Development of a High‐Pressure Reactor Based on Liquid‐Flow Pressurisation to Facilitate Enzymatic Hydroxylation of Gaseous Alkanes

Abstract

A new type of high‐pressure reactor based on liquid‐flow pressurisation using a HPLC pump has been developed. This high‐pressure reactor allows the easy and safe performance of reactions with gaseous alkanes under high‐pressures up to 10 MPa (100 atm), without the need for high‐pressure gas cylinders. The amount of substrate gas required for a single reaction is very small compared with reactions using a conventional autoclave, which, when using expensive substrate gasses, such as 13C‐labelled ethane, becomes critical. Employing this high‐pressure reactor in conjunction with cytochrome P450BM3 and the assistance of decoy molecules, the direct hydroxylation of gaseous alkanes was drastically improved. At 5 MPa the TOF of propane hydroxylation increased 10‐fold, reaching 2200 min−1. Hydroxylation of ethane was also substantially accelerated at 5 MPa, reaching a TOF of 28 min−1.

Kristalle in Minutenschnelle: Sofortige Mikrokristallisation verschiedenster Varianten der CYP102A1‐(P450BM3)‐Hämdomäne

“…Ähnlich dem nativen Substrat binden Täuschmoleküle an die Substratbindestelle von P450BM3 und regen so die Bildung der aktiven Sauerstoffspezies (Verbindung I, Cpd I; Abbildung unten, rot gepunkteter Kasten) an, ohne dabei selbst hydroxyliert zu werden. Cpd I kann dann genutzt werden um nichtnative Verbindungen, die in den durch Cpd I und dem Täuschmolekül gebildeten Zwischenraum passen, zu hydroxylieren, darunter Ethan, Propan, Zyklohexan und Benzol . Ein umfangreicher und ausführlicher Übersichtsartikel bezüglich Täuschmoleküle kann bei Shoji et al.…”

Designer Outer Membrane Protein Facilitates Uptake of Decoy Molecules into a Cytochrome P450BM3‐Based Whole‐Cell Biocatalyst

“…[13][14][15][16][17] The binding of decoy molecules reshapes the reaction pocket of P450BM3, to comfortably accommodate small exogenous molecules, such as gaseous alkanes and benzene derivatives, thereby enabling their oxidation by P450BM3 (Figure 1c). [13,14,[17][18][19][20] We have also observed preferable combinations of target substrates and decoy molecules, which affect hydroxylation rates and even the stereoselectivity of benzylic hydroxylations. [15] In a previous publication we demonstrated that decoy molecules can also be used in conjunction with whole-cell biotransformations, where decoy molecules added to the culture medium can accelerate the hydroxylation of benzene to phenol by P450BM3 expressed in Escherichia coli.…”

A Unique P450 Peroxygenase System Facilitated by a Dual-Functional Small Molecule: Concept, Application, and Perspective

“…The direct hydroxylation of small alkanes to alcohols is a long-standing challenge because of the higher bond dissociation energies (BDE) of their C-H bonds when compared with that of the corresponding hydroxylated products, the latter easily leads to overoxidation [111,112]. Natural oxidizing enzymes, such as methane monooxygenase, soluble butane monooxygenase (sBMO), fungal peroxygenase (AaeUPO), and engineered P450s, are promising biocatalysts for the selective hydroxylation of small alkanes [76,77,93,[113][114][115][116][117][118][119][120][121][122][123]. Recently, Chen et al reported the peroxide-driven hydroxylation of small alkanes (C 3 -C 6 ) by using engineered P450BM3 variants assisted by DFSMs [93].…”