首页|Functional analysis of recombinant menthone menthol reductase by chiral GC and GC-MS
Functional analysis of recombinant menthone menthol reductase by chiral GC and GC-MS
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NSTL
Elsevier
? 2022 Elsevier B.V.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 ~ 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 NCBIprot 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.
NSTL
Elsevier
