Akihiko Sakurai

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.

A new approach for improving cordycepin productivity in surface liquid culture of Cordyceps militaris using high-energy ion beam irradiation.

Aims:  To obtain a higher cordycepin production using Cordyceps militaris mutant obtained by a new mutagenesis technique called ‘ion beam’.

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 manganese peroxidase by white rot fungi from potato-processing wastewater: Role of amino acids on biosynthesis

The production of manganese peroxidase (MnP) by white rot fungus strain L-25 was carried out using potato-processing wastewater and the effects of amino acids in the potato-processing wastewater was investigated. The MnP was efficiently produced from the wastewater by the addition of glucose and the maximum MnP activity linearly increased with an increase in the glucose concentration. The initial pH affected the cell growth and also the production rate of the MnP. The maximum activity and the production rate of the MnP using the potato-processing wastewater-based medium were higher (ca. 2.5- fold) than that of the basal medium. Moreover, amino acids in the wastewater had significant effects on the MnP production. L-Glutamic acid, L-aspartic acid and L-serine induced the MnP secretion, on the other hand, L-phenylalanine, L-tyrosine, L-leucine and L-lysine inhibited it. The addition of L-leucine and L-lysine caused growth inhibition, while, L-phenylalanine and L-tyrosine blocked the MnP biosynthetic pathway. Ammonium ion released from the L-phenylalanine by the L-phenylalanine ammonia-lyase participated in the repression of the MnP biosynthetic pathway of the strain L-25.

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-His6-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 °C at pH 4.5 were 7.9 mM and 26.3 μmole/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.

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.