Cargando…
Engineering a Bacillus subtilis esterase for selective hydrolysis of d, l-menthyl acetate in an organic solvent-free system
Esterase/lipase-catalyzed selective hydrolysis of d, l-menthyl esters has become one of the promising approaches for producing l-menthol, one of the most important flavoring chemicals with extensive uses. However, the activity and l-enantioselectivity of the biocatalyst are not sufficient for meetin...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Royal Society of Chemistry
2023
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10068921/ https://www.ncbi.nlm.nih.gov/pubmed/37021103 http://dx.doi.org/10.1039/d3ra00490b |
Sumario: | Esterase/lipase-catalyzed selective hydrolysis of d, l-menthyl esters has become one of the promising approaches for producing l-menthol, one of the most important flavoring chemicals with extensive uses. However, the activity and l-enantioselectivity of the biocatalyst are not sufficient for meeting the industrial requirements. Herein, a highly active para-nitrobenzyl esterase from Bacillus subtilis 168 (pnbA-BS) was cloned and then engineered to enhance its l-enantioselectivity. On the basis of the strategy tailoring the steric exclusion effect and structural flexibility of the region adjacent to the substrate, the substitution of Ala400 to Pro caused a remarkable improvement in the E value from 1.0 to 466.6. The variant A400P was purified and further confirmed with strict l-enantioselectivity in the selective hydrolysis of d, l-menthyl acetate, whereas the improved l-enantioselectivity caused decreased activity. To develop an efficient, easy-to-use, and green methodology, organic solvent was omitted and substrate constant feeding was integrated into the whole-cell catalyzed system. During the catalytic process, the selective hydrolysis of 1.0 M d, l-menthyl acetate in 14 h offered a conversion of 48.9%, e.e.(p) value of >99%, and space-time yield of 160.52 g (l d)(−1). |
---|