Crude Fungal Extracts Research Articles

Batch Culture Fermentation of Endophytic Fungi and Extraction of Their Metabolites

Antibiosis is one of the possible modes of action shown by endophytic fungi having antifungal activity. To test if antifungal activity in endophytic fungi is due to antibiosis, assay of the metabolites of endophytic fungi was needed. To obtain metabolites for bioassay batch culture fermentation and extraction of metabolites was done. Fungus was multiplied on wickerham media at incubation temperature of 25 ± 2 °C for 4 weeks and then extracted with solvents of different polarity. All the solvent extracts were dried under vacuum rotary evaporator to get dried crude fungal extract, which was subjected to further fractionation and bioassay.

Optimisation of synergistic biomass-degrading enzyme systems for efficient rice straw hydrolysis using an experimental mixture design

Synergistic enzyme system for the hydrolysis of alkali-pretreated rice straw was optimised based on the synergy of crude fungal enzyme extracts with a commercial cellulase (Celluclast™). Among 13 enzyme extracts, the enzyme preparation from Aspergillus aculeatus BCC 199 exhibited the highest level of synergy with Celluclast™. This synergy was based on the complementary cellulolytic and hemicellulolytic activities of the BCC 199 enzyme extract. A mixture design was used to optimise the ternary enzyme complex based on the synergistic enzyme mixture with Bacillus subtilis expansin. Using the full cubic model, the optimal formulation of the enzyme mixture was predicted to the percentage of Celluclast™: BCC 199: expansin=41.4:37.0:21.6, which produced 769mg reducing sugar/gbiomass using 2.82FPU/g enzymes. This work demonstrated the use of a systematic approach for the design and optimisation of a synergistic enzyme mixture of fungal enzymes and expansin for lignocellulosic degradation.

Phytochemical analysis and enzyme analysis of endophytic fungi from Centella asiatica

ObjectiveTo evaluate the phytochemical properties, enzyme properties of an endophytic fungi from Centella asiatica plant. MethodsEndophyte was isolated by using potato dextrose agar medium from Centella asiatica plant. The qualitative chemicals screening were performed for the ethyl acetate extracts of fungi. Then the phytochemicals like phenols and flavonoids were quantitatively estimated. Then the ability of the fungal culture to produce different enzymes was analysed. Finally, High performance thin layer Chromatography was performed for alkaloid and flavonoid profiles of fungal extract. ResultsA Penicillium sp. was isolated from Centella asiatica plant and the fungus was identified by 18S rRNA analysis. The sequence of the fungus was submitted in NCBI with accession number (HM068965) and name Penicillium sp.nirjan22. The qualitative chemicals analysis of the fungus showed positive results for alkaloids, phenols, flavonoids, tannin and glycosides. It was found that the fungal crude extract contained 2.76g of phenol and 0.275 g of flavonoid. The Penicillium sp.nirjan22 produced cellulase enzyme. The HPTLC analysis of the ethyl acetate extract of fungi inferred that it contain both alkaloid and flavonoid. ConclusionsThe present study inferred that Penicillium sp. nirjan22 contain many bioactive compounds which can further isolate and check in vitro and in vivo models against different human ailments.

Biological activities of the fermentation extract of the endophytic fungus Alternaria alternata isolated from Coffea arabica L.

A total of 22 endophytic fungi isolated from coffee (Coffea arabica L.) were cultivated in vitro and their crude extracts tested. The screening was carried out using the agar diffusion method against Staphylococcus aureus, Escherichia coli and Candida albicans. The most effective isolate was Alternaria alternata, and subsequently, its extract was assayed. The total phenolic content was 3.44 μg GAE/mg of the crude extract. For the antibacterial and antifungal activity assays, minimum inhibitory concentrations (MIC) and minimum bactericidal and fungicidal concentrations (MBC and MFC) were determined. The ranges of MIC values were 50-100 μg/mL for S. aureus and 400-800 μg/mL for E. coli. The extract did not show activity in the tested concentrations for C. albicans. The fungal crude extract was assayed for antioxidant activities. Its ability to scavenge DPPH radicals and antioxidant activity by β-carotene/linoleic acid system oxidation was not significant. In addition, antitumor activity was studied using the MTT assay. At a dilution of 400 μg/mL, the extract displayed a cytotoxic activity of approximately 50% towards HeLa cells in vitro. The results indicate that endophytic fungi could be a promising source of bioactive compounds and warrant further study.

