46 research outputs found
Three-steps in one-pot: whole-cell biocatalytic synthesis of enantiopure (+)- and (−)-pinoresinol via kinetic resolution
Additional file 5. HPLC chromatograms of enantiomeric separations of reaction products. a Application of AtPrR2; b application of FiPLR. [3a] = (+)-pinoresinol 3a, [3b] = (−)-pinoresinol 3b, [4a] = (+)-lariciresinol 4a, [4b] = (−)-lariciresinol 4b, [5a] = (−)-secoisolariciresinol 5a
P450BM3-Catalyzed Oxidations Employing Dual Functional Small Molecules
A set of dual functional small molecules (DFSMs) containing different amino acids
has been synthesized and employed together with three different variants of the cytochrome
P450 monooxygenase P450BM3 from Bacillus megaterium in H2O2-dependent oxidation reactions.
These DFSMs enhance P450BM3 activity with hydrogen peroxide as an oxidant, converting these
enzymes into formal peroxygenases. This system has been employed for the catalytic epoxidation of
styrene and in the sulfoxidation of thioanisole. Various P450BM3 variants have been evaluated in
terms of activity and selectivity of the peroxygenase reactions.MINECO-CTQ2016-76908-C2-1,2-PComisión Europea de Investigación-ERC-648026Unión Europea-H2020-BBI-PPP-2015-2-1-720297Organización Holandesa de Investigación CientÃfica (VICI)-724.014.00
Regioselective biooxidation of (+)-valencene by recombinant E. coli expressing CYP109B1 from Bacillus subtilis in a two-liquid-phase system
<p>Abstract</p> <p>Background</p> <p>(+)-Nootkatone (<b>4</b>) is a high added-value compound found in grapefruit juice. Allylic oxidation of the sesquiterpene (+)-valencene (<b>1</b>) provides an attractive route to this sought-after flavoring. So far, chemical methods to produce (+)-nootkatone (<b>4</b>) from (+)-valencene (<b>1</b>) involve unsafe toxic compounds, whereas several biotechnological approaches applied yield large amounts of undesirable byproducts. In the present work 125 cytochrome P450 enzymes from bacteria were tested for regioselective oxidation of (+)-valencene (<b>1</b>) at allylic C2-position to produce (+)-nootkatone (<b>4</b>) via <it>cis</it>- (<b>2</b>) or <it>trans</it>-nootkatol (<b>3</b>). The P450 activity was supported by the co-expression of putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from <it>Pseudomonas putida </it>in <it>Escherichia coli</it>.</p> <p>Results</p> <p>Addressing the whole-cell system, the cytochrome CYP109B1 from <it>Bacillus subtilis </it>was found to catalyze the oxidation of (+)-valencene (<b>1</b>) yielding nootkatol (<b>2 </b>and <b>3</b>) and (+)-nootkatone (<b>4</b>). However, when the <it>in vivo </it>biooxidation of (+)-valencene (<b>1</b>) with CYP109B1 was carried out in an aqueous milieu, a number of undesired multi-oxygenated products has also been observed accounting for approximately 35% of the total product. The formation of these byproducts was significantly reduced when aqueous-organic two-liquid-phase systems with four water immiscible organic solvents – isooctane, <it>n</it>-octane, dodecane or hexadecane – were set up, resulting in accumulation of nootkatol (<b>2 </b>and <b>3</b>) and (+)-nootkatone (<b>4</b>) of up to 97% of the total product. The best productivity of 120 mg l<sup>-1 </sup>of desired products was achieved within 8 h in the system comprising 10% dodecane.</p> <p>Conclusion</p> <p>This study demonstrates that the identification of new P450s capable of producing valuable compounds can basically be achieved by screening of recombinant P450 libraries. The biphasic reaction system described in this work presents an attractive way for the production of (+)-nootkatone (<b>4</b>), as it is safe and can easily be controlled and scaled up.</p
Three-steps in one-pot: whole-cell biocatalytic synthesis of enantiopure (+)- and (−)-pinoresinol via kinetic resolution
Recent advances in oxygenase-catalyzed biotransformations
Oxygenases continue to be widely studied for selective biooxidation of organic compounds. Protein engineering has resulted in heme and flavin monooxygenases with widely altered substrate specificities, and attempts have been reported to scale up reactions catalyzed by these enzymes. Cofactor regeneration is still a key issue in these developments. Protein engineering contributed to understanding of structure vs. function in dioxygenases
Biotransformations using prokaryotic P450 monooxygenases
Recent studies on microbial cytochrome P450 enzymes cover several new areas. Advances have been made in structure-function analysis. New non-enzymatic/electrochemical systems for the replacement of NAD(P)H have been developed. The properties of some enzymes have been re-engineered by site-directed mutagenesis or by methods of directed evolution. New P450s have been functionally expressed and characterized. A combination of these approaches is believed to facilitate the use of isolated P450 monooxygenases in biocatalysis
Effect of detergents, salts and organic co-solvents on Ssl1.
<p>A: Relative activity of Ssl1 in presence of organic solvents, salts and detergents towards 2,6-dimethoxyphenol oxidation. Water immiscible solvents did not affect Ssl1 activity, with water miscible co-solvents the activity dropped to 20–40%. Ssl1 tolerated the addition of 10 mM sodium azide, with the detergents SDS and Triton-X-100 the activity was reduced to 60 or 80%. B: Residual activity of Ssl1 after 20 h incubation with organic solvents, salts and deteregents. DMSO stabilized Ssl1 whereas acetonitrile lead to a reduction in residual activity. All other studied additives did not lead to major changes of Ssl1 stability.</p
pH optima and pH stability of Ssl1.
<p>A: Stability of Ssl1 in buffers with different pH values was tested as residual oxidation activity towards 2,6-dimethoxy phenol. Ssl1 was inactivated within 30 min at pH 3 (values shown as diamonds), and within 3 days at pH 4 (circles). Stability at pH 5 (open circles) and pH 7 (squares) was similar with a half-time around 4 to 5 days. At pH 11 (triangles) Ssl1 was most stable with more than 70% residual activity after 7 days. B: Relative activities of Ssl1 at different pH values towards the substrates ABTS (values shown as diamonds), syringaldazine (circles), 2,6-dimethoxy phenol (triangles) and guaiacol (squares). All activities were normalized to the values at optimum pH with the respective substrate. Optimal pH values are 4 for ABTS, 8 for syringaldazine, and 9 for 2,6-dimethoxy phenol and guaiacol.</p