Multi-enzyme complex of white rot fungi in saccharification of lignocellulosic material

ABSTRACT The multi-enzyme complex (crude extract) of white rot fungi Pleurotus ostreatus, Pleurotus eryngii, Trametes versicolor, Pycnosporus sanguineus and Phanerochaete chrysosporium were characterized, evaluated in the hydrolysis of pretreated pulps of sorghum straw and compared efficiency with commercial enzyme. Most fungi complexes had better hydrolysis rates compared with purified commercial enzyme.

Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

Abstract An intronless endoglucanase from thermotolerant Aspergillus fumigatus DBINU-1 was cloned, characterized and expressed in the yeast Kluyveromyces lactis. The full-length open reading frame of the endoglucanase gene from A. fumigatus DBiNU-1, designated Cel7, was 1383 nucleotides in length and encoded a protein of 460 amino acid residues. The predicted molecular weight and the isoelectric point of the A. fumigatus Cel7 gene product were 48.19 kDa and 5.03, respectively. A catalytic domain in the N-terminal region and a fungal type cellulose-binding domain/module in the C-terminal region were detected in the predicted polypeptide sequences. Furthermore, a signal peptide with 20 amino acid residues at the N-terminus was also detected in the deduced amino acid sequences of the endoglucanase from A. fumigatus DBiNU-1. The endoglucanase from A. fumigatus DBiNU-1 was successfully expressed in K. lactis, and the purified recombinant enzyme exhibited its maximum activity at pH 5.0 and 60 °C. The enzyme was very stable in a pH range from 4.0 to 8.0 and a temperature range from 30 to 60 °C. These features make it suitable for application in the paper, biofuel, and other chemical production industries that use cellulosic materials.

Influence of iron and copper on the activity of laccases in Fusarium oxysporum f. sp. lycopersici

ABSTRACT Fusarium oxysporum f. sp. lycopersici is a phytopathogenic fungus that causes vascular wilt in tomato plants. In this work we analyze the influence of metal salts such as iron and copper sulphate, as well as that of bathophenanthrolinedisulfonic acid (iron chelator) and bathocuproinedisulfonic acid (copper chelator) on the activity of laccases in the intra (IF) and extracellular fractions (EF) of the wild-type and the non-pathogenic mutant strain (rho1::hyg) of F. oxysporum. The results show that laccase activity in the IF fraction of the wild and mutant strain increased with the addition of iron chelator (53.4 and 114.32%; respectively). With copper, it is observed that there is an inhibition of the activity with the addition of CuSO4 for the EF of the wild and mutant strain (reduction of 82 and 62.6%; respectively) and for the IF of the mutant strain (54.8%). With the copper chelator a less laccase activity in the IF of the mutant strain was observed (reduction of 53.9%). The results obtained suggest a different regulation of intracellular laccases in the mutant strain compared with the wild type in presence of CuSO4 and copper chelator which may be due to the mutation in the rho gene.

Comparative proteomic analyses of Hyphozyma roseonigra ATCC 20624 in response to sclareol

Abstract Sclareol is an important intermediate for ambroxide synthesis industries. Hyphozyma roseonigra ATCC 20624 was the only reported strain capable of degrading sclareol to the main product of sclareol glycol, which is the precursor of ambroxide. To date, knowledge is lacking about the effects of sclareol on cells and the proteins involved in sclareol metabolism. Comparative proteomic analyses were conducted on the strain H. roseonigra ATCC 20624 by using sclareol or glucose as the sole carbon source. A total of 79 up-regulated protein spots with a >2.0-fold difference in abundance on 2-D gels under sclareol stress conditions were collected for further identification. Seventy spots were successfully identified and finally integrated into 30 proteins. The up-regulated proteins under sclareol stress are involved in carbon metabolism; and nitrogen metabolism; and replication, transcription, and translation processes. Eighteen up-regulated spots were identified as aldehyde dehydrogenases, which indicating that aldehyde dehydrogenases might play an important role in sclareol metabolism. Overall, this study may lay the fundamentals for further cell engineering to improve sclareol glycol production.

Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism

Abstract Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669 bp) and pksT-2 (7901 bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase–acyltransferase domains through Agrobacterium -mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.