SEPARATION FROM USTILAGO SCITAMINEA OF DIFFERENT ELICITORS WHICH MODIFY THE PATTERN OF PHENOLIC ACCUMULATION IN SUGARCANE LEAVES

Smut caused by Ustilago scitaminea (Syd.) is a major disease of sugarcane. Different compounds from a crude fungal extract were separated by capillary electrophoresis. These compounds produced changes in the accumulation of free phenolics and enhanced phenylalanine ammonia-lyase and peroxidase activities in sugarcane leaves of the susceptible cv. Mayari 5514, and the resistant cv. Barbados 42231. Smut-elicitor fractions were resolved by capillary electrophoresis as different peaks. Those inducing the highest biological activity were resolved as three main peaks corresponding to negatively charged proteins, peptides or glycopeptides of medium molecular mass. These compounds enhanced the accumulation of free phenolics, mainly hydroxycinamic acids, by activation of phenylalanine ammonia- lyase in the resistant cultivar, and hydroxybenzoic acids in the susceptible cultivar. Another important difference was the enhancement in the resistant cultivar of peroxidase, an enzyme that uses free phenolics as substrates for the activation of important mechanisms of resistance of sugarcane leaves to the fungal pathogen.

Use of Recombinant Antigens for the Diagnosis of Invasive Candidiasis

Invasive candidiasis is a frequent and often fatal complication in immunocompromised and critically ill patients. Unfortunately, the diagnosis of invasive candidiasis remains difficult due to the lack of specific clinical symptoms and a definitive diagnostic method. The detection of antibodies against different Candida antigens may help in the diagnosis. However, the methods traditionally used for the detection of antibodies have been based on crude antigenic fungal extracts, which usually show low-reproducibility and cross-reactivity problems. The development of molecular biology techniques has allowed the production of recombinant antigens which may help to solve these problems. In this review we will discuss the usefulness of recombinant antigens in the diagnosis of invasive candidiasis.

Accelerating the Discovery of Biologically Active Small Molecules Using a High-Throughput Yeast Halo Assay

The budding yeast Saccharomyces cerevisiae, a powerful model system for the study of basic eukaryotic cell biology, has been used increasingly as a screening tool for the identification of bioactive small molecules. We have developed a novel yeast toxicity screen that is easily automated and compatible with high-throughput screening robotics. The new screen is quantitative and allows inhibitory potencies to be determined, since the diffusion of the sample provides a concentration gradient and a corresponding toxicity halo. The efficacy of this new screen was illustrated by testing materials including 3104 compounds from the NCI libraries, 167 marine sponge crude extracts, and 149 crude marine-derived fungal extracts. There were 46 active compounds among the NCI set. One very active extract was selected for bioactivity-guided fractionation, resulting in the identification of crambescidin 800 as a potent antifungal agent.

Fungal substances as modulators of NF-κB activation pathway

MCF7 breast cancer cell line, carrying a luciferase reporter gene under the control of nuclear factor kappa B (NF-kappaB)-responsive promoter, was established and used for the screening of fungal organic extracts for their ability to interfere with the NF-kappaB activation pathway. Twenty-eight crude fungal extracts, out of 242, were found to inhibit NF-kappaB reporter activity by more than 40%. Furthermore, positive extracts were used to evaluate their antiproliferative activity as well as their ability to influence the phosphorylation and degradation levels of IkappaBa. Fungal extracts prepared from Marasmius oreades and Cyathus striatus showed significant inhibitory effects on the NF-kappaB activation pathway. Taken together, our results support the notion of the presence of novel activities that might be utilized as cancer therapeutics.

The Relative and Absolute Configuration of PF1140.

A potentially general approach for elucidating the absolute configuration of N-hydroxypyridone antibiotics has been developed. One member of this family of antibiotics, PF1140, was efficiently purified from a crude fungal extract following allylation of its N-hydroxyl group. Removal of the resultant allyl group permitted regeneration of the N-hydroxyl group as well as conversion into the corresponding pyridone derivative. The stereochemistry of PF1140 including the absolute configuration was established by X-ray crystallographic analysis of the S-2-methoxy-2-(1-naphthyl)propionic ester derivative.

