Production of thermophilic endo-ß-1,4-xylanases by Aspergillus fumigatus FBSPE-05 using agro-industrial by-products.

In the present paper, endo-ß-1,4-xylanase production by Aspergillus fumigatus was evaluated in solid-state fermentation using low-cost substrates such as sugarcane bagasse (SCB), brewer's spent grain (BSG), and wheat bran (WB). The partial characterization of the crude enzyme was also performed. In the experimental conditions, the highest levels of endo-ß-1,4-xylanase production by A. fumigatus FBSPE-05 occurred within 8 days incubation when using SCB/liquid medium at 1:2 ratio (219.5 U g(-1)) and 4 days incubation when using WB/liquid medium at 1:1 ratio (215.6 U g(-1)). Crude enzyme from this last condition was used to enzyme characterization, showing best enzyme activity at 60 °C and pH 6.0, which suggests a thermophilic endoxylanase. The crude enzyme retained 73% of its activity after 1 h at 60 °C, and zymogram has shown three bands of endo-ß-1,4-xylanase activity, with different molecular masses. A. fumigatus FBSPE-05 was able to grow and produce good levels of endo-ß-1,4-xylanase using agro-industrial by-products, making this strain worthy for further investigation. To our knowledge, this is the first study reporting the use of SCB and/or BSG as sole substrates for endoxylanase production by solid-state fermentation using A. fumigatus.

Brewer's spent grain and corn steep liquor as substrates for cellulolytic enzymes production by Streptomyces malaysiensis.

AIMS: To evaluate cellulase production by Streptomyces malaysiensis in submerged fermentation using brewer's spent grain (BSG) and wheat bran (WB) as carbon source, and corn steep liquor (CSL) as nitrogen source, as compared to yeast extract (YE), and partial characterization of the crude enzyme. METHODS AND RESULTS: Maximum cellulase production by Streptomyces malaysiensis (720 U l(-1)) occurred within 4 days incubation when using a growth medium containing BSG 0.5% (w/v) and CSL1.2% (w/v). CMCases activity showed to be stable over an acidic pH range (2.0-7.0) and in temperatures of 40-60 degrees C. Zymogram indicated three bands of CMCase activity, with different molecular masses. CONCLUSION: S. malaysiensis was able to grow and produce good levels of CMCases using solely brewer's spent grain and corn steep liquor as low-cost substrates, making this strain and these low cost by-product worthy for further investigation, and potentially feasible for biotechnological applications in different areas. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first study reporting the use of the low-cost by-products brewer's spent grain and corn steep liquor, as sole substrates for microbial enzyme production.

Aspergillus fumigatus thermophilic and acidophilic endoglucanases.

This study evaluated the production of cellulolytic enzymes by an Aspergillus fumigatus strain, isolated from sugar cane bagasse, according to its ability to grow on microcrystalline cellulose as the sole carbon source. The effect of the carbon source (brewer's spent grain, sugarcane bagasse, and wheat bran) and of the nitrogen source (corn steep liquor and sodium nitrate) on cellulase production was studied using submerged and solid state cultivations at 30 degrees C. The highest levels of endoglucanase (CMCase) corresponded to 365 U L(-1) and was obtained using sugarcane bagasse (1%) and corn steep liquor (1.2%) in submerged fermentation within 6 days of cultivation. This supernatant was used to run a sodium dodecyl sulfate polyacrylamide gel electrophoresis that showed six bands with endoglucanase activity. CMCase activity was higher at 65 degrees C and pH 2.0, indicating that this microorganism produces a thermophilic and acid endoglucanase. Solid state cultivation favored FPase production, that reached 47 U g(-1) of dry substrate (wheat bran and sugarcane bagasse) within 3 days.

Nitrogen regulation of Saccharomyces cerevisiae invertase. Role of the URE2 gene.

The regulation of extracellular enzymes is of great biotechnological interest. We studied the regulatory role of the URE2 gene on the periplasmic invertase of Saccharomyces cerevisiae, because its periplasmic asparaginase is regulated by the URE2/GLN3 system. Enzymatic activity was measured in the isogenic strains P40-1B, the ure2 mutant P40-3C, and the P40-3C strain transformed with the pIC-CS plasmid carrying the URE2 gene. The assays were performed using midlog and stationary phase cells and nitrogen-starved cells from these growth phases. During exponential growth, the level of invertase in both wild-type and ure2 mutant cells was comparable. However, the invertase activity in ure2 mutant cells from stationary phase was sixfold lower than in the wild-type cells. When P40-3C cells were transformed with the pIC-CS plasmid, the wild-type phenotype was restored. On nitrogen starvation in the presence of sucrose, the invertase activity in wild-type cells from midlog phase decreased three times, whereas in stationary cells, the activity decreased eight times. However, invertase activity doubled in ure2 mutant cells from both phases. When these cells were transformed with the aforementioned plasmid, the wild-type phenotype was restored, although a significant invertase decrease in stationary cell was not observed. These results suggested that the URE2 protein plays a role in invertase activity.

