Simple and efficient isolation of cordycepin from culture broth of a Cordyceps militaris mutant.

Isolation of cordycepin from the culture broth of Cordyceps militaris mutant was investigated. Based on the solubility curve, three crystallizing processes, temperature shift (process I), pH shift (process II), and pH shift followed by temperature shift (process III) were carried out. Process III was the most promising method regarding both purity and yield.

Formate oxidase, an enzyme of the glucose-methanol-choline oxidoreductase family, has a His-Arg pair and 8-formyl-FAD at the catalytic site.

Formate oxidase of Aspergillus oryzae RIB40 contains an 8-replaced FAD with molecular mass of 799 as cofactor. The ¹H-NMR spectrum of the cofactor fraction obtained from the enzyme indicated that the 8-replaced FAD in the fraction was 8-formyl-FAD, present in open form and hemiacetal form. The oxidation-reduction potentials of the open and hemiacetal forms were estimated by cyclic voltammetry to be -47 and -177 mV vs. Normal Hydrogen Electrode respectively. The structure of the enzyme was constructed using diffraction data to 2.24 Å resolution collected from a crystal of the enzyme. His511 and Arg554 were situated close to the pyrimidine part of the isoalloxazine ring of 8-formyl-FAD in open form. The enzyme had 8-formyl-FAD, the oxidation potential of which was approximately 160 mV more positive than that of FAD, and the His-Arg pair at the catalytic site, unlike the other enzymes belonging to the glucose-methanol-choline oxidoreductase family.

Production of cordycepin by a repeated batch culture of a Cordyceps militaris mutant obtained by proton beam irradiation.

Cordycepin (3'-deoxyadenosine) is one of the most versatile metabolites of Cordyceps militaris due to its broad spectrum of biological activity. In our previous study, the C. militaris mutant G81-3, which produces higher levels of cordycepin, was obtained by high-energy proton beam irradiation. In this study, the effects of adenosine on cordycepin production in a surface liquid culture of the mutant and the wild type strains were investigated. For the mutant strain, the optimum dose of adenosine yielded a 30% increase in cordycepin production; the maximum levels of production with adenosine and without adenosine were 8.6g/l and 6.7 g/l, respectively. In contrast, the increase due to adenosine supplementation for the wild type strain was only 15% (3.1g/l with adenosine and 2.7 g/l without adenosine). Furthermore, a repeated batch culture, an efficient production method, was carried out to eliminate the relatively long lag phase of the mutant culture. Over four cycles, both the mutant and the wild type strain maintained a production level of more than 85% of that of the initial cycle. As a result, the disadvantage of the mutant was successfully overcome, resulting in a productivity (0.48 g/(ld)) higher than that of the batch culture (0.29 g/(ld)). The productivity for cordycepin obtained in this study is the highest reported value to date, and this method could be applied to large-scale production of cordycepin at industrial levels.

Medicinal uses of the mushroom Cordyceps militaris: current state and prospects.

Cordyceps militaris is a potential harbour of bio-metabolites for herbal drugs and evidences are available about its applications for revitalization of various systems of the body from ancient times. Amongst all the species, C. militaris is considered as the oldest source of some useful chemical constituents. Besides their popular applications for tonic medicine by the all stairs of the community, the constituents of C. militaris are now used extensively in modern systems of medicine. The current survey records the mysterious potentials of C. militaris are boosting up the present herbal treatments, as well as gearing up the green pharmacy revolution, in order to create a friendly environment with reasonable safety. Evidence showed that the active principles of C. militaris are beneficial to act as pro-sexual, anti-inflammatory, anti-oxidant/anti-aging, anti-tumour/anti-cancer/anti-leukemic, anti-proliferative, anti-metastatic, immunomodulatory, anti-microbial, anti-bacterial, anti-viral, anti-fungal, anti-protozoal, insecticidal, larvicidal, anti-fibrotic, steroidogenic, hypoglacaemic, hypolipidaemic, anti-angiogenetic, anti-diabetic, anti-HIV, anti-malarial, anti-fatigue, neuroprotective, liver-protective, reno-protective as well as pneumo-protective, let alone their other synergistic activities, which let it be marketable in the western countries as over-the-counter medicine. A number of culture techniques for this mushroom have been noticed, for example, storage/stock culture, pre-culture, popular/indigenous culture (spawn culture, husked rice culture and saw dust culture) and, special/laboratory culture (shaking culture, submerged culture, surface liquid culture and continuous/repeated batch culture). The prospects for herbal biotechnology regarding drug discovery using C. militaris delivering what it has promised are high, as the technology is now extremely more powerful than before. This study chiefly highlights the medicinal uses of the mushroom C. militaris including its culture techniques, also aiming to draw sufficient attention of the researchers to the frontier research needs in this context.