Production of 2-(2-phenylethyl) Chromones in Cell Suspension Cultures of Aquilaria sinensis

2-(2-Phenylethyl) chromones are the major constituents responsible for the quality of agarwood, which is one of the most valuable non-timber products used as incenses, perfumes, traditional medicines and other products. In this study, cell suspension culture of Aquilaria sinensis (Lour) Gilg was used to monitor the eliciting effects of crude fungal extracts on cell growth and chromones production. Crude extracts of Melanotus flavolivens (B. etc) Sing. prepared with different solvents were used to elicit the production of 2-(2-phenylethyl) chromones in cell suspension cultures of A. sinensis. Four 2-(2-phenylethyl) chromones,␣6,7-dimethoxy-2-(2-phenylethyl) chromone (1), 6,7-dimethoxy-2-[2-(4′-methoxyphenyl)ethyl] chromone (2), 6-methoxy-2-[2-(4′-methoxyphenyl)ethyl] chromone (3) and 6-methoxy-2-(2-phenylethyl) chromone␣(4),␣were detected by LC–MS in the cell suspension culture of A. sinensis elicited with crude extracts of M. flavolivens. Three hundred and seventy eight, 196 and 31 μg g−1 DW of 2-(2-phenylethyl) chromones were obtained in the cell cultures induced by water extracts, 50 and 95% ethanol extracts of M. flavolivens, respectively. The results show that water-soluble materials in the crude extracts are the main components inducing the production of 2-(2-phenylethyl) chromones in the cell cultures.

Synthesis and characterization of a new cutinase substrate, 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate

Phytopathogenic fungi penetrate plants by breaking down the cuticular barrier with cutinase. Cutinases are extracellular hydrolytic enzymes that degrade cutin, a polyester composed of hydroxy and epoxy fatty acids. Until now, cutinase has been recognized by its ability to release labeled cutin monomers or by a non-specific esterase assay based on the hydrolysis of p-nitrophenyl esters of short fatty acids. In this work, an insoluble p-nitrophenyl derivative was synthesized and purified, and its structure was determined to be 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate (pNMSEH) by nuclear magnetic resonance (H + NMR) analysis. pNMSEH was tested as a new cutinase substrate with Pseudomonas mandocino cutinase and porcine liver esterase. While a linear release over time of p-nitrophenol (pNP) was recorded in the presence of cutinase, no response was obtained with the esterase. The calculated kinetic parameters of pNMSEH hydrolysis by cutinase revealed a high specificity ( K m = 1.8 mM), albeit a low catalytic rate ( V max = 10.5 μmol min −l l −1). This new synthetic substrate may be helpful for detecting and assaying cutinase activity in mixed solutions, such as crude fungal extracellular extracts.

UV‐Guided Isolation of Fungal Metabolites by HSCCC

Analytical standardised reversed phase liquid chromatography (RPLC) data can be helpful in finding a suitable solvent combination for isolation of fungal metabolites by high‐speed counter current chromatography. Analysis of the distribution coefficient (K D) of fungal metabolites in a series of biphasic systems consisting of n‐heptane–ethyl acetate (EtOAc)–methanol (MeOH)–water has been used to illustrate the relationship between the RPLC retention indexes and K D's. The relationship can be used to select a suitable solvent combination for HSCCC, as demonstrated by the isolation of two new cyclic peptides from a crude fungal extract.

The Relative and Absolute Configuration of PF1140

A potentially general approach for elucidating the absolute configuration of N-hydroxypyridone antibiotics has been developed. One member of this family of antibiotics, PF1140, was efficiently purified from a crude fungal extract following allylation of its N-hydroxyl group. Removal of the resultant allyl group permitted regeneration of the N-hydroxyl group as well as conversion into the corresponding pyridone derivative. The stereochemistry of PF1140 including the absolute configuration was established by X-ray crystallographic analysis of the S-2-methoxy-2-(1-naphthyl)propionic ester derivative.

