The earth-star basidiomycetous mushroom Astraeus odoratus produces polyhydroxyalkanoates during cultivation on malt extract.

Polyhydroxyalkanoates (PHAs) including poly-3-hydroxybutyrate (P3HB) as secondary metabolisms were in vitro produced by the edible basidiomycetous mushroom Astraeus odoratus during its growth on malt agar extract. Various carbon and nitrogen sources containing cellulose, glucose, glycerol, rice straw powder, soybean meal and peptone were investigated for the growth of basidiomycetous mushrooms. During cultivation, the A. odoratus culture exudated the considerably extracellular fluid up to approx. 2.3 ml on 2% malt extract agar plate within 7 days. The chemical compounds of the exudated fluid were further investigated by Fourier-transform infrared spectroscopy (FTIR) and gas chromatography/mass spectrometry (GC/MS); and its morphology of the lyophilized sample was observed by scanning electron microscope (SEM). FTIR results showed the characteristic bands of OH at 3445 cm-1, CH/CH2/symmetric CH3 (stretch) at 2923 and 2852 cm-1, C=O at 1730 cm-1, asymmetric CH3 (bend) at 1454 and 1414 cm-1, C-O of COO- at 1396 cm-1 and C-O-C at 1223, 1160, 1116, 1058 and 1019 cm-1 which were similar to the absorptive characteristics of P3HB. Methyl ester derivatives of GC/MS results identified 7 compounds including: 3-hydroxybutanoic (monomer of PHB), aminobenzoic, salicylic, hexadecenoic, octadecadienoic, octadecenoic and octadecanoic acids. SEM images revealed a fibriform and porous materials. Hence, the occurrence of PHAs was first described in a basidiomycetous mushroom A. odoratus. Thus, PHAs could be found not only in bacteria and but also in basidiomycetous mushroom, which can be promising target for bioplastics and green environmental studies.

A colorimetric assay for determination of methyl parathion using recombinant methyl parathion hydrolase.

A simple, rapid and sensitive colorimetric dipstick assay for the detection of the organophosphorous insecticide methyl parathion (MPT) residue in vegetables was developed. The assay was based on the hydrolysis of MPT by a recombinant methyl parathion hydrolase (recMPH), the encoding gene of which was isolated from Burkholderia cepacia, a soil bacterium indigenous to Thailand. This reaction generates protons leading to a change in pH that correlates with the amount of MPH present. Hence, the pH indicator bromothymol blue was used to monitor the MPH hydrolysis as the associated color changes can be observed by the naked eye. The recMPH was immobilized on a PVDF membrane to establish a dipstick assay format. The assays could detect MPT residues in spiked vegetable samples at the concentration of 1 mg/L without using analytical instrumentation. The test is reusable and stable for up to 3 months in the absence of any preservatives.

The coprophilous mushroom Coprinus radians secretes a haloperoxidase that catalyzes aromatic peroxygenation.

Coprophilous and litter-decomposing species (26 strains) of the genus Coprinus were screened for peroxidase activities by using selective agar plate tests and complex media based on soybean meal. Two species, Coprinus radians and C. verticillatus, were found to produce peroxidases, which oxidized aryl alcohols to the corresponding aldehydes at pH 7 (a reaction that is typical for heme-thiolate haloperoxidases). The peroxidase of Coprinus radians was purified to homogeneity and characterized. Three fractions of the enzyme, CrP I, CrP II, and CrP III, with molecular masses of 43 to 45 kDa as well as isoelectric points between 3.8 and 4.2, were identified after purification by anion-exchange and size exclusion chromatography. The optimum pH of the major fraction (CrP II) for the oxidation of aryl alcohols was around 7, and an H2O2 concentration of 0.7 mM was most suitable regarding enzyme activity and stability. The apparent Km values for ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)], 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, and H2O2 were 49, 342, 635, 88, and 1,201 microM, respectively. The N terminus of CrP II showed 29% and 19% sequence identity to Agrocybe aegerita peroxidase (AaP) and chloroperoxidase, respectively. The UV-visible spectrum of CrP II was highly similar to that of resting-state cytochrome P450 enzymes, with the Soret band at 422 nm and additional maxima at 359, 542, and 571 nm. The reduced carbon monoxide complex showed an absorption maximum at 446 nm, which is characteristic of heme-thiolate proteins. CrP brominated phenol to 2- and 4-bromophenols and selectively hydroxylated naphthalene to 1-naphthol. Hence, after AaP, CrP is the second extracellular haloperoxidase-peroxygenase described so far. The ability to extracellularly hydroxylate aromatic compounds seems to be the key catalytic property of CrP and may be of general significance for the biotransformation of poorly available aromatic substances, such as lignin, humus, and organopollutants in soil litter and dung environments. Furthermore, aromatic peroxygenation is a promising target of biotechnological studies.