Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp.

In this study, three feather degrading bacterial strains were isolated from agroindustrial residues from a Brazilian poultry farm. Three Gram-positive, spore-forming, rod-shaped bacteria and were identified as B. subtilis 1271, B. licheniformis 1269 and B. cereus 1268 using biochemical, physiologic and molecular methods. These Bacillus spp. strains grew and produced keratinases and peptidases using chicken feather as the sole source of nitrogen and carbon. B. subtilis 1271 degraded feathers completely after 7 days at room temperature and produced the highest levels of keratinase (446 U ml^?1). Feather hydrolysis resulted in the production of serine, glycine, glutamic acid, valine and leucine as the major amino acids. Enzymography and zymography analyses demonstrated that enzymatic extracts from the Bacillus spp. effectively degraded keratin and gelatin substrates as well as, casein, hemoglobin and bovine serum albumin. Zymography showed that B. subtilis 1271 and B. licheniformis 1269 produced peptidases and keratinases in the 15?C140 kDa range, and B. cereus produced a keratinase of ~200 kDa using feathers as the carbon and nitrogen source in culture medium. All peptidases and keratinases observed were inhibited by the serine specific peptidase inhibitor phenylmethylsulfonyl fluoride (PMSF). The optimum assay conditions of temperature and pH for keratinase activity were 40?C50°C and pH 10.0 for all strains. For gelatinases the best temperature and pH ranges were 50?C70°C and pH 7.0?C11. These isolates have potential for the biodegradation of feather wastes and production of proteolytic enzymes using feather as a cheap and eco-friendly substrate.     

Production of cellulases by Thermomucor indicae-seudaticae: characterization of a thermophilic β-glucosidase

Abstract

The current study evaluated the production and characterization of β-glucosidase by the thermophilic fungus Thermomucor indicae-seudaticae in solid-state fermentation of wheat bran. Isolated fungi have significant amounts of β-glucosidase, an enzyme that may be applied to different industrial processes, such as the production of fuels, food, and other chemical compounds. Maximal enzyme activity occurred in pH 3.5–4.5 and at 70?°C. The enzyme exhibited high thermostability, for 1?h, up to 60?°C, and good tolerance to glucose (10?mM) and ethanol (10%). The optimization of fermentative parameters on the production of β-glucosidase was carried out by evaluating the best supplementary nutrient source, pH of nutrient solution, initial substrate moisture and fermentation temperature. The optimization of the above fermentation parameters increased enzyme activity by 120.0%. The highest enzymatic activity (164.0?U/g) occurred with wheat bran containing 70% initial moisture, supplemented with 1.0% (NH₄)₂SO₄ solution at pH 5.5–6.0 and fungus incubated at 40?°C. A more detailed study of β-glucosidase suggested that Sulfur is an important component of the main amino acid present in this enzyme. The enhancer of the enzyme activity occurred when the fungus was grown on wheat bran supplemented with a sulfur-containing solution. In fact, increasing the concentration of sulfur in the solution increased its activity.     

Genomic and exoproteomic analyses of cold‐ and alkaline‐adapted bacteria reveal an abundance of secreted subtilisin‐like proteases

Proteases active at low temperature or high pH are used in many commercial applications, including the detergent, food and feed industries, and bacteria specifically adapted to these conditions are a potential source of novel proteases. Environments combining these two extremes are very rare, but offer the promise of proteases ideally suited to work at both high pH and low temperature. In this report, bacteria from two cold and alkaline environments, the ikaite columns in Greenland and alkaline ponds in the McMurdo Dry Valley region, Antarctica, were screened for extracellular protease activity. Two isolates, Arsukibacterium ikkense from Greenland and a related strain, Arsukibacterium sp. MJ3, from Antarctica, were further characterized with respect to protease production. Genome sequencing identified a range of potential extracellular proteases including a number of putative secreted subtilisins. An extensive liquid chromatography–tandem mass spectrometry analysis of proteins secreted by A. ikkense identified six subtilisin‐like proteases as abundant components of the exoproteome in addition to other peptidases potentially involved in complete degradation of extracellular protein. Screening of Arsukibacterium genome libraries in Escherichia coli identified two orthologous secreted subtilisins active at pH 10 and 20°C, which were also present in the A. ikkense exoproteome. Recombinant production of both proteases confirmed the observed activity.     