Preliminary studies of new strains of Trametes sp. from Argentina for laccase production ability

Abstract Oxidative enzymes secreted by white rot fungi can be applied in several technological processes within the paper industry, biofuel production and bioremediation. The discovery of native strains from the biodiverse Misiones (Argentina) forest can provide useful enzymes for biotechnological purposes. In this work, we evaluated the laccase and manganese peroxidase secretion abilities of four newly discovered strains of Trametes sp. that are native to Misiones. In addition, the copper response and optimal pH and temperature for laccase activity in culture supernatants were determined.The selected strains produced variable amounts of laccase and MnP; when Cu2+ was added, both enzymes were significantly increased. Zymograms showed that two isoenzymes were increased in all strains in the presence of Cu2+. Strain B showed the greatest response to Cu2+ addition, whereas strain A was more stable at the optimal temperature and pH. Strain A showed interesting potential for future biotechnological approaches due to the superior thermo-stability of its secreted enzymes.

Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan

Abstract This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.

Purified dextransucrase from Pediococcus pentosaceus CRAG3 as food additive.

The extracellular crude dextransucrase (0.67 U/mg) from P. pentosaceus CRAG3 (GenBank accession number JX679020) after PEG-1500 fractionation gave specific activity, 20.0 U/mg which by gel filtration resulted in 46.0 U/mg. The purified dextransucrase displayed molecular size of approximately, 224 kDa. The optimum assay conditions for dextransucrase activity were 5% sucrose in 20 mM sodium acetate buffer (pH 5.4) and 30 oC. The dextransucrase was stable up to 40 oC and at pH range of 5.4-7.0. The metal ions such as Co2+, Ca2+, Mg2+ and Zn2+ stimulated the dextransucrase activity by 56, 44, 14 and 12%, respectively. It was most stable at -20 oC with half-life of 307 days. Amongst various additives used, glycerol and Tween 80 provided significant stability to the enzyme with half-life 15.5 and 85.5 h, respectively as compared to control (6.9 h). The solidification of sucrose supplemented milk by purified dextransucrase due to dextran synthesis displayed its application as additive for improving the texture of dairy products.

Purification and characterization of a ribonuclease from the wild edible mushroom Armillaria luteo-virens.

A 15 kDa ribonuclease (RNase) was purified from dried fruiting bodies of the wild edible mushroom Armillaria luteo-virens. The simple 4-step purification protocol involved ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on SP-Sepharose and a final gel filtration by FPLC on Superdex-75. The RNase was unadsorbed on Affi-gel blue gel, but adsorbed on DEAE-cellulose and SP-Sepharose. The N-terminal amino acid sequence of purified RNase was AGVQYKLTILLV, which showed low sequence homology to those of previously reported RNases. The optimal pH and temperature of the enzyme were very close to 4.0 and 70°C, respectively. The enzyme showed considerably high ribonucleolytic activity and broad specificity towards polyhomoribonucleotides, with a specificity of poly(U)>poly(C)>poly (G)>poly(A). The ribonucleolytic activities towards poly(U), poly(C), poly(G) and poly(A) were 279.5, 184.1, 69.9 and 52.3 U/mg, respectively.

Purification and characterization of mycoferritin from Aspergillusflavus MTCC 873.

The fungus Aspergillus flavus MTCC 873, a non-toxigenic isolate demonstrated its capability to synthesize mycoferritin (MF) upon induction with iron in yeast extract sucrose (YES) medium. The molecular mass, yield, iron and carbohydrate contents of the MF were 440 kDa, 0.015 mg/g of wet mycelia, 0.8 and 30.4%, respectively. Native gel-electrophoresis revealed a band corresponding to dimeric form of equine spleen ferritin (ESF). Subunit analysis by SDS-PAGE revealed a single protein band with an apparent molecular mass of 24 kDa, suggesting similar sized subunits in the structure of apoferritin shell. Immunological cross-reactivity was observed with the anti-fish liver ferritin. Transmission electron microscopy (TEM) revealed an apparent particle size of 100 Å. N-terminal amino acid sequence of MF revealed a sequence of SLPLQDYA, which showed identities with other eukaryotic ferritin sequences. The spectral characteristics (UV/VIS, fluorescence and circular dichroic spectra) were similar to ESF. The fungus, unlike A. parasiticus 255 (non-toxigenic) was incapable of producing aflatoxins, when grown in YES media.

