Making the scent of ambergris without relying on naturally occurring lumps: one step toward that goal is to synthesize its major component, ambrein, with enzymes and then use light to turn it into fragrance compounds.
In a 2020 study, researchers modified an enzyme from bacteria and combined it with another enzyme, raising the in-vitro yield of ambrein to 46.0%. They also investigated how different light treatments changed the composition of the volatile compounds produced. Alongside fragrance production, the study examined the effects of the resulting ambrein on cultured cells.
Making a fragrance precursor, then using light to create scent
Ambrein in ambergris undergoes photooxidation, which breaks the molecule apart to produce smaller, volatile compounds. The researchers aimed to build an artificial route that would efficiently supply this starting material and convert it into fragrance compounds.
The starting point was squalene. Using the cyclase BmeTC, derived from the bacterium Bacillus megaterium, they carried out reactions that form ring structures at both ends of the elongated molecule. They produced the enzyme in Escherichia coli, extracted it, and used gas chromatography, mass spectrometry, and other methods to examine how amino-acid substitutions affected the products.
Combining a modified enzyme with the original enzyme increased the yield
Adding another substitution, Y167A, to the previously modified enzyme D373C increased the ambrein yield from 2.2% to 21.5% in a comparison using purified enzymes. This double-mutant enzyme was particularly effective at converting an intermediate into ambrein.
The researchers therefore combined it with the wild-type, or original, enzyme, which promotes the first step. Even with the total amount of enzyme held constant, the yield reached 46.0%, approximately 20 times that of the earlier modified enzyme. Dividing the work between enzymes suited to different steps helped improve the result.
These were in-vitro results obtained using purified enzymes. The paper also discusses the prospect of increasing production in yeast, but productivity in large-scale cultures remains to be tested. Nor does this artificial route identify the reaction pathway operating inside sperm whales.
Light-treatment conditions can also change fragrance composition
The researchers tested two methods on the synthesized ambrein: six weeks of ultraviolet irradiation, and four hours of visible-light irradiation with an added photosensitizer. A photosensitizer is a substance that uses light energy to help drive a reaction. They compared three: rose Bengal, tetraphenylporphine, and methylene blue. Solvents and other conditions also differed between photosensitizers, so this was not a comparison that isolated the photosensitizer alone.
Under ultraviolet irradiation, the yield of the measured volatile compounds was approximately 1%. With photosensitizers and visible light, it reached 8–15%, and less unreacted ambrein remained. These values indicate compound production, not odor intensity itself.
The products included four known compounds, such as ambrox, and six compounds whose structures had not been determined. The ultraviolet and rose Bengal conditions produced compositions relatively close to those of the two natural ambergris samples used for comparison. The other photosensitizers increased the proportions of particular compounds, changing the composition.
In scent comparisons using filter paper after the solvent had evaporated, the ultraviolet and rose Bengal products were also reported to resemble the natural samples, whereas products from the other conditions differed. However, the number of assessors and statistical tests were not reported for this scent comparison. At this stage, the results point both toward approximating natural ambergris and toward creating different compositions.
Two different effects were also investigated in cultured cells
Making ambrein easier to obtain also makes its biological activities easier to study. The researchers used bone-related cells and human-derived neuroblastoma cells.
In the bone experiments, no significant change was seen in an indicator of osteoblasts, the cells that build bone. In contrast, adding ambrein promoted differentiation into osteoclasts, the cells that resorb bone. Thus, no change in osteoblasts was confirmed, whereas increased differentiation into osteoclasts was observed.
In the neuroblastoma-cell experiments, ambrein was applied for 24 hours, after which the culture medium was removed and amyloid β was added. Amyloid-β-induced cell death was reduced. Both experiments involved adding a substance to cultured cells and must be distinguished from trials of effects on people smelling the fragrance or of therapeutic benefits. The molecular mechanisms responsible for these effects also remain unclear.
Summary
This study advanced both the process of making ambrein and the process of generating volatile compounds from it. Combining the modified and wild-type enzymes increased the yield compared with the earlier modified enzyme, while reaction conditions involving photosensitizers changed the composition of the fragrance compounds produced.
Ambrein also promoted osteoclast differentiation in cultured cells and, in a separate experiment, reduced amyloid-β-induced cell death. Technology that provides a reliable supply of a fragrance precursor can support both fragrance creation and research into the molecule’s biological activities.
The research received support from JSPS KAKENHI and Niigata University’s interdisciplinary research program. ADEKA supplied some ambrein produced and isolated using the method described in this study. The authors declared no competing interests.
Reference
Yamabe Y, Kawagoe Y, Okuno K, et al. Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein. Scientific Reports. 2020;10:19643. Original research paper