Expression in Escherichia coli of an unnamed protein gene from Aspergillus oryzae RIB40 and cofactor analyses of the gene product as formate oxidase.

An unnamed protein of Aspergillus oryzae RIB40 (accession no. XP_001727378), the amino acid sequence of which shows high similarity to those of formate oxidase isoforms produced by Debaryomyces vanjiriae MH201, was produced in Escherichia coli in C-His(6)-tagged form. The gene product, purified by affinity column chromatography, catalyzed the oxidation of formate to yield hydrogen peroxide but showed no evidence of activity on the other substrates tested. The K(m) and V(max) values at 30 degrees C at pH 4.5 were 7.9 mM and 26.3 micromole/min mg respectively. The purified enzyme showed UV-visible spectra atypical of ordinary flavoproteins. The UV-visible spectra of the enzyme and the UV-visible spectra, fluorescence spectra, and mass spectrometry of the extract obtained by boiling the purified enzyme suggested that the enzyme has a non-covalently bound FAD analog, which is expected to be 8-formyl-FAD.

Degradation of bisphenol A using sonochemical reactions.

The sonochemical degradation of bisphenol A in aqueous solution, a suspected endocrine disruptor, which can cause several damages for humans, animals and the environment, was investigated at different ultrasonic intensities under air atmosphere. Bisphenol A (0.50mM) was completely degraded after 10, 3 and 2h of ultrasonic irradiation at a frequency of 404kHz, and intensities of 3.5, 9.0 and 12.9kW/m(2), respectively. During ultrasonic irradiation, some aromatic intermediates such as 2-(4-hydroxyphenyl)-2-(3,4-dihydroxyphenyl)propane, commonly known as 3-hydroxybisphenol A were detected. Further cleavage of the aromatic rings resulted in other products, like formaldehyde and organic acids, also being detected. The proposed pathways of bisphenol A degradation by ultrasonic irradiation are based on the above-mentioned intermediates. The relationship between bisphenol A degradation and formation of hydrogen peroxide and nitric acid was taken into account, correlating this to the radicals that take part in the degradation process. In order to optimize the performance of the ultrasonic system, additional experiments using Fenton-like reactions were also carried out. However, the addition of iron (II) sulfate (FeSO(4)) did not increase bisphenol A degradation rates. Compared with the system without iron (II) sulfate, the total organic carbon concentration (TOC) was reduced by about 30%, at 404kHz and 9.0kW/m(2).

A new development of dyestuffs degradation system using ultrasound.

Dyestuffs are often present in industrial wastewaters and can consist of hazardous substances which have a serious impact on the environment and personal health. This report describes a system developed to degrade these substances using sonochemical reactions. Ultrasonic frequencies of 118, 224, 404 and 651 kHz and power input values of 11.4, 29.0 and 41.5 W were tested on Rhodamine B and Orange II dyestuff solutions in order to find the best degradation conditions. The ultrasonic irradiation of air-saturated solutions produces free radicals that combine and generates hydrogen peroxide, and compared to the production of hydrogen peroxide when irradiating water, a decrease was found during the irradiation to dyestuff solutions, indicating that some of the free radicals were consumed in the dyestuffs degradation process. The effects of the ultrasonic irradiation conditions on the pH, nitric and nitrous acid formations as well as the total organic carbon value (TOC) were also investigated. For the ultrasonic frequencies of 224, 404 and 651 kHz, the degradation rates were very similar, however, the 118 kHz system presented a degradation rate of about one-third that of the higher frequencies for both dyestuffs. The Rhodamine B solutions were decolorized within 2 h of ultrasonic irradiation for all systems with the exception of the 118 kHz one. For Orange II, except for the 118 kHz system, all solutions were decolorized within 4 h of ultrasonic irradiation. All reactions were carried out at 25 degrees C and the total ultrasonic irradiation time was 10 h.