4.7 Article

Functional analysis of recombinant menthone menthol reductase by chiral GC and GC-MS

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INDUSTRIAL CROPS AND PRODUCTS
卷 184, 期 -, 页码 -

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ELSEVIER
DOI: 10.1016/j.indcrop.2022.115075

关键词

Recombinant Menthone Menthol Reductase (MMR); Chiral GC; GC-MS; MALDI-TOF MS; Enantioselective (-)-Menthol

资金

  1. Medicinal Plants and Drugs Research Instituite

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In this study, a cell-free reaction for the production of (-)-menthol was introduced. Recombinant MMR protein was expressed in Escherichia coli and purified with high efficiency. The reductase activity and stereospecific activity of recombinant MMR were analyzed, and (-)-menthol with expected chirality was successfully synthesized.
Menthol in its L-isomer is a key flavoring additive mainly due to its cold-mimicking characteristics. Production of menthol in preferred L-isoform has been studied in different ways through cell-based or multiple-step biocatalyst processes. Herein, we introduce a single-step, cell-free reaction for (-)-menthol production. In this study, recombinant MMR (Menthone Menthol Reductase) was codon-optimized and expressed in Escherichia coli (E. coli) BL21 (DE3). The His-tagged protein was purified with similar to 90% efficiency using Ni-NTA affinity chromatography. The identity of the recombinant MMR was confirmed using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-TOF MS) through in-gel digestion process and analyzing the extracted peptides in NCBI-prot database. The reductase activity of recombinant MMR in the presence of NADPH (as a cofactor) was examined through monitoring the UV-Vis absorption by NADPH at 340 nm during NADP(+)/ NADPH oxidation-reduction reaction. The stereospecific activity of recombinant MMR was analyzed through its interaction with (-)-menthone substrate using Chiral GC and GC-MS techniques. We observed the expected chirality (minus) in resulted menthol with 68% yield.

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