Mycotoxins and Invertase Enzyme of the Mycoflora of Molasses in Upper Egypt

A total of twenty-nine species and one species variety belonging to 12 genera was isolated from 30 samples of molasses on 1% glucose (10 genera, 22 species and 1 variety) and 50% sucrose (7, 21 and 1) Czapek’s agar at 25°C media. Aspergillus, Mucor, Mycosphaerella and Pénicillium were the most common genera on the two types of media. From the above genera, the most prevalent species were: Aspergillus flavus, A fumigatus, A niger, Mycosphaerella tassiana, Pénicillium chrysogenum, P oxalicum and P purpurogenum Also, some species were only isolated on 50% sucrose such as Eurotium amstelodami, E. chevalieri, E. repens, Humicola fuscoatra, Penicillium aurantiogriseum and P puberulum About 65 fungal isolates isolated from 50% sucrose agar were tested for their ability to produce invertase enzyme in liquid medium and 93.8% of the isolates could produce this enzyme. From the positive isolates, 32 showed high invertase activity, 21 had moderate activity and the remaining 8 isolates were of weak activity. Sixty isolates of Aspergillus, Emericella, Eurotium, Mycosphaerella and Penicillium from the preceding study were screened for the presence of their respective mycotoxins. Larva of brine shrimp (Artema salina L.) were used for toxicity test of the fungal crude extracts. Three isolates out of 60 tested were toxic. Using thin-layer chromatographic technique, 5 different known mycotoxin were detected aflatoxins : B1, B2, G1, G2 and citrinin.

APPLICATION OF pH-ZONE-REFINING CCC TO THE ISOLATION OF ANTIFUNGAL FERMENTATION PRODUCTS

The application of pH-zone-refining countercurrent chromatography (CCC) to the purification and bioassay guided isolation of novel antifungal agents from fungal fermentation extracts is demonstrated with separations of three families of acidic natural products. The separations were carried out using two types of commercially available multilayer coil planet centrifuges. Using this methodology as the final isolation step, three new glycosylated polyketides, arthrinosides A-C, were isolated from fermentation extracts of an Arthrinium sp. The three compounds differ in the length and hydroxylation of the polyketide chain and were not easily separable using normal or reverse phase column chromatography. Mycoparasitic acids A and B, new natural products differing only in epoxidation of an endocyclic double bond, were separated directly from crude fungal culture extracts. This CCC method was applied to the purification of a new biotransformation product of sordarin resulting from hydroxylation at C-11. Several hundred milligrams of this new derivative were easily separated in a single chromatographic run.

CDNA cloning and immunologic characterization of Pen o 18, the vacuolar serine protease major allergen of Penicillium oxalicum

Penicillium species are prevalent indoor airborne fungi that have been identified as causative agents of human extrinsic bronchial asthma. In the preparation of standardized diagnostic reagents, it is imperative to define the allergens of these ubiquitous fungi. Results from our previous study on P. oxalicum suggest that the 34-kd major immunoglobulin E–reacting component of this prevalent Penicillium species is probably a vacuolar serine protease. The purpose of the present study was to define this major P. oxalicum allergen (Pen o 18) through cDNA cloning and immunologic characterization. The cDNA of Pen o 18 was isolated through a combination of reverse transcriptase–polymerase chain reaction and 5′- and 3′-rapid amplification cDNA ends reactions. The primers used in these reactions were constructed according to the internal amino acid sequences of Pen o 18 and the conserved amino acid sequences of fungal serine proteases. Our results showed that a 1897-bp cDNA with an open reading frame of 503 residues was isolated for the proenzyme of Pen o 18. The encoded protein has a 16-residue signal peptide and a 119-residue prosequence. On maturation, the protein has an N-terminal glutamate that is the 136th residue encoded by the cDNA. Apparently the precursor also undergoes C-terminal processing with the cleavage of about 47 amino acids. The cDNA for Pen c 18 (the vacuolar serine protease allergen from P. citrinum ) was also isolated for comparison. Contrary to a previous report, the C-terminal region of Pen c 18 is similar to that of Pen o 18. Recombinant proteins (rPen o 18 and rPen c 18) with the putative mature N-termini and a his-tag were obtained by expressing the corresponding cDNAs in Escherichia coli. Serum samples from 7 asthmatic patients with immunoglobulin E reactivity to the 34-kd component of P. oxalicum also react to his-tagged recombinant Pen o 18. The presence of immunoglobulin E cross-reactivity between rPen o 18 and rPen c 18 was detected by immunoblot inhibition. Two monoclonal antibodies (PCM39 and FUM20) against fungal serine proteases react with rPen o 18, rPen c 18, and the 35/34-kd components in the corresponding crude fungal extracts. These components also react with immunoglobulin E antibodies in serum samples from asthmatic patients. In conclusion, results obtained confirm that the 34-kd major allergen of P. oxalicum is a vacuolar serine protease. The cDNAs of Pen o 18 and Pen c 18 encode precursor molecules that appear to undergo both N-terminal and C-terminal processing. Constructs beginning with mature N-terminal can be expressed in E. coli to produce recombinant polypeptides that are reactive to monoclonal antibodies or immunoglobulin E antibodies in serum samples from asthmatic patients. Results obtained may provide useful information and materials for preparation of standardized diagnostic reagents in clinical mold allergy. (J Lab Clin Med 2001;137:115-24)

