Biochemical characterization and insights into the potency of the acidic Aspergillus niger NRC114 purified α-galactosidase in removing raffinose family oligosaccharides from soymilk yogurt.

BACKGROUND: Because humans lack α-galactosidase, foods containing certain oligosaccharides from the raffinose family, such as soybeans and other legumes, may disrupt digestion and cause flatulence. RESULTS: Aspergillus niger NRC114 α-galactosidase was purified using protein precipitation, gel filtration, and ion exchange chromatography steps, which resulted in a 123-fold purification. The purified enzyme was found to be 64 kDa using the SDS-PAGE approach. The optimum pH and temperature of the purified α-galactosidase were detected at pH 3.5 and 60 ºC, respectively. The pure enzyme exhibited potent acidic pH stability at pH 3.0 and pH 4.0 for 2 h, and it retained its full activity at 50 ºC and 60 ºC for 120 min and 90 min, respectively. The enzyme was activated using 2.5 mM of K+, Mg2+, Co2+, or Zn2+ by 14%, 23%, 28%, and 11%, respectively. The Km and Vmax values of the purified enzyme were calculated to be 0.401 µM and 14.65 µmol min-1, respectively. The soymilk yogurt showed an increase in its total phenolic content and total flavonoids after enzyme treatment, as well as several volatile compounds that were detected and identified using GC-MS analysis. HPLC analysis clarified the enzymatic action in the hydrolysis of raffinose family oligosaccharides. CONCLUSION: The findings of this study indicate the importance of A. niger NRC114 α-galactosidase enzyme for future studies, especially its applications in a variety of biological fields.

Therapeutic impact of purified Trichoderma viride L-asparaginase in murine model of liver cancer and in vitro Hep-G2 cell line.

BACKGROUND: Hepatocellular carcinoma (HCC) is among the common cancers, but difficult to diagnose and treat. L-asparaginase has been introduced in the treatment protocol of pediatric acute lymphoblastic leukemia (ALL) since the 1960s with a good outcome and increased survival rates to nearly 90%. Moreover, it has been found to have therapeutic potential in solid tumors. Production of glutaminase-free-L-asparaginase is of interest to avoid glutaminase-related toxicity and hypersensitivity. In the current study, an extracellular L-asparaginase that is free of L-glutaminase was purified from the culture filtrate of an endophytic fungus Trichoderma viride. The cytotoxic effect of the purified enzyme was evaluated in vitro against a panel of human tumor cell lines and in vivo against male Wister albino mice intraperitoneally injected with diethyl nitrosamine (200 mg/kg bw), followed by (after 2 weeks) oral administration of carbon tetrachloride (2 mL/kg bw). This dose was repeated for 2 months, and after that, the blood samples were collected to estimate hepatic and renal injury markers, lipid profiles, and oxidative stress parameters. RESULTS: L-asparaginase was purified from T. viride culture filtrate with 36 purification folds, 688.1 U/mg specific activity, and 38.9% yield. The highest antiproliferative activity of the purified enzyme was observed against the hepatocellular carcinoma (Hep-G2) cell line, with an IC50 of 21.2 g/mL, which was higher than that observed for MCF-7 (IC50 34.2 g/mL). Comparing the DENA-intoxicated group to the negative control group, it can be demonstrated that L-asparaginase adjusted the levels of the liver function enzymes and the hepatic injury markers that had previously changed with DENA intoxication. DENA causes kidney dysfunction and altered serum albumin and creatinine levels as well. Administration of L-asparaginase was found to improve the levels of the tested biomarkers including kidney and liver function tests. L-asparaginase treatment of the DENA-intoxicated group resulted in a significant improvement in the liver and kidney tissues to near normal similar to the healthy control group. CONCLUSION: The results suggest that this purified T. viride L-asparaginase may be able to delay the development of liver cancer and may be used as a potential candidate for future application in medicine as an anticancer medication.

Physiological studies on carboxymethyl cellulase formation by Aspergillus terreus DSM 826.

