In order to produce cordycepin, the liquid surface culture of
G81-3 was carried out as previously reported (13). The medium
contained 86.2 g/L glucose, 93.8 g/L yeast extract (Difco) and Vogel’s
medium diluted to a 1/10 concentration. The culture was continued
for 33 days at a temperature of 25 1C and a humidity of 30 2%
until the cordycepin production reached a plateau. The pH values of
culture filtrate before and after the cultivation were 6.4 and 7.5,
respectively. The concentration of cordycepin was determined by a
Shimadzu HPLC apparatus (LC-10AT system) with a photodiode
array detector (SPD-M10A) and a reverse phase column (TSK-gel
ODS-80Ts, Tosoh Corp., Japan). The mobile phase consisted of
methanol and 0.1% (v/v) phosphoric acid (2/98, v/v). The flow rate
was 1.0 mL/min, and the column temperature was 40C. The
chromatogram was monitored by the absorbance range between
200 and 800 nm. The authentic standard of cordycepin was purchased from SigmaeAldrich Co. As a result of the surface culture,
more than 99% of the synthesized cordycepin was secreted to the
liquid medium. Some products other than cordycepin (retention
time, approximately 12 min) were detected as shown in Fig. 1A (no
peak was detected between 400 and 800 nm).