Production and characterization of monoclonal antibodies to serine proteinase allergens in Penicillium and Aspergillus species.

Alkaline and/or vacuolar serine proteinases are major allergens in prevalent airborne Penicillium and Aspergillus species. The object of this study is to generate and characterize monoclonal antibodies against these serine proteinase allergens. BALB/c mice were immunized individually with the Penicillium citrinum culture medium or the crude extract and culture medium preparations of Aspergillus fumigatus. Hybridoma cells that secrete monoclonal antibodies against serine proteinase allergens were selected by immunoblotting. Antigens in three different Penicillium (P. citrinum, P. notatum and P. oxalicum) and two different Aspergillus species (A. fumigatus, and A. flavus) recognized by these monoclonal antibodies were analysed by sodium dodecyl sulphate and two-dimensional polyacrylamide gel electrophoresis immunoblotting and N-terminal amino acid sequence analysis. Four (PCM8, PCM10, PCM16 and PCM39) and one (FUM20) monoclonal antibodies against serine proteinase allergens were generated after fusion of NS-1 cells with spleen cells obtained from BALB/c mice immunized with antigens from P. citrinum and A. fumigatus, respectively. Immunoblotting results showed that PCM8 reacted with an alkaline serine proteinase allergen in P. citrinum and P. notatum. PCM10 and PCM39 reacted with the alkaline serine proteinase in two Penicillium (P. citrinum, P. notatum) and two Aspergillus species (A. fumigatus, and A. flavus) tested. PCM16 reacted with the alkaline serine proteinase allergen in P. citrinum, A. fumigatus and A. flavus but not with that in P. notatum. MoAb FUM20 reacted with the alkaline serine proteinase allergen in two Aspergillus species (A. fumigatus and A. flavus) but not with that in two different Penicillium species (P. citrinum, P. notatum) tested. Among these five monoclonal antibodies generated, only PCM39 and FUM20 can react with the vacuolar serine proteinase allergen in P. notatum, P. oxalicum and in A. fumigatus. The 35 kDa P. citrinum component that reacted with FUM20 has an N-terminal amino acid sequence of DSPSVEKNAP. Five monoclonal antibodies against different epitopes of the serine proteinase major allergens in prevalent Penicillium and Aspergillus species were generated in the present study. Antibodies obtained may be useful in the characterization and standardization of serine proteinase allergens in crude fungal extracts.

Reduction of raffinose oligosaccharides in chickpea (Cicer arietinum) flour by crude extracellular fungal α-galactosidase

The efficiency of crude extracelluar α-galactosidases from Cladosporium cladosporides, Aspergillus oryzae and A niger in reducing the raffinose and stachyose content in chickpea flours was studied and compared with other traditional treatments. The optimum pH for α-galactosidase activity was found to be 4·5 for A oryzae and 5·0 for Cl cladosporides and A niger, while the optimum temperature of enzyme activity was 40°C for Cl cladosporides and 50°C for A oryzae and A niger. The specific activities of α-galactosidase from Cl cladosporides, A oryzae and A niger were 3·35, 3·94 and 5·94 units μg−1 protein, respectively. The enzyme activity was stable between pH 4·0 and 7·0 for A oryzae and A niger and between pH 5·0 and 7·0 for Cl cladosporides. The enzymes were thermostable when incubated at temperature ranges of 40–60°C for Cl cladosporides and 40–50°C for A oryzae and A niger. The optimum conditions for removing the raffinose and stachyose were obtained by incubating chickpea flours with 30 ml of crude fungal α-galactosidase extract (290, 210 and 130 units ml−1 for Cl cladosporides, A oryzae and A niger, respectively) for 3 h at the optimum conditions of each strain. Crude fungal α-galactosidases reduced the raffinose oligosaccharides content in chickpea flours by 100%, while germination reduced the raffinose content by 69% and stachyose content by 75%. Other traditional techniques reduced the raffinose content by 13–49% and stachyose content by 10–32%. © 1998 Society of Chemical Industry