Physiological studies were conducted to determine the optimum cultural conditions for maximal carboxymethyl cellulase (CMCase) formation by Aspergillus terreus DSM 826. Shaking condition at 150 rpm is favorable for the production of CMCase from rice straw and sugar cane bagasse. The highest enzyme yield was obtained at the third day of incubation at 30 °C for both cases; however CMCase formation occurred at a broad range of pH values, with maximal formation of A. terreus DSM 826 CMCase at pH 4.5 and 5.0 when rice straw and sugar cane bagasse were used as sole carbon source, respectively. Carboxymethyl cellulose (CMC) was found to be a good inducer for CMCase formation in both agricultural wastes with CMC concentrations of 0.5 and 1.0 % (w/v) in case of rice straw and sugar cane bagasse, respectively. High level of enzyme formation was obtained with the addition of ammonium chloride as nitrogen source in both cases and at a concentration of 0.4 % (v/v Tween-80) as an addition to medium containing rice straw. However this addition did not influence the production of CMCase in case of using sugar cane bagasse as carbon source.

A statistical strategy for optimizing the production of α-galactosidase by a newly isolated Aspergillus niger NRC114 and assessing its efficacy in improving soymilk properties.

BACKGROUND: α-Galactosidase is widely distributed in plants, microorganisms, and animals, and it is produced by different fungal sources. Many studies have confirmed the valuable applications of α-galactosidase enzymes for various biotechnological purposes, like the processing of soymilk. RESULTS: Aspergillus niger NRC114 was exploited to produce the extracellular α-galactosidase. One factor per time (OFT) and central composite design (CCD) approaches were applied to determine the optimum parameters and enhance the enzyme production. The CCD model choices of pH 4.73, 1.25% mannose, 0.959% meat extract, and 6-day incubation period have succeeded in obtaining 25.22 U/mL of enzyme compared to the 6.4 U/mL produced using OFT studies. Treatment of soymilk by α-galactosidase caused an increase in total phenols and flavonoids by 27.3% and 19.9%, respectively. Antioxidant measurements revealed a significant increase in the enzyme-treated soymilk. Through HPLC analysis, the appearance of sucrose, fructose, and glucose in the enzyme-treated soymilk was detected due to the degradation of stachyose and raffinose. The main volatile compounds in raw soymilk were acids (45.04%) and aldehydes (34.25%), which showed a remarkable decrease of 7.82% and 20.03% after treatment by α-galactosidase. CONCLUSIONS: To increase α-galactosidase production, the OFT and CCD approaches were used, and CCD was found to be four times more effective than OFT. The produced enzyme proved potent enough to improve the properties of soymilk, avoiding flatulence and undesirable tastes and odors.

Correction to: Biosynthesis and characterization of silver nanoparticles induced by fungal proteins and its application in different biological activities.

In the publication of this article [1], the title of Figure 6 was missing. The original article has been corrected.

Purification and properties of an endoglucanase of Aspergillus terreus DSM 826.

Endoglucanase (EG) from A. terreus DSM 826 grown on sugar cane bagasse as a carbon source was purified using acetone fractionation, then a Sepharose-4B chromatographic column, with purification of about 27-fold and 10.5% recovery. The optimum temperature and pH for activity of the purified EG were found to be 50 degrees C and pH 4.8, respectively. The purified enzyme can stand heating up to 50 degrees C for 1 h without apparent loss of activity. However, the enzyme, incubated at 80 degrees C for 5 min, showed about 56% loss of activity. Optimum EG activity was recorded with a citrate buffer system (pH 4.8; 0.05 M). Co2+ (2.5 x 10(-2) M) and Zn2+ (5 x 10(-2) M) were found to activate the purified EG of A. terreus DSM 826 by about 83 and 25%, respectively. On the other hand, Hg2+ inhibited the activity of the purified EG by about 50 and 71% at a concentration of 2.5 x 10(-2) and 5 x 10(-2) M, respectively. Carboxymethyl cellulose was found to be the best substrate for the purified EG, with V(max) values of 4.35 micrpmol min(-1) mg(-1) protein.

Biosynthesis and characterization of silver nanoparticles induced by fungal proteins and its application in different biological activities.

BACKGROUND: The present study aims to apply an efficient eco-friendly and inexpensive process for green synthesis of silver nanoparticles (AgNPs) through the mediation of fungal proteins from Aspergillus fumigatus DSM819, characterization, and its application as antimicrobial finishing agent in textile fabrics against some infectious microorganisms. RESULTS: Optimum conditions for AgNP biosynthesis could be achieved by means of using 60% (v/v) of cell-free filtrate (CFF) and 1.5 mM of AgNO3 at pH 10.0 after 90 min. The obtained AgNPs were of spherical shape with 90% of distribution below than 84.4 nm. The biosynthesized AgNPs exerted an antimicrobial activity against the studied pathogenic microorganisms (E. coli, B. mycoides, and C. albicans). In addition, IC50 values against in vitro tumor cell lines were found to be 31.1, 45.4, 40.9, and 33.5 µg/ml for HCT116, A549, MCF7, and PC3, respectively. Even with a very low concentration (0.25%), the treated PET/C fabrics by AgNPs exerted an antimicrobial activity against E. coli, B. mycoides, and C. albicans to give inhibition zone diameter of 15, 15, and 16 mm, respectively. CONCLUSIONS: The green biosynthesis approach applied in this study is a non-toxic alternative to the traditional chemical and physical methods, and would be appropriate for biological large-scale production and prospective treatments.

