Sclareol synthesis

Yield:-

Reaction Conditions:

with N glutinosa sclareol synthase NgSs₃ in aq. buffer at 28;Catalytic behavior;Enzymatic reaction;

Steps:

7.B B. Enzyme Activity Assay
B. Enzyme Activity Assay (0433) The N glutinosa sclareol synthase (NgSs3) was further characterized by assaying its ability to convert labdenediol diphosphate (LPP) to sclareol. An LPP enzyme reaction mix was generated by mixing 100 μM geranylgeranyl diphosphate (GGPP), 1 mM dithiothreitol (DTT) in LPP assay buffer (described in Example 4B) containing 10 μl of LPP synthase, purified from S. Sclarea (see Example 4A) and incubated for 4 hours at 28° C. Five hundred (500) μL aliquots of the prepared LPP reaction mix were then incubated with twenty microliters of NgSs3 or SsSs3, affinity-purified enzyme or crude extracts thereof, or a negative control crude extract, overnight at 28° C. (0434) The reaction product was extracted with ethyl acetate and analyzed by gas chromatography-mass spectrometry (GC-MS). GC-MS analysis was performed as described in Example 4B. The initial oven temperature was 80° C., which was increased to 275° C. with a ramping rate of 10° C./min. The identity of the product(s) was confirmed by concordance of the retention times and comparison of the spectra obtained from each of the samples with those obtained from known standards. (0435) The crude extracts, containing either Ss sclareol synthase or Ng sclareol synthase, S3F2-3, produced a main product with a retention time of 17.95 and 17.94 min, respectably, which was similar to the retention time of the sclareol standard (17.96 min), while the sample treated with the negative control crude extract did not show any visible sclareol peak. The main product of the reaction using affinity-purified SsSs and NgSs3 and enzymes also generated sclareol peaks, on the chromatogram, at 17.94 min. The MS spectra, corresponding to the retention time of 17.95 min were identical throughout samples and for the sclareol standard, demonstrating that the NgSs3 enzyme is a sclareol synthase.

References:

Evolva, Inc.;Park, Grace Eunyoung;Julien, Bryan N.;Burlingame, Richard US9353385, 2016, B2 Location in patent:Page/Page column 83