Reduction of raffinose oligosaccharides in chickpea (Cicer arietinum) flour by crude extracellular fungal α-galactosidase

The efficiency of crude extracelluar α-galactosidases from Cladosporium cladosporides, Aspergillus oryzae and A niger in reducing the raffinose and stachyose content in chickpea flours was studied and compared with other traditional treatments. The optimum pH for α-galactosidase activity was found to be 4·5 for A oryzae and 5·0 for Cl cladosporides and A niger, while the optimum temperature of enzyme activity was 40°C for Cl cladosporides and 50°C for A oryzae and A niger. The specific activities of α-galactosidase from Cl cladosporides, A oryzae and A niger were 3·35, 3·94 and 5·94 units μg−1 protein, respectively. The enzyme activity was stable between pH 4·0 and 7·0 for A oryzae and A niger and between pH 5·0 and 7·0 for Cl cladosporides. The enzymes were thermostable when incubated at temperature ranges of 40–60°C for Cl cladosporides and 40–50°C for A oryzae and A niger. The optimum conditions for removing the raffinose and stachyose were obtained by incubating chickpea flours with 30 ml of crude fungal α-galactosidase extract (290, 210 and 130 units ml−1 for Cl cladosporides, A oryzae and A niger, respectively) for 3 h at the optimum conditions of each strain. Crude fungal α-galactosidases reduced the raffinose oligosaccharides content in chickpea flours by 100%, while germination reduced the raffinose content by 69% and stachyose content by 75%. Other traditional techniques reduced the raffinose content by 13–49% and stachyose content by 10–32%. © 1998 Society of Chemical Industry

Identification of an endogenous NADPH-regenerating system coupled to nitrate reduction in vitro in plant and fungal crude extracts

A low molecular weight factor obtained from the crude extract of nit-1 mutant of Neurospora crassa (NEF) was capable of stimulating the activity of reconstituted NADPH specific nitrate reductase (NR, EC 1.6.6.3; Z.Z. Alikulov, N.A. Savidov, H.S. Lips, A low molecular weight regulator of nitrate reductase in higher plants. 4th International Symposium on Inorganic Nitrogen Assimilation and 1st Fohs Biostress Symposium. Seeheim/Darmstadt, Germany, 1995, p. 9). This factor eluted from a G15 column as glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P). Like the hexose-phosphates, NEF allowed rapid regeneration of NADPH from the NADP +, resulting from nitrate reduction. Sequential treatment of the NEF-containing fraction with commercial preparations of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.43) and phosphoglucose isomerase (PGI, EC 5.3.1.9) resulted in the oxidation of NEF and the reduction of NADP +. NEF, therefore, was a substrate of G6PDH and 6PGDH in a reaction which reduced NADP + to NADPH. It was concluded that the low molecular weight factor enhancing NR activity isolated from fungal and plant crude extracts (Z.Z. Alikulov, N.A. Savidov, H.S. Lips, A low molecular weight regulator of nitrate reductase in higher plants. 4th International Symposium on Inorganic Nitrogen Assimilation and 1st Fohs Biostress Symposium. Seeheim/Darmstadt, Germany, 1995, p. 9) may be a mixture of hexose phosphates. G6PDH, 6PGDH and PGI were active in crude extracts of plants and fungi used for NR assays. Oxidation of one mole of G6P led to the production of approximately two moles of NADPH. The activities of PGI, G6PDH and 6PGDH exceeded that of NR in both fungal and plant crude extract. Fungal crude extracts exhibited a considerably higher capacity to regenerate NADPH than plant extracts. The addition of G6P during the in vitro NR assay increased the stability of the reaction allowing detection of very low NR activity levels. The stimulation of NR in vitro by NADPH generating systems points out the physiological importance of these systems in nitrate assimilation and in the nitrate reduction assays in situ and in vivo.