N-demethylation of methylene blue by lignin peroxidase from Phanerochaete chrysosporium. Stoichiometric relation for H2O2 consumption.

Phanerochaete chrysosporium lignin peroxidase (LiP) can degrade synthetic dyes such as heterocyclic, azo, and triphenylmethane on its activation by H2O2. Analysis of the reaction products indicated that N-demethylation reactions are involved in the degradation of crystal violet and methylene blue (MB). We studied LiP oxidation of methylene blue and azure B (AB) in reaction mixtures containing different dye:H2O2 stoichiometric relations aiming at the selective formation of N-demethylated derivatives. High yields, about 70%, of the mono- and didemethylated derivatives, azure B and azure A, were obtained with the use of 1:1 and 1:2 MB:H2O2, respectively. Using azure B as substrate in reaction mixtures containing 1:1 AB:H2O2, a yield of 70% was also observed in azure A. Reaction mixtures containing 1:3 MB:H2O2 and 1:2 AB:H2O2, originated several oxidation products in similar proportions. These results indicated that the process of enzymatic degradation of methylene blue and azure B initiates via N(CH3)2 oxidation. According to the yields that were obtained for azure B and azure A, this enzymatic route can be used for the synthesis of these dyes since these data compare favorably to the chemical route that has a yield of 35%. The use of a dye:H2O2 relation of 1:10 resulted in a decoloration level of about 85%, showing the usefulness of this procedure for wastewater treatment. The reaction products were followed by spectrophotometric analysis within the wavelength of 500-700 nm. The product identifications were performed using a reverse-phase high-performance liquid chromatography (HPLC) C-18 column and thin-layer chromatography.

Endocellulase and exocellulase activities of two Streptomyces strains isolated from a forest soil.

Two Streptomyces strains, M7a and M23, from a Brazilian forest soil were evaluated for the cellulase production of their supernatants after growth in a microcrystalline cellulose medium, using carboxymethylcellulose and filter paper as substrates at different temperatures and pH values. Endoglucanase and exoglucanase activities were compared to a commercial Trichoderma reesei cellulase using fluorogenic conjugated substrates. Similar specific activities were observed for the enzyme preparations of strain M23 and T. reesei. For M7a the activities were about seven times higher than those obtained for T. reesei. Extracellular or cell-associated cellobiase activities were not detected in both strains.

Brazilian bioethanol program.

Brazil is the largest producer of bioethanol, and sugarcane is the main raw material. Bioethanol is produced by both batch and continuous processes, and in some cases, flocculating yeast is used. This article analyzes the Brazilian Ethanol Program. For the 1996-1997 harvest, Brazil produced 14.16 billion L of ethanol and 13.8 million metric t of sugar, from 286 million metric t of sugarcane. These products were produced by 328 industries in activity, with 101 autonomous ethanol plants producing only ethanol, and 227 sugar mills producing sugar and ethanol. The sugar-ethanol market reaches about 7.5 billion US$/yr, accounting for direct and indirect revenues.

L-asparaginase II of saccharomyces cerevisiae. Activity profile during growth using an ure2 mutant P40-3C and a P40-3C + URE2p strain.

The activity profile of the periplasmic asparaginase of Saccharomyces cerevisiae was determined during cell growth in an ure2 mutant; in an ure2 transformed with a plasmid containing the gene URE2 and, for comparison, in the strain D273-10B. Cells were cultivated in media presenting variable quantitative and qualitative nitrogen availability and the enzyme activity was evaluated in fresh and in nitrogen-starved cells. Nitrogen affected the asparaginase II level in fresh and starved cells of all strains. In the best condition, enzyme was produced by the wild-type cells at the late log-phase in the glucose/ammonium medium with a carbon to nitrogen ratio 4.3:1. Upon starvation, the activity doubled. The overall profile of the transformed strain was similar to that of the wild-type strain. In the ure2 mutant, high-enzyme levels were observed during growth, as expected. However the activity level, upon starvation, in proline grown cells, increased sixfold, suggesting that in addition to the Ure2p-Gln3p system, another system regulates asparaginase II biosynthesis.

