Purification and characterization of an acid phosphatase from the commercial mushroom Agaricus bisporus

Acid phosphatase [AP; EC 3.1.3.2], a key enzyme involved in the synthesis of mannitol in Agaricus bisporus, was purified to homogeneity and characterized. The native enzyme appeared to be a high molecular weight type glycoprotein. It has a molecular weight of 145 kDa and consists of four identical 39-kDa subunits. The isoelectric point of the enzyme was found at 4.7. Maximum activity occurred at 65°C. The optimum pH range was between 3.5 and 5.5, with maximum activity at pH 4.75. The enzyme was unaffected by EDTA, and inhibited by tartrate and inorganic phosphate. The enzyme exhibits a K _m for p-nitrophenylphosphate and fructose-6-phosphate of 370 M and 3.1 mM, respectively. A broad substrate specificity was observed with significant activities for fructose-6-phosphate, glucose-6-phosphate, mannitol-1-phosphate, AMP and -glycerol phosphate. Only phosphomonoesters were dephosphorylated. Antibodies raised against the purified enzyme could precipitate AP activity from a cell-free extract in an anticatalytic immunoprecipitation test.     

Purification and characterization of NADP-dependent glutamate dehydrogenase from the commercial mushroom Agaricus bisporus

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent glutamate dehydrogenase (NADP-GDH) of Agaricus bisporus, a key enzyme in ammonia assimilation, was purified to apparent electrophoretic homogeneity with 27% recovery of the initial activity. The molecular weight of the native enzyme was 330 kDa. The enzyme is probably a hexamer, composed of identical subunits of 48 kDa. The isoelectric point of the enzyme was found at pH 4.8. The N-terminus appeared to be blocked. The enzyme was specific for NADP(H). The K_m-values were 2.1, 3.2, 0.074, 27.0, and 0.117mM for ammonia, 2-oxoglutarate, NADPH, L-glutamate, and NADP respectively. The pH optima for the amination and deamination reactions were found to be 7.6 and 9.0, respectively. The temperature optimum was 33°C. The effect of several metabolites on the enzyme's activity was tested. Pyruvate, oxaloacetate, ADP, and ATP showed some inhibitory effect. Divalent cations slightly stimulated the aminating reaction. Antibodies raised against the purified enzyme were able to precipitate NADP-GDH activity from a cell-free extract in an anticatalytic immunoprecipitation test. Analysis of a Western blot showed the antibodies to be specific for NADP-GDH.     

Purification and characterization of RNA polymerase II resistant to alpha-amanitin from the mushroom Agaricus bisporus

The DNA-dependent RNA polymerases II or B (ribonucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from the mushroom Agaricus bisporus has been purified to apparent homogeneity. The purification procedures involve precipitation with polyethylenimine, selective elution of RNA polymerase II from the polyethylenimine precipitate, ammonium sulfate fractionation, DEAE-cellulose chromatography, CM-cellulose chromatography, and exclusion chromatography on Bio-Gel A-1.5M. With this procedure 11 mg of RNA polymerase II is recovered from 1.5 kg of mushroom tissue. RNA polymerase II from Agaricus bisporus has 12 subunits with the following molecular weights: 182,000, 140,000, 89,000, 69,000, 53,000, 41,000, 37,000, 31,000, 29,000, 25,000, 19,000, and 16,500. Purified RNA polymerase II from Agaricus bisporous was half-maximally inhibited by the mushroom toxin alpha-amanitin at a concentration of 6.5 microgram/mL (7 X 10(-6) M), which is 650-fold more resistant than mammalian RNA polymerases II. The apparent Ki for the alpha-amanitin-RNA polymerase complex was estimated to be 12 X 10(-6) M. The activity of purified RNA polymerase II from the mushroom was quite typical of other eukaryotic RNA polymerase II with regard to template preference, salt optima, and divalent metal cation optima.     