A novel high molecular weight thermo-acidoactive β-glucosidase from <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

An extracellular high molecular weight β-glucosidase was secreted by a local strain P1 of Beauveria bassiana. The enzyme was produced in the presence of various carbon sources, namely glucose, maltose, lactose, glycerol, starch, wheat bran and gruel. The highest level of β-glucosidase activity was produced with wheat bran at the concentration of 3%. Glucose caused a repressor effect on the β-glucosidase expression in a dose-dependent manner. The highest enzyme production level was obtained at initial pH of 6.0 and 7.0 in the culture medium. The zymography analysis revealed that B. bassiana secreted a β-glucosidase with high molecular weight between 400 and 600 kDa. The enzymatic preparation was characterized and showed temperature and pH optima of 55°C and 5.0, respectively. The enzyme was stable at 40 and 50°C but its stability declined at 60°C. Interestingly, this β-glucosidase had high stability at acid and basic pH saving its initial activity after 24 h incubation at pH from 3.0 to 11.0. It was stable also in presence of monovalent Na⁺ and K⁺ ions saving 60% of its initial activity at 2 M salts. Bivalent metal ions preserved totally or partially the enzymatic activity; in addition, Ba²⁺ was revealed as an activator. This is the first report that focuses on the production and the biochemical characterization of a β-glucosidase from the entomopathogenic fungus, B. bassiana.     

Novel cellulases from an extremophilic filamentous fungi <Emphasis Type="Italic">Penicillium citrinum</Emphasis>: production and characterization

The enzymatic hydrolysis of cellulose has potential economical and environment-friendly applications. Therefore, discovery of new extremophilic cellulases is essential to meet the requirements of industry. Penicillium citrinum (MTCC 6489) that was previously isolated from soil in our laboratory, produced alkali tolerant and thermostable cellulases. Endoglucanase and filter paper activity hydrolase (FPAse) production of P. citrinum were studied using wheat bran substrate in solid state and submerged culture. Zymogram analysis of endoglucanase revealed the presence of two isoforms differing in molecular weight. One of them was 90 kDa and other one was 38 kDa. Partially purified endoglucanase showed two different peaks at pH 5.5 and 8.0, respectively, in its pH optima curve. But FPase showed only one peak (at pH 6.5) in its pH optima curve. Cellulase of P. citrinum is thermostable in nature. The present work reports for the first time, the alkali stable cellulase from alkali tolerant fungus Penicillium citrinum. Thermostable endoglucanase from P. citrinum may have potential effectiveness as additives to laundry detergents.     

Signal peptide of <Emphasis Type="Italic">Aureobasidium</Emphasis><Emphasis Type="Italic">pullulans</Emphasis> xylanase: use for extracellular production of a fungal xylanase by <Emphasis Type="Italic">Escherichia coli</Emphasis>

An extracellular xylanase XynI of glycoside hydrolase family 11 from the dimorphic fungus Aureobasidium pullulans ATCC 20524 possesses an N-terminal extension of 34 amino acids (Ohta et al., J. Biosci. Bioeng. 92:262–270, 2001). The N-terminal extension includes three sites (Ala-X-Ala-X-Ala-X-Ala) that are potentially cleavable by signal peptidase I of Escherichia coli. The A. pullulans xynI signal sequence was fused in frame to the mature protein region of the equivalent xylanase gene xynA from the filamentous fungus Penicillium citrinum. The gene fusion xynI::A was inserted into the plasmid pET-26b(+) to yield pEXP401. An E. coli BL21(DE3) transformant harboring the pEXP401 exhibited xylanase activity (per ml of the culture) of 16.8 U in the fraction of culture supernatant as well as 4.29 U in the fraction of cell-free extract after 12 h of growth with isopropyl-β-d-thiogalactopyranoside at 30°C. N-terminal amino acid sequence analysis of the secreted recombinant proteins revealed cleavage at four distinct sites within the N-terminal extension of XynI, two of which conformed to the Ala-X-Ala motif prior to the cleavage site. The XynA proteins secreted into the culture medium showed high specific activities from 506 to 651 U/mg, which were twofold higher than that of the native enzyme.     