Purification and biochemical characterization of two isolated laccase isoforms from Agaricus bisporus CU13 and their potency in dye decolorization.

Agaricus bisporus CU13 laccase was purified using ammonium sulfate precipitation (40-80%), Sephadex G100, and DEAE Sephadex A50 anion exchange column chromatography, respectively. Two laccase isoenzymes (Lacc1 & Lacc2) with purification folds of 1.40 and 5.81 respectively, were obtained from DEAE Sephadex A50 column. Optimal temperature and pH were recorded at 55 °C and pH 5.0 for both laccase isoenzymes using ABTS as substrate. Lacc1 was more thermostable than Lacc2 with residual activity of 95, 80 and 6%, while Lacc2 only retained 72, 25 and 0.4% of its activity after incubation for 90 min. at 50, 60 and 70 °C, respectively. Lacc2 retained about 93 and 86% of the initial activity at pH 9.0 and 7.0, whereas Lacc1 was stable at pH 7.0 and 5.0 followed by pH 9.0 and retained about 87, 76, and 36% of its activity respectively, after 4 h of incubation. Lacc1 was activated by 40% in the presence of Cu2+ (10 mM). Km and Vmax values found to be 0.394 and 0.158 µM, and 0.1351 and 0.4755 µmol min-1 for Lacc1 and Lacc2, respectively. The efficiency of both isoenzymes to decolorize Acid blue dye, make the enzyme seems to be a prospective for further biotechnological applications.

Optimization of silver nanoparticles biosynthesis mediated by Aspergillus niger NRC1731 through application of statistical methods: enhancement and characterization.

The fungal-mediated silver nanoparticles (AgNPs) biosynthesis optimization via the application of central composite design (CCD) response surface to develop an effective ecofriendly and inexpensive green process was the aim of the current study. Nanosilver biosynthesis using the Aspergillus niger NRC1731 cell-free filtrate (CFF) was studied through involving the most parameters affecting the AgNPs green synthesis and its interactions effects. The statistical optimization models showed that using 59.37% of CFF in reaction containing 1.82 mM silver nitrate for 34 h at pH 7.0 is the optimum value to optimize the AgNPs biosynthesis. The obtained AgNPs were characterized by means of electron microscopy, UV/visible spectrophotometry, energy dispersive X-ray analysis and infrared spectroscopy to elucidate its almost spherical shape with diameter of 3-20 nm. The produced AgNPs exhibited a considerable antimicrobial activity against Bacillus mycoides, Escherichia coli in addition to Candida albicans.

Application of response surface methodology to optimize the extracellular fungal mediated nanosilver green synthesis.

This study aims to optimize the biosynthesis of nanosilver particles mediated by Trichoderma viride ATCC36838 using response surface methodology (RSM). Silver nanoparticles (AgNPs) were biosynthesized effectively in terms of the factors impacting silver ion (Ag+) reduction to metallic nanosilver (Ag0) using culture filtrate under shaking condition. The results of statistics calculations revealed that 2 mM silver nitrate and 28% (v/v) of culture filtrate at pH 7.0 for 34 h were the optimum values for AgNPs biosynthesis. The characterization of the produced AgNPs was conducted using electron microscopy, energy dispersive X-ray analysis, UV/visible spectrophotometry, and Fourier transform infrared spectroscopy. Round to oval AgNPs were detected with aspects of TEM within diameter range of 4-16 nm. The results of this study could help in developing a reliable ecofriendly, simple, and low cost process for microbial assisted AgNPs green synthesis especially with the continuous increase in its application fields.