The effect of some osmolytes on the activity and stability of mushroom tyrosinase.

The thermodynamical stability and remained activity of mushroom tyrosinase (MT) from Agaricus bisporus in 10 mM phosphate buffer, pH 6.8, stored at two temperatures of 4 and 40 degrees C were investigated in the presence of three different amino acids (His, Phe and Asp) and also trehalose as osmolytes, for comparing with the results obtained in the absence of any additive. Kinetics of inactivation obey the first order law. Inactivation rate constant (kinact) value is the best parameter describing effect of osmolytes on kinetic stability of the enzyme. Trehalose and His have the smallest value of kinact (0.7x10(-4) s-1) in comparison with their absence (2.5x10(-4) s-1). Moreover, to obtain effect of these four osmolytes on thermodynamical stability of the enzyme, protein denaturation by dodecyl trimethylammonium bromide (DTAB) and thermal scanning was investigated. Sigmoidal denaturation curves were analysed according to the two states model of Pace theory to find the Gibbs free energy change of denaturation process in aqueous solution at room temperature, as a very good thermodynamic criterion indicating stability of the protein. Although His, Phe and Asp induced constriction of MT tertiary structure, its secondary structure had not any change and the result was a chemical and thermal stabilization of MT. The enzyme shows a proper coincidence of thermodynamic and structural changes with the presence of trehalose. Thus, among the four osmolytes, trehalose is an exceptional protein stabilizer.

Chemical properties and NMR spectroscopic identification of certain fungal siderophores.

Siderophores of six fungi viz. Aspergillus sp. ABp4, Aureobacidium pullulans, Penicillium oxalicum, P. chrysosporium, Mycotypha africana and Syncephalastrum racemosum were examined for their (1) electrophoretic mobilities to determine the acidic, basic or neutral charge; (2) Fe (III) binding nature viz., mono-, di-, or trihydroxamate; (3) amino acid composition; and (4) NMR (nuclear magnetic resonance) spectroscopy to determine their structure. Electrophoretic mobilities of siderophores of 3 fungi (P. oxalicum, P. chrysosporium, and M, africana) exhibited net basic charge, siderophores of 2 fungi (Aspergillus sp. ABp4 and S. racemosum) were acidic and 1 fungus (A. pullullans) was neutral. Electrophoresis of ferrated siderophore at pH 2 and colour of the spots indicated that siderophores of Aspergillus sp. ABp4 and P. oxalicum and A. pullulans were trihydroxamates, whereas siderophore of P. chrysosporium was dihydroxamate. Amino acid composition of siderophores purified by XAD-2 column chromatography, revealed the presence of asparagine, histidine, and proline in Aspergillus sp. ABp4, serine and alanine in P. chrysosporium, and valine in M. africana. The structure of purified siderophores as revealed by NMR spectroscopy identified siderophore of AB - 2670 (A. pullulans) as asperchrome F1, and AB-513 (M. africana) as rhizoferrin. The peak obtained for siderophore AB-5 (Aspergillus sp. ABp4) did not show resemblance to any known siderophore, therefore may be an exception.

The transcriptional activator GAL4-VP16 regulates the intra-molecular interactions of the TATA-binding protein.

Binding characteristics of yeast TATA-binding protein (yTBP) over five oligomers having different TATA variants and lacking a UASGAL, showed that TATA-binding protein (TBP)-TATA complex gets stabilized in the presence of the acidic activator GAL4-VP16. Activator also greatly suppressed the non-specific TBP-DNA complex formation. The effects were more pronounced over weaker TATA boxes. Activator also reduced the TBP dimer levels both in vitro and in vivo, suggesting the dimer may be a direct target of transcriptional activators. The transcriptional activator facilitated the dimer to monomer transition and activated monomers further to help TBP bind even the weaker TATA boxes stably. The overall stimulatory effect of the GAL4-VP16 on the TBP-TATA complex formation resembles the known effects of removal of the N-terminus of TBP on its activity, suggesting that the activator directly targets the N-terminus of TBP and facilitates its binding to the TATA box.

Mercury induced modifications in the stereochemistry of the active site through Cys-73 in a serine protease--crystal structure of the complex of a partially modified proteinase K with mercury at 1.8 A resolution.