Optimization of Culture Medium for Sporulation and Biomass Production of a Nematophagous Fungus: Consideration of Nutritional and Environmental Conditions

This study reports the effects of various nutritional and environmental factors on sporulation and biomass of Paecilomyces lilacinus IPC‐P. These factors included carbon and nitrogen sources, carbon‐to‐nitrogen ratios, mineral elements and vitamins together with water potentials, temperatures, dark/light cycles and pH. On the basis of these results, together with a ‘two‐step’ cultivation and orthogonal method, the culture conditions for sporulation of this fungus were optimized. The spore suspension was inoculated on a basal medium (sucrose 19.00 g/l, soy peptone 4.06 g/l, K₂HPO₄ 1.00 g/l, KCl 0.50 g/l, MgSO₄ 0.50 g/l, FeSO₄ 0.01 g/l, agar 13.00 g/l) for 4 days, before being transferred to a sporulation medium (dextrin 2.27 g/l, urea 2.13 g/l, CaCl₂ 3.00 g/l, ZnSO₄·7H₂O 0.01 g/l, agar 13.00 g/l) for a further 4 days under the following environmental conditions: ?3.9 MPa/pH 7/light 24 h/temperature 29°C; these conditions were altered to ?0.3 MPa/pH 6/light 24 h/temperature 23°C in order to obtain better biomass yields. The data presented provide information on the nutrient and environmental requirements of this fungus, which will be essential for its commercial production.     

Proteins and Peptidases from Conidia and Mycelia of <Emphasis Type="Italic">Scedosporium apiospermum</Emphasis> Strain HLPB

The conidia–mycelia transformation is an essential step during the life cycle of the fungal human pathogens of the Pseudallescheria boydii complex. In the present study, we have analyzed the protein and peptidase profiles in two distinct morphological stages, conidia and mycelia, of Scedosporium apiospermum sensu stricto. Proteins synthesized by the mycelia, migrating at the ranges of 62–48 and 22–18 kDa, were not detected from the conidial extract. Conidia produced a single cellular peptidase of 28 kDa able to digest copolymerized albumin, while mycelia yielded 6 distinct peptidases ranging from 90 to 28 kDa. All proteolytic enzymes were active at acidic pH and fully inhibited by 1,10-phenanthroline, characterizing these activities as metallo-type peptidases. Quantitative peptidase assay, using soluble albumin, showed a high metallopeptidase production in mycelial cells in comparison with conidia. The regulated expression of proteins and peptidases in different morphological stages of S. apiospermum represents a potential target for isolation of stage-specific markers for biochemical and immunological analysis. Martha Machado Pereira and Bianca Alcantara Silva contributed equally to this work.     

Catalytic and thermodynamic properties of β-glucosidases produced by Lichtheimia corymbifera and Byssochlamys spectabilis

Abstract

The objective of the present study was to optimize parameters for the cultivation of Lichtheimia corymbifera (mesophilic) and Byssochlamys spectabilis (thermophilic) for the production of β-glucosidases and to compare the catalytic and thermodynamic properties of the partially purified enzymes. The maximum amount of β-glucosidase produced by L. corymbifera was 39?U/g dry substrate (or 3.9?U/mL), and that by B. spectabilis was 77?U/g (or 7.7?U/mL). The optimum pH and temperature were 4.5 and 55?°C and 4.0 and 50?°C for the enzyme from L. corymbifera and B. spectabilis, respectively. β-Glucosidase produced by L. corymbifera was stable at pH 4.0–7.5, whereas the enzyme from B. spectabilis was stable at pH 4.0–6.0. Regarding the thermostability, β-glucosidase produced by B. spectabilis remained stable for 1?h at 50?°C, and that from L. corymbifera was active for 1?h at 45?°C. Determination of thermodynamic parameters confirmed the greater thermostability of the enzyme produced by the thermophilic fungus B. spectabilis, which showed higher values of ΔH, activation energy for denaturation (E_a), and half-life t_(1/2). The enzymes were stable in the presence of ethanol and were competitively inhibited by glucose. These characteristics contribute to their use in the simultaneous saccharification and fermentation of vegetable biomass.     

