| Yazarlar |
Dr. Öğr. Üyesi Yunus ENSARİ
Kafkas Üniversitesi, Türkiye |
Gaurao V Dhoke
Türkiye |
|
Mehdi Dolatabadi Davari
Türkiye |
|
Marco Bocola
Türkiye |
|
Anna Joelle Ruff
Türkiye |
|
Ulrich Schwaneberg
Türkiye |
| Özet |
| Expanding the substrate scope of enzymes opens up new routes for synthesis of valuable chemicals. Ketone-functionalized fatty acid derivatives and corresponding chiral alcohols are valuable building blocks for the synthesis of a variety of chemicals including pharmaceuticals. The alcohol dehydrogenase from Candida parapsilosis (cpADH5) catalyzes the reversible oxidations of chiral alcohols and has a broad substrate range; a challenge for cpADH5 is to convert alcohols with small substituents (methyl or ethyl) next to the oxidized alcohol moiety. Molecular docking studies revealed that W286 is located in the small binding pocket and limits the access to substrates that contain aliphatic chains longer than ethyl substituent. In the current manuscript, we report that positions L119 and W286 are key residues to boost oxidation of medium chain methyl 3-hydroxy fatty acids; interestingly the enantiopreference toward methyl 3-hydroxybutyrate was inverted. Kinetic characterization of W286A showed a 5.5 fold increase of Vmax and a 9.6 fold decrease of Km values toward methyl 3-hydroxyhexanoate (Vmax: 2.48 U mg− and Km: 4.76 mm). Simultaneous saturation at positions 119 and 286 library yielded a double mutant (L119M/W286S) with more than 30-fold improved activity toward methyl 3-hydroxyoctanoate (WT: no conversion; L119M/W286S: 30 %) and inverted enantiopreference (S-enantiomer ≥99 % activity decrease and R-enantiomer >20-fold activity improvement) toward methyl 3-hydroxybutyrate. |
| Anahtar Kelimeler |
| alcohol dehydrogenase | directed evolution | fatty acid methyl ester | methyl 3-hydroxyalkanoate | protein engineering |
| Makale Türü | Özgün Makale |
| Makale Alt Türü | SSCI, AHCI, SCI, SCI-Exp dergilerinde yayımlanan tam makale |
| Dergi Adı | CHEMISTRY-A EUROPEAN JOURNAL |
| Dergi ISSN | 0947-6539 |
| Dergi Tarandığı Indeksler | SCI |
| Makale Dili | İngilizce |
| Basım Tarihi | 09-2017 |
| Cilt No | 23 |
| Sayı | 51 |
| Sayfalar | 12636 / 12645 |
| Doi Numarası | 10.1002/chem.201702581 |
| Atıf Sayıları | |
| WoS | 5 |
| SCOPUS | 5 |
| Google Scholar | 6 |