Purification and characterization of four isolectins of mushroom (Agaricus bisporus)

Four lectins were purified from a mushroom (Agaricus bisporus) by ammonium sulfate fractionation, anion-exchange chromatography, affinity chromatography on bovine submaxillary mucin-Sepharose 4B and preparative isoelectric focusing. They were designated as ABA-I (pI 6.70), II (pI 5.98), III (pI 5.69) and IV (pI 5.53). Polyacrylamide gel electrophoresis of each lectin in the presence of sodium dodecyl sulfate gave a single band with an apparent molecular mass of 16 000 Da. Sedimentation equilibrium analysis suggested that each lectin is a tetramer of subunits. The four lectins were found to have quite similar carbohydrate-binding specificities. The hemagglutination activities of the lectins were effectively inhibited by bovine and porcine submaxillary mucins (BSM and PSM), and NH2-terminal glyco-octapeptides obtained by cyanogen bromide cleavage of human erythrocyte glycophorin A. In addition, desialylated PSM-glycopeptides were more potent inhibitors than untreated PSM-glycopeptides. Among monosaccharides and their glycosides, only methyl N-acetyl-alpha-galactosaminide inhibited lectin binding at a high concentration, but a synthetic oligosaccharide, O-beta-galactopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-alpha-D- galactopyranosyl)-N-tosyl-L-serine, was a strong inhibitor.     

Purification and characterization of an extracellular prolyl endopeptidase from Agaricus bisporus

Prolyl endopeptidase [EC 3.4.21.26] was purified to homogeneity from the culture filtrate of Agaricus bisporus by a procedure that comprised ammonium sulfate fractionation, anion-exchange chromatographies on DEAE-Toyopearl and DEAE-Sephadex, hydroxylapatite chromatography, and high-performance liquid chromatography (HPLC) on a TSKgel G 2000 SW column. The overall activity recovery was 8.6%. The enzyme was most active at or around pH 7.5 and was stable in the range of pH 5-9 when checked with Z-Gly-Pro-beta-naphthylamide as a substrate. The isoelectric point of the enzyme was about 4.8. The enzyme was a monomeric protein of molecular weight 78,000 +/- 2,000 as judged by gel permeation chromatography on Sephadex G-150 and electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide gel. The enzyme hydrolyzed Pro-X bonds and at least five subsites (S3, S2, S1, S1', and S2') were found to be involved in enzyme-substrate binding. Among them, S2, S1, and S1' subsites of the enzyme showed high stereospecificity. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP), Z-Gly-Pro-CH2Cl, Z-Pro-prolinal, Z-Pro-pyrrolidine, Z-Thiopro-pyrrolidine, Z-Pro-thiazolidine, Z-Thioprothiazolidine, and p-chloromercuribenzoate (PCMB), while it was not inhibited by phenyl-methylsulfonyl fluoride (PMSF), E-64, iodoacetamide, or metal chelators. Although the A. bisporus enzyme showed no immunological cross reaction with anti-bovine prolyl endopeptidase antiserum, the other characteristics were quite similar to those of mammalian and plant enzymes.     

Purification and characterization of glutamine synthetase from Rhodomicrobium vannielii

Abstract Glutamine synthetase (GS) from the purple non-sulfur bacterium Rhodomicrobium vannielii has been purified to electrophoretic homogeneity by affinity chromatography. Molecular weight and catalytic properties of the enzy,e are similar to those described for other species of Rhodospirillaceae. However, the enzyme from this organism appears to be antigenically different from the glutamine synthetases of other species of Rhodospirillaceae studied.     

Volatile compounds from the mycelium of the mushroom Agaricus bisporus

The pattern of volatiles from the mycelium of two commercial strains of Agaricus bisporus, grown in axenic culture on a semi-synthetic medium, was found to be broadly similar to that of the volatiles identified from sporophores. Tetrachloro-1,4-dimethoxybenzene, a known secondary metabolite of several Basidiomycetes, was found in the mycelium though not in the sporophores. [³⁶Cl]Tetrachloro-1,4-dimethoxybenzene was obtained when sodium [¹³Cl]chloride was added to the medium.     

