin glucan layer. This sheath appeared to be involved in both mycelial adhesion to the straw cell wall during degradation and AAO immobilization on hyphal surfaces, with the latter evidenced by double labelling. AAO distribution during differential degradation of straw tissues was observed by immunofluorescence microscopy. Finally, TEM immunogold studies confirmed that AAO penetrates the plant cell wall during P. eryngii degradation of wheat straw.

Immunodetection and characterization of tomato endo-beta-1,4-glucanase Cel1 protein in flower abscission zones.

Tomato (Lycopersicon esculentum Mill.) endo-beta-1,4-glucanase Cel1 mRNA accumulation was previously correlated with abscission of flower explants. Cel1 antibodies were raised against a fusion protein encoding a portion of the Cel1 polypeptide and was shown to react specifically with three polypeptides with molecular masses ranging between 51 and 53 kD in flower abscission zones induced to abscise. All three polypeptides were clearly suppressed in two transgenic lines expressing an antisense Cel1 gene that specifically suppressed the accumulation of Cel1 mRNA, indicating that all three polypeptides are products of the Cel1 gene. Cel1 protein accumulation was correlated with flower abscission. Breakstrength and Cel1 protein content were also analyzed in flower explants, indicating that Cel1 protein accumulation is correlated with the final stages of flower shedding, which suggests that Cel1 is involved in the late stage of abscission. These results support the involvement of Cel1 in the abscission of flower explants and suggest that other hydrolase activities also participate in that process.

Differential ethylene-inducible expression of cellulase in pepper plants.

Ethylene promotes the abscission of leaves and the ripening of fruits in pepper plants, and in both events an increase in cellulase activity is observed. However, two enzyme isoforms (pI 7.2 and 8.5, respectively) are differentially involved in the two physiological phenomena. The pI 8.5 form has been purified from ripe fruits. It is a glycoprotein with an apparent molecular mass of 54 kDa. Two short peptides were sequenced and a very high homology to a tomato cellulase was observed. Polyclonal antibodies, raised against the purified enzyme, have allowed us to demonstrate that the observed ethylene-induced increase in cellulase activity is paralleled by de novo synthesis of protein. Three cDNAs (CX1, CX2 and CX3), encoding different cellulases, were obtained and characterized and their expression investigated. Accumulation of all three mRNAs is induced by ethylene treatment, though to different levels. CX1 is mainly expressed in ripe fruits while CX2 is especially found in abscission zones. CX3 accumulates at very low levels in activated abscission zones. Comparisons with other known cellulases demonstrate clear heterogeneity within the higher plant cellulases. Differences in ethylene inducibility and molecular structure suggest different physiological roles for cellulase in pepper plants.

Endo-1,4-beta-glucanase in the cell wall of stems of auxin-treated pea seedlings.

Endo-1,4-beta-glucanase induced by treatment of pea seedlings with 2,4-D was extracted from a preparation of the walls of epicotyl cells. The beta-glucanase was purified by chromatography on DEAE-cellulose, affinity chromatography on Con A-Sepharose and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The activity of beta-glucanase was retained after removal of SDS and extraction from polyacrylamide gels. The band of a protein (46 kDa), that corresponded to the activity of endo-1,4-beta-glucanase, was injected directly into mice for preparation of antiserum and the protein was also subjected to amino acid sequencing after blotting onto a membrane. Western blot analysis showed that the antiserum obtained bound to a 46-kDa polypeptide and recognized endo-1,4-beta-glucanase. The N-terminal sequence of the 46-kDa polypeptide revealed some homology to abscission endo-1,4-beta-glucanases of bean and avocado fruit.

Electron microscopic observation of cotton cellulose degradation by exo- and endo-type cellulases from Irpex lacteus.

The interaction of two highly purified cellulases, exo- and endo-type cellulases from Irpex lacteus, with pure cotton and amorphous cellulose was investigated by electron microscopy. The morphological observations indicated that exo- and endo-type cellulases are both strongly adsorbed on the internal microfibril of cotton fiber before enzymatic hydrolysis, and then initiate their action toward the internal cellulose microfibrils with retention of the original shape. The two cellulases, however, caused considerably different morphological changes in cotton cellulose, and each cellulase seems to degrade native cellulose with a distinct mode of action. In the hydrolysis of cotton with exo-type cellulase, deep transverse cracks were produced and they extended from the fiber surface to the lumen structure located inside the fibers. In contrast, it was found that there were no deep cracks on fibers treated with endo-type cellulase, but severe internal erosion and cavitation occurred along fibril or microfibril layers inside the fibers. Thus, the degradation of cotton by exo- and endo-type cellulases yielded quite different morphological patterns, while little difference was found for regenerated celluloses. The mode of enzymatic hydrolysis of cellulose shown by cellulases with different degrees of randomness (exo and endo types) appears to be markedly affected by the fine structure of cellulose fibers.

