Identification and Isolation of Glucosytransferases (GT) Expressed Fungi Using a Two-Photon Ratiometric Fluorescent Probe Activated by GT.

As is well-known, fungi are an important biocatalysis model of glucosylation and have been widely applied for bioactive compounds glucosylation mediated by the intracellular glucosytransferases (GTs). However, there is no efficient method for the real-time detection of GTs and the rapid isolation of the target fungi strains with the high expression of GTs. In the present work, we first developed a two-photon ratiometric fluorescent probe N-( n-butyl)-4-hydroxy-1,8-naphthalimide (NHN) for detecting the glucosyltransferases activity and intracellular imaging of GTs. Under UV light (365 nm), the transformed product of NHN mediated by intracellular glucosyltransferase displayed blue emission to guide the rapid isolation of fungal strains possessing overexpression of GTs from complex soil samples. Finally, by using the fluorescent probe, two target fungi were isolated and identified to be Rhizopus oryzae and Mucor circinelloides by molecular analysis, and they exhibited a robust capability for regio- and stereospecific O-glycosylation. Our results fully demonstrated that NHN may be a promising tool for guiding real-time GTs activity in fungal strains and even for developing natural fungal strains with GTs overexpression.

Diterpenoids from the roots of Euphorbia ebracteolata and their inhibitory effects on human carboxylesterase 2.

A chemical investigation of the roots of Euphorbia ebracteolata identified eighteen diterpenoids and glycosides. On the basis of spectroscopic data, they were determined to be ent-kauranes, ent-atisanes, tigliane derivatives, ingenane, and ent-abietanes, among which were eleven previously undescribed diterpenoids. The inhibitory effects of the isolated compounds against human carboxylesterase 2 (hCE-2) were evaluated in vitro, which revealed moderate inhibitory effects with IC50 values < 50 µM. Next, the inhibitory kinetics were evaluated for the putative hCE-2 inhibitor 4ß,9α,16,20-tetrahydroxy-14(13 → 12)-abeo-12αH-1,6-tigliadiene-3,13-dione (IC50 3.88 µM), and results indicated competitive inhibition with Ki 4.94 µM. Additionally, none of the diterpenoids showed cytotoxic effects against five human tumor cell lines as determined by MTT assays.