Statistical Optimization of Alkaline Protease Production from Penicillium citrinum YL-1 Under Solid-State Fermentation

Proteases from halotolerant and halophilic microorganisms were found in traditional Chinese fish sauce. In this study, 30 fungi were isolated from fermented fish sauce in five growth media based on their morphology. However, only one strain, YL-1, which was identified as Penicillium citrinum by internal transcribed spacer (ITS) sequence analysis, can produce alkaline protease. This study is the first to report that a protease-producing fungus strain was isolated and identified in traditional Chinese fish sauce. Furthermore, the culture conditions of alkaline protease production by P. citrinum YL-1 in solid-state fermentation were optimized by response surface methodology. First, three variables including peptone, initial pH, and moisture content were selected by Plackett–Burman design as the significant variables for alkaline protease production. The Box–Behnken design was then adopted to further investigate the interaction effects between the three variables on alkaline protease production and determine the optimal values of the variables. The maximal production (94.30 U/mL) of alkaline protease by P. citrinum YL-1 took place under the optimal conditions of peptone, initial pH, and moisture content (v/w) of 35.5 g/L, 7.73, and 136%, respectively.     

Collagenolytic activity in several species of deuteromycetes under various storage conditions

The ability of deuteromycetes of the genera Penicillium, Aspergillus, and Botrytis to retain collagenolytic activity was studied after both 2 and 10 years of storage on a Czapek medium under a layer of mineral oil at 4°C, as well as in silica gel granules at 20 and ?60°C. The enzymatic activity of several species, including Botrytis terrestris, Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum, was retained under both conditions of storage. Aspergillus repens retained enzymatic activity only if stored under a layer of mineral oil. The viability of conidia and the collagenolytic activity of Botrytis terrestris, P. janthinellum, P. chrysogenum, and Penicillium citrinum, maintained on silica gel for 10 years, depended on the storage temperature. The viability of the test strains improved after storage on a silica gel at ?60°C. A strain of Aspergillus repens lost its ability to dissolve collagen at various storage tempeatures on the silica gel. The index of lysis for three strains of Penicillium deuteromycetes (Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum) increased after a 10-year storage on silica gel at ?60°C.     

Cysteine peptidases in the tomato trypanosomatid Phytomonas serpens: influence of growth conditions, similarities with cruzipain and secretion to the extracellular environment

We have characterized the cysteine peptidase production by Phytomonas serpens, a tomato trypanosomatid. The parasites were cultivated in four distinct media, since growth conditions could modulate the synthesis of bioactive molecules. The proteolytic profile has not changed qualitatively regardless the media, showing two peptidases of 38 and 40 kDa; however, few quantitative changes were observed including a drastic reduction (around 70%) on the 40 and 38 kDa peptidase activities when parasites were grown in yeast extract and liver infusion trypticase medium, respectively, in comparison with parasites cultured in Warren medium. The time-span of growth did not significantly alter the protein and peptidase expression. The proteolytic activities were blocked by classical cysteine peptidase inhibitors (E-64, leupeptin, and cystatin), being more active at pH 5.0 and showing complete dependence to reducing agents (dithiothreitol and l-cysteine) for full activity. The cysteine peptidases were able to hydrolyze several proteinaceous substrates, including salivary gland proteins from Oncopeltus fasciatus, suggesting broad substrate utilization. By means of agglutination, fluorescence microscopy, flow cytometry and Western blotting analyses we showed that both cysteine peptidases produced by P. serpens share common epitopes with cruzipain, the major cysteine peptidase of Trypanosoma cruzi. Moreover, our data suggest that the 40 kDa cysteine peptidase was located at the P. serpens cell surface, attached to membrane domains via a glycosylphosphatidylinositol anchor. The 40 kDa peptidase was also detected in the cell-free culture supernatant, in an active form, which suggests secretion of this peptidase to the extracellular environment.     