Lignin peroxidase production by Streptomyces viridosporus T7A: nitrogen nutrition optimization using glucose as carbon source.

The production of lignin peroxidase by Streptomyces viridosporus T7A was studied in submerged batch fermentations using growth media containing 6.5 g/L yeast extract and 2.5-10.0 g/L glucose, corresponding to carbon to nitrogen (C/N) ratios from 7.1-12.4. The kinetics for biomass and enzyme accumulation and glucose consumption were followed allowing definition of optimized conditions for enzyme production. Considering the physiological response of the microorganism in relation to enzyme production, a sharp increase on enzyme activity was consistently observed upon glucose depletion, indicating glucose regulation. In accordance to that the plot of maximal enzyme vs maximal enzyme per gram of glucose consumption showed a linear inversely proportional relationship, indicating that the characteristics of the metabolic pool at the studied C/N ratios affected enzyme biosynthesis even after glucose depletion.

Lignin peroxidase and protease production by Streptomyces viridosporus T7A in the presence of calcium carbonate. Nutritional and regulatory carbon sources.

Streptomyces are good producers of enzymes of industrial interest, such as lignin peroxidase (LiP) and proteases. To optimize production of these enzymes by Streptomyces viridosporus T7A, two parameters were evaluated: carbon sources and calcium carbonate. Shake-flask fermentations were performed using culture media, with and without CaCO3, contained yeast extract, mineral salts and either glucose, lactose, galactose, or corn oil. In the absence of calcium carbonate, the maximum values for LiP and protease activities occurred during the idiophase with LiP activity being favored by glucose, corn oil, and galactose, and protease activity being favored only by corn oil. Calcium carbonate affected the cell morphology by reducing the size of the pellets. Moreover, in the presence of the salt, LiP production was growth-associated in all media but the glucose medium. Higher enzyme levels were observed when galactose and glucose were used as carbon sources. Protease activity was repressed by both glucose and galactose, whereas corn oil was the best carbon source for the enzyme production. Calcium carbonate increased LiP production by up to 2.6-fold. Such improvement was not observed for protease production, suggesting a selective effect of CaCO3 on LiP activity.

Lignin peroxidase production by Streptomyces viridosporus T7A: use of corn oil as a carbon source.

Lignin peroxidase (LiP) production cost should be reduced to justify its use in the control of environmental pollution. In this work, we studied the enzyme production by Streptomyces viridosporus T7A using glucose or corn oil as a carbon source having 0.65% yeast extract as a nitrogen source. Enzyme activity, observed using either 0.65% glucose or corn oil at 0.1, 0.5, and 1.0% concentration, was 300, 150, 300, and 200 U/L, respectively. Although higher enzyme activity was obtained in both media containing 0.65% glucose and 0.5% corn oil, the use of corn oil resulted in a better LiP stability. When combined carbon sources were used, higher values of enzyme activity (360, 350, and 225 U/L) were observed in media with 0.65% glucose and supplemented with 0.1, 0.5, and 1.0% corn oil, respectively. Although the presence of both glucose and 0.5% corn oil is favorable for LiP production, satisfactory results in terms of enzyme production and stability could be also observed using 0.5% corn oil as a sole carbon source, which may lead to reduced production costs of the LiP enzyme.

Extracellular proteolytic processing of Aspergillus awamori GAI into GAII is supported by physico-chemical evidence.

The proportion of glucoamylases, GAI and GAII, in the culture supernatant of Aspergillus awamori fermentations depends on the medium C/N ratio in such a way that the transformation of GAI into GAII is favored by the existence of a surplus of the carbon source in the growth medium. This condition also favors the appearance of the proteolytic activity. The authors report the observation that the shift in the isoenzyme proportion was concomitant to the peak of proteolytic activity. A peptide that may have resulted from the continuous degradation of the GAI C-terminal peptide, Gp-1, was isolated by gel filtration and purified by reverse-phase chromatography. This peptide matched with the region G14-A34 of the substrate-binding domain of GAI, thus reinforcing the hypothesis of the extracellular proteolytic processing of GAI.

Production and characterization of Phanerochaete chrysosporium lignin peroxidases for pulp bleaching.