Cellulose-binding polypeptides from Cellulomonas fimi: endoglucanase D (CenD), a family A beta-1,4-glucanase.

Five cellulose-binding polypeptides were detected in Cellulomonas fimi culture supernatants. Two of them are CenA and CenB, endo-beta-1,4-glucanases which have been characterized previously; the other three were previously uncharacterized polypeptides with apparent molecular masses of 120, 95, and 75 kDa. The 75-kDa cellulose-binding protein was designated endoglucanase D (CenD). The cenD gene was cloned and sequenced. It encodes a polypeptide of 747 amino acids. Mature CenD is 708 amino acids long and has a predicted molecular mass of 74,982 Da. Analysis of the predicted amino acid sequence of CenD shows that the enzyme comprises four domains which are separated by short linker polypeptides: an N-terminal catalytic domain of 405 amino acids, two repeated sequences of 95 amino acids each, and a C-terminal domain of 105 amino acids which is > 50% identical to the sequences of cellulose-binding domains in Cex, CenA, and CenB from C. fimi. Amino acid sequence comparison placed the catalytic domain of CenD in family A, subtype 1, of beta-1,4-glycanases. The repeated sequences are more than 40% identical to the sequences of three repeats in CenB and are related to the repeats of fibronectin type III. CenD hydrolyzed the beta-1,4-glucosidic bond with retention of anomeric configuration. The activities of CenD towards various cellulosic substrates were quite different from those of CenA and CenB.

Respiratory allergy to Aspergillus-derived enzymes in bakers' asthma.

Baking and food industry workers are exposed to several powdered Aspergillus-derived enzymes with carbohydrate-cleaving activity that are commonly used to enhance baked products. We describe a retrospective study of sensitization to fungal alpha-amylase and cellulase on bakers. Five bakers in whom respiratory allergy symptoms developed when they were exposed to bread "improvers" that contained fungal alpha-amylase and cellulase were investigated by in vivo and in vitro tests. Type I hypersensitivity to these enzymes was demonstrated in the five patients by means of skin testing, histamine release test, positive reverse enzyme-immunoassay for specific IgE antibodies, and bronchial provocation test response to alpha-amylase or cellulase or both. Isolated immediate and dual responses to the bronchial challenge tests with these enzymes were observed. Immunoblot analysis with use of a pooled serum identified IgE-binding components in both enzymes. In the reverse-enzyme immunoassay-inhibition assays cross-reactivity between alpha-amylase and cellulase was not found, but some degree of cross-reactivity between alpha-amylase and A. oryzae, and between cellulase and A. niger was demonstrated. Four of the patients were also sensitized to cereal flour. Aspergillus-derived enzymes used as flour additives can elicit IgE-mediated respiratory allergy, and this fact has to be considered in the diagnosis and clinical management of bakers' asthma.

Cross-reactive and specific monoclonal antibodies against cellobiohydrolases I and II and endoglucanases I and II of Trichoderma reesei.

Splenocytes derived from mice inoculated with a commercial cellulase preparation or purified cellulases were fused with a stable myeloma cell line (SP2/0). Specific monoclonal antibodies to cellobiohydrolases I and II and endoglucanases I and II were established. In addition to specific monoclonal antibodies, we were also able to establish stable hybridoma cell lines which produced monoclonal antibodies that recognized similar epitopes possessed by two or more of the above cellulases. By obtaining monospecific antibodies for all four individual cellulases, the role and function of the individual cellulases can thus be studied in greater detail.

Allergic reactions to enzymes used in plant cloning experiments.

We report a case of rhinitis and asthma resulting from exposure to enzymes used in an experimental plant pathology laboratory. Immediate skin-test responses were elicited to both "Cellulase" and "Macerozyme" products, and a large late onset skin reaction occurred at the Cellulase site beginning in 6 hr. Special radioallergosorbent test (RAST) substrates were prepared that showed a strong positive response to Cellulase. By the RAST method other laboratory workers were screened and another symptomatic individual was found who also showed evidence of type I hypersensitivity to both enzymes. Skin biopsy and immunodiffusion data are presented. We conclude that enzymes used to digest cell wall structures of plants are capable of eliciting both immediate and late onset skin-test reactions and positive RAST responses in susceptible persons. These reactions are thought to be mediated by IgE antibodies and can be associated with symptoms of rhinitis and asthma.