Proteinese K (PK) isolated from Tritirachium album Limber was crystallized with HgCl2 in excess, under microgravity conditions. The intensity data were collected at 4 degrees C to 1.8 A resolution and the final R-factor after refinement for all the reflections was 0.164. Mercury has been found at two sites with partial occupancies (0.4 and 0.6) which are at distances of 2.48 A and 2.58 A respectively from Cys-73 Sgamma. The Cys-73 in the enzyme structure is located close to the active site residue, His-69. This region is completely buried and is not accessible to the solvent. It is rather tightly packed. Therefore, the binding of mercury distorts the stereochemistry of the neighbouring residues including those belonging to the catalytic triad. As a result of this, the Ogamma of Ser-224 is displaced by 0.6 A which causes the inactivation of proteinase K by increasing the H-bond distance to 3.7 A between Ser-224 Ogamma and His-69 Nepsilon2.

Modification of arginine residues at the substrate binding site of yeast glutathione reductase.

Yeast glutathione reductase (GR) was inactivated by phenylglyoxal (PG), which specifically modifies arginine residues of the enzyme. Inactivation followed psuedo-first order rate kinetics. There was no reversible complex formation prior to inactivation. Analysis of the kinetic data showed the order of reaction to be unity with respect to the modifier. Inactivation of GR was completely prevented by the presence of oxidised glutathione (GSSG), whereas NADP gave only partial protection. Stoichiometric studies showed that around four arginine residues per subunit were modified by PG in the absence of GSSG, whereas only one was modified in its presence. From these observations, it is concluded that essential arginine residues are present at the substrate binding site.

Isolation and characterization of a copper containing protein from blue cell walls of Neurospora crassa.

Culturing Neurospora crassa in presence of toxic amounts of copper (0.63 mM) resulted in blue coloured mycelia and cell walls. Significant amounts (approximately 45%) of total mycelial copper were associated with cell wall isolates under conditions of copper toxicity. Hence, such blue cell walls were analysed to identify specific ligands involved in copper binding. While decuprification of the blue cell walls with 8-hydroxy quinoline (8 HQ) did not alter their copper binding abilities, similar treatment with EDTA (10 mM) decreased such abilities indicating that EDTA treatment lead to loss of copper binding ligands from cell walls. Treatment of blue cell walls with 8 HQ followed by EDTA resulted in the solubilization of a copper binding protein (relative MW approximately 14 kDa) which was associated with phosphate and carbohydrate moieties. On amino acid analysis, this protein was found to be devoid of free thiol groupings but enriched in acidic and basic amino acids, distinguishing it from classical intracellular metal binding proteins such as metallo-thioneins and phytochelatins that are inducively synthesized under conditions of metal toxicity. The biological significance of the isolated wall-bound copper binding protein, which appears to be a normal constituent of cell walls, is discussed in relation to cytoplasmic metal binding proteins and mechanism(s) adapted by fungi in countering metal toxicity.

A hemolytic protein from cultured mycelia of mushroom, Termitomyces clypeatus.

A hemolytic protein was purified from cultured mycelia of T. clypeatus. Some of the physico-chemical properties of the hemolysin were studied. The protein was analysed to be a lipoprotein and delipidation removed its hemolytic property. The monomeric protein subunit of the lipoprotein had a molecular weight of 64,000. Mode of action of the hemolysin were studied by observing protections of sugar and lipid components to hemolysin mediated lysis of red blood cells. It was observed that the hemolysin possibly interacted with the phospholipid components of the blood cells causing lysis.

Características químicas y bioquímicas de la biomasa microbiana / Chemical and biochemical characteristics of the microbial biomass

La composición química y bioquímica de la biomasa microbiana determinará su potencial nutricional, el cual varía según las diferentes especies de microorganismos, la fuente de nutrientes, y la forma empleada para su crecimiento, así como también por efectos del proceso durante el propio crecimiento o posteriores durante la recuperación de la biomasa. Se detalla la comkposición química de los productos de biomasa microbiana producidos comercialmente o en instalaciones piloto demostrativas, clasificándolos según procedencia de algas, bacterias, levaduras u hongos. Finalmente, se reseña información sobre el efecto que algunos parámetros ejercen en la composición de la biomasa, que ilustra tendencias observadas experimentalmente, sin llegar a constituir una revisión exhaustiva de la literatura técnica