Optimization of physico‐chemical and nutritional parameters for a novel pullulan‐producing fungus,Eurotium chevalieri

Aims: To isolate the novel nonmelanin pullulan‐producing fungi from soil and to optimize the physico‐chemical and nutritional parameters for pullulan production. Methods and Results: A selective enrichment method was followed for the isolation, along with development of a suitable medium for pullulan production, using shake flask experiments. Pullulan content was confirmed using pure pullulan and pullulanase hydrolysate. Eurotium chevalieri was able to produce maximum pullulan (38 ± 1·0 g l^?1) at 35°C, pH 5·5, 2·5% sucrose, 0·3% ammonium sulfate and 0·2% yeast extract in a shake flash culture medium with an agitation rate of 30 rev min^?1 for 65 h. Conclusions: The novel pullulan‐producing fungus was identified as E. chevalieri (MTCC no. 9614), which was able to produce nonmelanin pullulan at from poorer carbon and nitrogen sources than Aureobasidium pullulans and may therefore be useful for the commercial production of pullulan. Significance and Impact of the Study: Eurotium chevalieri could produce pullulan in similar amounts to A. pullulans. Therefore, in future, this fungus could also be used for commercial pullulan production, because it is neither polymorphic nor melanin producing, hence its handling during pullulan fermentation will be easier and more economical.     

Secreted protease of the entomopathogenic fungus <Emphasis Type="Italic">Cordyceps militaris</Emphasis>. II. Enzyme properties and adsorption on the insect cuticle

Electrophoretically pure preparation of a secreted subtilisin-like protease was isolated from the culture liquid of an entomopathogenic fungus C. militaris strain KP-97-2. The enzyme had an optimum activity at 45°C and pH 7–9. Data were collected on the substrate specificity of the enzyme and its stability at different values of pH and temperature. Adsorption and desorption of the extracellular protease on the cuticle of Nauphoeta cinerea was studied in function of pH.     

Purification and characterization of x-prolyl-dipeptidyl aminopeptidase from Lactococcus lactis subsp. cremoris NRRL 634

Summary An X-prolyl-dipeptidylaminopep tidase (Pep-XP) was purified from the crude intracellular extract of Lactococcus lactis subsp. cremoris NRRL 634 by ion exchange and gel filtration chromatographies. The enzyme was purified 80-fold with a recovery of 6%, and appeared as a single band with a molecular weight of about 80 kDa on polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS-PAGE). The peptidase showed its maximal activity on arginyl-proline-p-nitroanilide at pH 7.0 and at a temperature of 45 °C, although there was a good activity of Pep-XP in the pH range of 5.5–7.0 and temperatures between 40 and 50 °C. The Michaelis constant (K _m) and the maximum reaction velocity (V _max) values were 0.92 mM and 7.9 U/mg protein min, respectively. The activity of Pep-XP was completely inhibited by phenylmethanesulphonyl fluoride, an inhibitor of serine peptidases, and metal chelators had little effect on enzyme activity. The purified enzyme hydrolyzed synthetic substrates whose structure is X-Pro-Y like Lys-Pro-pNA, but did not hydrolyse Pro-pNA or azocasein, showing that the enzyme did not have aminopeptidase or endopeptidase activities.     

Production of Conidia by Botrytis fabae grown in vitro

Conidiation in Botrytis fabae was stimulated by irradiating 1 to 3 day old, but not 4 to 5 day old mycelium. Three cycles of 12 h irradiation + 12 h darkness stimulated the production of about twice as many spores compared with only 12 h irradiation. At 18°C all the spores had been produced within 3 days but not within 2 days from the start of irradiation. Near-u.v. irradiation at wavelengths of 375–400 nm induced most sporulation. Red light at 600–650 nm also stimulated conidiation but irradiation at other wavelengths from 300 to 700 nm was ineffective. Fewer conidia were produced when the fungus was kept in darkness at 4°C between periods of irradiaton at 18°C compared with continuous 18°C. The optimum osmotic potential of the culture, medium for conidiation was about-27 bar although more mycelium grew at even lower osmotic potentials. Abundant spore production occurred when the fungus was grown in media with a wide range of pH values.     