The production of lignin peroxidase from Phanerochaete chrysosporium was studied using immobilized mycelia in nylon-web cubes in semicontinuous fermentation using glucose pulses or ammonium tartrate pulses. Consistent enzyme production was achieved when glucose pulses were used, leading to an average activity of 253 U/L. The crude enzyme was added to eucalyptus kraft pulp before conventional and ECF bleaching sequences. Optimization of the enzymatic pretreatment led to the following operational conditions: enzyme load of 2 U/g of pulp, hydrogen peroxide addition rate of 10 ppm/h, and reaction time of 60 min. Pulp final characteristics were dependent on the chemical treatment sequence that followed enzymatic pretreatment. The chief advantage of enzymatic pretreatment was pulp viscosity preservation, which was observed in most of the experiments carried out with seven different chemical treatment sequences.

Asparaginase II of Saccharomyces cerevisiae. GLN3/URE2 regulation of a periplasmic enzyme.

The production of some extracellular enzymes is known to be negatively affected by readily metabolized nitrogen sources such as NH4+ although there is no consensus regarding the involved mechanisms. Asparaginase II is a periplasmic enzyme of Saccharomyces cerevisiae encoded by the ASP3 gene. The enzyme activity is not found in cells grown in either ammonia, glutamine, or glutamate, but it is found in cells that have been subjected to nitrogen starvation or have been grown on a poor source of nitrogen such as proline. In this report it is shown that the formation of this enzyme is dependent upon the functional GLN3 gene and that the response to nitrogen availability is under the control of the URE2 gene product. In this respect the expression of ASP3 is similar to the system that regulates the GLN1, GDH2, GAP1, and PUT4 genes that codes for glutamine synthetase, NAD-linked glutamate dehydrogenase, general amino-acid permease, and high affinity proline permease, respectively.

Glucoamylase isoenzymes tailoring through medium composition.

Two major glucoamylase isoenzymes (GAI and GAII) have been identified in culture supernatants of Aspergillus awamori. It has been suggested that a stepwise degradation of a native enzyme during the fermentation by proteases and/or glucosidases results in the formation of isoenzymes that have different characteristics concerning substrate specificity and stability to pH and temperature. In this study, the glucoamylase isoenzymes produced by Aspergillus awamori using liquid media with C/N 10 (2.0% starch, 0.45% (NH4)2 SO4) and C/N 26 (5.2% starch, 0.45% (NH4)2 SO4) were analyzed. In both cases, GAI and GAII were characterized concerning its hydrolitic activities, mol wt, and isoeletric point. Using HPLC gel filtration and FPLC chromatofocusing, it was obtained for GAI a mol wt of 110,000 Da, pi 3.45 and for GAII a mol wt of 86,000 Da, pI 3.65. A different isoenzymes proportion was observed by the use of the two C/N ratios. In the lower carbohydrate content, fermentation of the GAI form predominated, whereas in the C/N 26 medium, GAII was prevalent. Gel eletrophoresis, amino acid analysis, and structural data confirmed that both preparations were glucoamylases with a high degree of homogeneity.

Lipase production by Penicillium restrictum in a bench-scale fermenter : effect of carbon and nitrogen nutrition, agitation, and aeration.

A preliminary screening work selected Penicillium restrictum as a promising micro-organism for lipase production. The physiological response of the fungus towards cell growth and enzyme production upon variable carbon and nitrogen nutrition, specific air flow rate (Qa) and agitation (N) was evaluated in a 5-L bench-scale fermenter. In optimized conditions for lipase production meat peptone at 2% (w/v) and olive oil at 1% (w/v) were used in a growth medium with a C/N ratio of 9.9. Higher C/N ratios favored cell growth in detriment of enzyme production. Low extracellular lipase activities were observed using glucose as carbon source suggesting glucose regulation. Final lipase accumulation of 13,000 U/L was obtained, using optimized specific air flow rate (Qa) of 0.5 wm and an impeller speed (N) of 200 rpm. Agitation showed to be an important parameter to ensure nutrient availability in a growth medium having olive oil as carbon source.

Assay for in vivo yeast invertase activity using NaF.

The methods used for invertase activity determination are based on the measurement of glucose or reducing sugars produced by the enzymatic hydrolysis of sucrose into glucose and fructose. When whole yeast cells are used in these assays, the monosaccharides formed by the action of the periplasmic enzyme can be taken up and metabolized, leading to errors on the enzyme activity determination. This study reports a method for a more accurate invertase activity measurement by blocking the glycolytic pathway. In this method the cells were preincubated with 50 mM sodium fluoride, and inhibitor of enolase. This in vivo measurement of the enzyme activity, under initial rate conditions, was performed using cell concentrations up to 64 mg cell/ml. The results obtained showed that this method is particularly useful for cells with low invertase activity.