RAPD discrimination of Agaricus bisporus mushroom cultivars

Cultivars of the white button mushroom Agaricus bisporus are difficult to differentiate, which has made strain protection problematic for this crop species. We have used RAPDs to discriminate between 26 strains of A. bisporus, 24 of which were commercial cultivars, and to characterise the genetic relatedness of these strains. Using 20 primers, 211 RAPD markers were identified and used in hierarchical cluster, patristic distance and parsimony analyses. All strains could be differentiated using the aggregated primer data. Although no one primer could differentiate all 26 strains, several individual primers yielded unique fingerprints for a variety of strains. The greatest differences (up to 28% variation) were observed in comparisons with or between two wild collections of A. bisporus. Quondam cultivars, commercial brown and off-white varieties proved more variable than the widely grown 'hybrid' types. Of the 15 hybrid varieties analysed, only one differed substantially (20% or more variable). The patristic and parsimony analyses both demonstrated the gross similarity of the hybrids, many of which appear to be essentially derived varieties from two original hybrid cultivars. RAPD analyses can assist mushroom strain identification and could play a role in the protection of novel cultivars.     

Purification and characterization of glutamine synthetase from the archaebacterium Methanobacterium ivanovi.

Glutamine synthetase (GS) was purified to electrophoretic homogeneity from the obligate anaerobic archaebacterium Methanobacterium ivanovi. The 130-fold-purified enzyme was obtained by heat treatment, ion-exchange chromatography, and gel filtration. Like all other eubacterial GSs known so far, the GS of M. ivanovi was found to be a dodecamer of about 600,000 daltons composed of a single type of subunit. The enzyme was stable at 63 degrees C for 10 min and was not sensitive to oxygen. The isoelectric point was 4.6, and the optimum pH of gamma-glutamyltransferase activity was 8.0. The Km values for hydroxylamine, glutamine, and ADP in the transferase reaction were 6.8, 22.7, and 0.35 mM, respectively. L-Methionine-DL-sulfoximine strongly inhibited the activity. Like the GS from gram-positive bacteria, Anabaena sp., several yeasts, and mammals, the enzyme from M. ivanovi was not regulated by adenylylation as demonstrated by snake venom phosphodiesterase treatment. Inhibition of the transferase activity by L-alanine, glycine, L-histidine, and L-tryptophan was observed. L-Glutamine alone or in the presence of AMP did not inhibit the GS synthetic activity. The GS of Methanobacterium ivanovi did not cross-react with a variety of antisera against GS from Escherichia coli, Anabaena strain 7120, or Bacillus megaterium. Archaebacterial GS appears to be structurally and functionally similar to eubacterial GS in gram-positive bacteria.     

Isolation and properties of chitin synthetase from Agaricus bisporus mycelium

Using the standard procedure for the isolation of chitosomes (S. Bartnicki-Garcia, C. E. Bracker, E. Reyes, and J. Ruiz-Herrera, 1978, Exp. Mycol. 2, 173) a 50-fold purification has been achieved of chitin synthetase (ChS) from Agaricus bisporus hyphae grown in stirred, pellet-free liquid culture. Some properties of the enzyme were determined and compared with those of chitosomal ChS of other organisms. Effects of some antifungal compounds upon enzyme activity were also investigated. Inclusion of digitonin into the extraction medium afforded a second species of chitosomes, which have a higher sedimentation coefficient than the “standard” chitosomes. As regards ultrastructure, behavior in sucrose density gradients, level of activities, zymogenicity, cofactor requirements, kinetics, apparent K_m values for UDPGlcNAc and GlcNAc, inhibitory constants for nikkomycin X, nikkomycin Z, and amphotericin B methyl ester (AME), as well as modulation by digitonin, the mushroom chitosomal ChS displayed basically the same features as the corresponding enzyme from Mucor rouxii yeast cells. Some differences were, however, observed: Rennilase was almost ineffectual in overcoming latency of the enzyme, and trypsin performed satisfactorily only in a narrow concentration range, whereas pronase was very good. Furthermore, stimulation of ChS by digitonin in low concentrations was considerably higher (attaining more than 300%) with the mushroom than with the Mucor enzyme. The strong stimulatory effect of digitonin seems to be specific for the spirostanol glycoside since it could be produced by neither the closely related saponin α-tomatine, nor by AME—both compounds, like digitonin, sterol-complexing agents. The suggestion is made that stimulation of ChS by digitonin may be causally related to its antimycotic activity. Several possibilities are discussed as to its mode of action in stimulating ChS.     