Novel thermoactive glucoamylases from the thermoacidophilic Archaea Thermoplasma acidophilum,Picrophilus torridus and Picrophilus oshimae

The thermoacidophilic Archaea Thermoplasma acidophilum (optimal growth at 60 °C and pH 1–2), Picrophilus torridus and Picrophilus oshimae (optimal growth at 60 °C and pH 0.7) were able to utilize starch as sole carbon source. During growth these microorganisms secreted heat and acid-stable glucoamylases into the culture fluid. Applying SDS gel electrophoresis activity bands were detected with appearent molecular mass (Mw) of 141.0, 95.0 kDa for T. acidophilum, 133.0, 90.0 kDa for P. torridus and 140.0, 85.0 kDa for P. oshimae. The purified enzymes were incubated with various polymeric substrates such as starch, pullulan, panose and isomaltose. The product pattern, analyzed by HPLC, showed that in all cases glucose was formed as the sole product of hydrolysis. The purified glucoamylases were optimally active at pH 2.0 and 90 °C and have an isoelectric points (pI) between 4.5 and 4.8. Enzymatic activity was detected even at pH 1.0 and 100 °C. The glucoamylases were thermostable at elevated temperature with a half-life of 24 h at 90 °C for both P. torridus and T. acidophilum, and 20 h at 90 °C for P. oshimae. The enzyme system of T. acidophilum has a lower K _m value for soluble starch (1.06 mg/ml) than the enzymes from P. oshimae and P. torridus (4.35 mg/ml and 2.5 mg/ml), respectively. Enzyme activity was not affected by Na⁺, Mg⁺⁺, Ca⁺⁺, Ni⁺⁺, Zn⁺⁺, Fe⁺⁺, EDTA and DTT. This revised version was published online in August 2006 with corrections to the Cover Date.     

The characterization of a pectin-degrading enzyme from Aspergillus oryzae grown on passion fruit peel as the carbon source and the evaluation of its potential for industrial applications

An extracellular pectinase (PEC-I) was isolated from the crude extract of Aspergillus oryzae when grown on passion fruit peel (PFP) as the carbon source and partially purified by ultra filtration, gel filtration and ion-exchange chromatography procedures. Pectinase activity was predominantly found in the retentate. The pectinase from retentate (PEC-Ret) was most active at 50?°C and pH 7.0 and stable at 50?°C with a half-life of approximately 8?h. PEC-I showed higher activity at pH 4.5 and 55?°C, 70?°C and 75?°C and was inhibited by cations (Ag⁺, Fe²⁺, Fe³⁺, Co²⁺, Ca²⁺ and Hg²⁺), EDTA, tannic acid and vanillin. On the other hand, PEC-I was activated by Cu²⁺, ferulic acid, cinnamic acid and 4-hydroxybenzoic acid. The gel under denaturing conditions of PEC-Ret and PEC-I samples showed a protein band of ～45?kDa coincident with that found by staining for pectinase activity. In the bioscouring of cotton fabric the PEC-Ret pectinase preparation led to a better wettability and removed more pectin from the cotton fibers than the commercial enzyme preparation Viscozyme L, but was less effective than a commercial alkaline pectate lyase preparation and alkaline scouring. The incubation of PEC-Ret with guava juice resulted in a 4.15% decrease in juice viscosity.     

Optimisation of medium and culture conditions for the production of antifungal substances to Colletotrichum musae by Trametes elegans SR06

An endophytic fungus SR06 was isolated from a leaf of Amomum villosum Lour., which had a high antagonistic effect on Colletotrichum musae with an inhibition ratio of 41.20%. The antifungal substances could be secreted into fermentation broth, which had a high inhibitory activity. Strain SR06 was identified as Trametes elegans according to internal transcribed spacer sequence analysis. Response surface methodology (RSM) was used to optimise the process parameters of antifungal substances production. Using the Plackett–Burman design, three variables (glucose, yeast extract and MgSO₄·7H₂O) exerted significant effects on antifungal substances production. Then RSM experiments were conducted to further optimise the three variables. The optimal medium components were 26.45?g/L glucose, 10?g/L peptone, 14.96?g/L yeast extract and 1.49?g/L MgSO₄·7H₂O, and the optimal initial pH was 6.0, with a culture temperature of 28°C and a shaking speed of 180?rpm. Under the optimised conditions, a significant improvement in the production of antifungal substances by T. elegans SR06 was accomplished, and the inhibition zone diameter was up to 29.2?mm after culturing for 7d. The average control efficacy of the fermentation supernatant of SR06 against C. musae was 51.29% on banana fruits, which was significantly higher than that of the fungicide carbendazim.