Physiological studies on carboxymethyl cellulase formation by Aspergillus terreus DSM 826

Physiological studies were conducted to determine the optimum cultural conditions for maximal carboxymethyl cellulase (CMCase) formation by Aspergillus terreus DSM 826. Shaking condition at 150 rpm is favorable for the production of CMCase from rice straw and sugar cane bagasse. The highest enzyme yield was obtained at the third day of incubation at 30ºC for both cases; however CMCase formation occurred at a broad range of pH values, with maximal formation of A. terreus DSM 826 CMCase at pH 4.5 and 5.0 when rice straw and sugar cane bagasse were used as sole carbon source, respectively. Carboxymethyl cellulose (CMC) was found to be a good inducer for CMCase formation in both agricultural wastes with CMC concentrations of 0.5 and 1.0 % (w/v) in case of rice straw and sugar cane bagasse, respectively. High level of enzyme formation was obtained with the addition of ammonium chloride as nitrogen source in both cases and at a concentration of 0.4 % (v/v Tween-80) as an addition to medium containing rice straw. However this addition did not influence the production of CMCase in case of using sugar cane bagasse as carbon source.

Cytidine deaminase activity in Penicillium politans NRC-510.

Cell-free extracts of nitrate-grown Penicillium politans NRC-510 catalyzes the hydrolytic deamination of cytidine to uridine. Uridine was chromatographically identified in cell-free extracts. The enzyme exhibited optimum pH and temperature activities at 6.5 and 80 degrees C respectively. Thermal stability experiments indicated that the enzyme restored its activity at 80 degrees C for at least 60 minutes. When cell-free extracts were incubated at 90 degrees C for 5 minutes enzyme activity was inhibited by about 33%. The involvement of sulfhydryl group(s) in the catalytic site of the enzyme was shown. HgCl2 (5 x 10(-3) M) and CuSO4 (10(-2) M) caused a complete inhibition of enzyme activity. Ethylene diamine tetraacetate at a concentration of 5 x 10(-3) M and 10(-2) M inhibited the enzyme as well. Whereas, MgCl2, CoSO2 and MnCl2 had a remarkable activating effect. Dialysis of the cell-free extracts resulted to an increase in enzyme activity by about 30%. To our knowledge the thermophilic nature of the cytidine deaminase of P. politans NRC-510 is unique.

Deamination of adenosine by extracts of Penicillium politans NRC-510.

Cell-free extracts of nitrate-grown Penicillium politans NRC-510 could catalyze the hydrolytic deamination of adenosine to inosine maximally at pH 6.0 and 45 degrees C. However the same extracts could not catalyze the N-glycosidic bond cleavage of adenosine at pH 4.0, 6.0 and 8.0. Incubation of the extracts at 55 degrees C for 30 minutes caused about 31% loss in activity whereas incubation of the extracts at 60 degrees C for 15 minutes caused a complete loss of enzyme activity. Results indicated the absence of the involvement of sulfhydryl groups in the catalytic site of adenosine deaminase. The enzyme is inhibited by ethylene diamine tetraacetate indicating that adenosine deaminase is a metalloenzyme. MnCl2 and MgCl2 had a remarkable activating effect, whereas HgCl2, CaCl2 and ZnSO4 showed an inhibitory effect on enzyme activity. Dialyzing the extracts for 24 hours significantly increase deaminase activity by about 33%. The apparent K(m) value was calculated for adenosine and found to be 3.63 x 10(-3) M, which indicates high affinity of adenosine deaminase for its substrate adenosine.

Immunization of mice with crude extract of Saccharomyces boulardii yeast induces cross-reactive immune responses with antigenic preparations from different developmental stages of the Schistosoma mansoni and reduces the parasite worm burden.