Purine degradation in the edible mushroom Agaricus bisporus

Agaricus bisporus is able to use urate, allantoin, allantoate, urea and alloxanate as nitrogen sources for growth. The presence of urate oxidase, allantoinase, ureidoglycolase and urease activities, both in fruit bodies and mycelia, points to a degradative pathway for urate similar to that found in various microorganisms. So far all efforts, to demonstrate the enzyme responsible for allantoate degradation failed. A urease inhibitor appeared to be present in cell-free extracts, from fruit bodies.     

Microbial ecology of the Agaricus bisporus mushroom cropping process

Applied Microbiology and Biotechnology - Agaricus bisporus is the most widely cultivated mushroom species in the world. Cultivation is commenced by inoculating beds of semi-pasteurised composted...     

Purification and partial characterization of glutamine synthetase from the photosynthetic bacterium Rhodospirillum rubrum

Glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) from the photosynthetic bacterium Rhodospirillum rubrum grown under nitrogen fixing conditions has been purified to homogeneity. The purification procedure involves affinity chromatography on ADP-agarose type 2 as the major purification step. The recovery in the purification is 70%. The specific activity of the purified enzyme is about 10-times higher in the gamma-glutamyl transferase assay than in the coupled biosynthetic assay. The molecular weight was determined to 530,000 by native gradient polyacrylamide gel electrophoresis and to 500,000 by gel filtration. The subunits have an apparent molecular weight of 52,000. Glutamine synthetase isolated from Rsp. rubrum which had been exposed to ammonium ions ('switch-off') before harvest had about 20% of the transferase activity compared with the enzyme purified from nitrogen-starved cells. The low-activity form showed two bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Submerged growth of the cultivated mushroom,Agaricus bisporus

Agaricus bisporus grew well in submerged culture in a medium containing malt extract, phosphate, and casein. Moderate growth occurred in defined media containing glucose, asparagine, phenylalanine, vitamins and minerals. Other amino acids did not stimulate growth. Growth was stimulated by vegetable oils, partly due to utilization of the oils, and partly to a more complex mechanism. Oleates had the same effect as vegetable oils; palmitates a lesser one. In shaken flasks maximum yield was reached after 22–24 days and in stirred and aerated fermentors after 8–10 days. Besides dry weight of mycelium, laccase activity was determined. The latter determination is suitable for a rapid estimation of the growth in routine experiments. The flavour of the mycelium was like that of mushrooms but weaker. It was strongest in standing liquid cultures and on solid media. The mycelium grown in submerged culture was suitable as spawn for mushroom culture. Presented at the First International Mycological Congress, Exeter, 7–16 September 1971, and at the Meeting of the Netherlands Society for Microbiology, Rotterdam, 8 December 1971. We thank the Mushroom Experiment Station, Horst, the Netherlands, for kindly supplying the compost for fructification experiments and Mr. P. Arntz, M.Sc., for advice in the fermentor work. F. IJ. Dijkstra is indebted to the Royal Netherlands Fermentation Industries (Gist-Brocades), Delft, for a research grant.