Biotransformation of kaurenoic acid by Psilocybe cubensis

Abstract

Biotransformation of ent-kaur-16-en-19-oic acid (1), the bioactive kaurene diterpenoid isolated from the stem bark of Croton oblongifolius Roxb., was carried out using Psilocybe cubensis. The incubation resulting in three hydroxylated products; ent-16β,17-dihydroxy-kauran-19-oic acid (2) was isolated after 2 days of incubation and the two novel metabolites; ent-12α,16β,17-trihydroxy-kauran-19-oic acid (3) and ent-11α,16β,17-trihydroxy-kauran-19-oic acid (4) were obtained after incubation for 9 days. The metabolites were identified by spectroscopic methods and X-ray crystallography. The biological activities of each compound were evaluated by cytotoxicity test against 6 tumor cell lines; K562 (human promyelocytic leukemia cells), HEP-G2 (hepatoma), SW620 (colon), Chago (lung), KATO-3 (gastric) and BT474 (breast) and anti-bacteria activity against 4 bacteria; Bacillus cereus, Staphylococcus aureus ATTC 25923, Escherichia coli ATTC 25922 and Pseudomonas aeruginosa ATTC 27853. The results revealed that all products exerted lower levels of biological activities than their parent compound in cytotoxicity testing towards 6 cell lines and were inactive for anti-bacteria activity. The enzyme responsible for compound 2 production was inducible in 2-day grown culture supplemented with ent-kaurenoic acid. The enzyme has been identified as an induced oxygenase requiring NADPH and FAD as cofactors and located in the microsomal fraction. The 45 kDa protein from SDS-PAGE was selected for further analysis by MALDI/Tof MS and peptide mass mapping. The result showed the correlation of the peptide fragment with cyt P450 monooxygenase from Aspergillus fumigatus Af293 at sequence coverage of 12 %.