Immunization of mice with Saccharomyces boulardii crude extract caused a significant reduction in Schistosoma mansoni worm burden (23.7%; P = 0.02) in comparison to control mice. Mice sera collected after the 1st immunization showed a significant increase (P < 0.05) in both IgM and IgG against parasite soluble cercarial antigenic preparation (CAP) than unimmunized mice sera (UMS). Upon 2nd immunization, sera from yeast extract immunized mice (YIM) showed increase of IgM and IgG (1.6 and 1 fold) against CAP than UMS. Sera collected 2, 4 and 6 weeks (W) after infection of YIM with S. mansoni cercariae showed higher IgM (1.4, 1.5 and 1 folds) and IgG (0.9, 1.2 and 1.1 folds) levels against CAP than sera of infected UM (IUM). When compared to sera from UM, a week post 1st and 2nd immunization sera showed a significant elevation (P < 0.05) of both IgM and IgG to parasite soluble adult worm antigenic preparation (SWAP). Sera collected at 2, 4 and 6W after infection of YIM with parasite cercariae showed higher IgM (1.1, 1.3 and 1.2 folds) and IgG (1.2, 1.1 and 1.3 folds) reactivities against SWAP than sera of IUM. One week post 1st and 2nd immunization with the yeast extract, sera showed a significant increase (P < 0.05) of both IgM and IgG levels against parasite soluble egg antigen (SEA) when compared with sera from UM. At 2 and 4W post infection, sera from immunized infected mice showed a significant decrease in IgM (P < 0.05) but the decrease of IgG (0.7 fold) was not significant when compared to IUM. Six weeks post infection the decrease in both antibodies remained but it was significant for IgG (P < 0.05) but not for IgM (2.1 folds) when compared with IUM. Immunization with S. boulardii extract caused a non-significant increase (1.2 folds) in mesenteric lymph node (MLN)-CD4+T cells, significant elevation of MLN-CD8+T cells (P < 0.05) and a non-significant increase (0.9 folds) of B-cells when compared to that of UM. Also, Immunization with S. boulardii extract caused 1 fold elevation in both CD4+ and CD8(+)-T lymphocytes in thymus of YIM when compared with their counts in thymus of UM.

Some properties of two aldolases in extracts of Aspergillus oryzae.

Fructose 1,6-diphosphate (FDP) aldolase and 2-keto-3-deoxy-D-gluconate (KDG) aldolase the two key enzymes of Embden-Meyerhof-Parnas (EMP) and the nonphosphorolytic Entner-Doudoroff (ED) pathways respectively, were identified in cell-free extracts of four Aspergillus oryzae strains grown on D-glucose as sole source of carbon. A. oryzae NRRL 3435 gave the highest enzymatic activity for the two enzymes and selected for further studies. Studies on the properties of the two key enzymes indicated that the optimum conditions for the activities of FDP aldolase and KDG aldolases occurred at pH 8.5, 45 degrees C and pH 8.0, 55 degrees C, respectively. Tris-acetate buffer and phosphate buffer showed the highest enzymatic activity for these two enzymes respectively. KDG aldolase was stable at 55 degrees C for 60 minutes however FDP aldolase was found to be less stable above 45 degrees C. On the other hand the two aldolases showed a high degree of stability towards frequent freezing and thawing. Dialysis of the extracts caused a decrease in the enzymatic activity of KDG aldolase, and an increase in FDP aldolase activity. The addition of ethylene diamine tetraacetate to the crude extracts caused an inhibition of KDG aldolase, whileas FDP aldolase was not affected. Addition of MnCl(2), CoSO(4), MgCl(2) and ZnSO(4) to the dialyzed extracts increased the activity of KDG aldolase by 67%, 54%, 61% and 37%, respectively. On the other hand the addition of some metal salts caused an inhibition of FDP aldolase. The results obtained indicate the absence of evidence for the involvement of sulfhydryl groups in the catalytic sites of the two aldolases.

Hydrolytic cleavage of purine ribonucleosides in Aspergillus phoenicis.

Cell-free extracts of nitrate-grown Aspergillus phoenicis could catalyze the hydrolytic cleavage of the N-glycosidic bond of inosine, guanosine and adenosine to the corresponding base and ribose by the nucleoside hydrolase. No evidence was obtained concerning the hydrolytic degradation of N-glycosidic bond of pyrimidine ribonucleosides namely cytidine and uridine by the same extracts. Optimum pH and temperature for adenosine, guanosine and inosine hydrolysis were the same at pH 3.5 and 55 degrees C, respectively. Citrate buffer showed the highest hydrolase activity when compared to the analogous activity obtained with the other buffers used. The rate of hydrolysis of the three nucleosides was in the order inosine > guanosine > adenosine. Incubation of extracts at 55 degrees C for 15 minutes caused about 85%, 75% and 62% loss of activity with adenosine, guanosine and inosine respectively. Dialyzing the extract caused a decrease in enzyme activity. Addition of inorganic arsenate to the reaction mixture (containing adenosine, guanosine or inosine) did not affect the amount of ribose liberated. Addition of EDTA at a concentration of 5 x 10(-3) M caused an inhibition of about 50%, however a complete inhibition for enzyme activity was obtained at 10(-2) M EDTA. MgSO4, CoSO4 and ZnSO4 at a final concentration of 5 x 10(-3) M showed activation of ribonucleoside hydrolase.