Enantioselective Transamination in Continuous Flow Mode with Transaminase Immobilized in a Macrocellular Silica Monolith

Abstract

ω-Transaminases have been immobilized on macrocellular silica monoliths and used as heterogeneous biocatalysts in a continuous flow mode enantioselective transamination reaction. The support was prepared by a sol-gel method based on emulsion templating. The enzyme was immobilized on the structured silica monoliths both by adsorption, and by covalent grafting using amino-functionalized silica monoliths and glutaraldehyde as a coupling agent. A simple reactor set-up based on the use of a heat-shrinkable Teflon tube is presented and successfully used for the continuous flow kinetic resolution of a chiral amine, 4-bromo-α-methylbenzylamine. The porous structure of the supports ensures effective mass transfer and the reactor works in the plug flow regime without preferential flow paths. When immobilized in the monolith and used in the flow reactor, transaminases retain their activity and their enantioselectivity. The solid biocatalyst is also shown to be stable both on stream and during storage. These essential features pave the way to the successful development of an environmentally friendly process for chiral amines production.

Biocatalysis Using Immobilized Enzymes in Continuous Flow for the Synthesis of Fine Chemicals

“…Debecker and co-workers recently reported the continuous kinetic resolution of methyl benzylamine with an ω-transaminase (ATA-113 Codexis inc. USA) covalently bound to macrocellular silica monoliths. 40 The reactors were prepared inside heat-shrinkable Teflon tubes, and the porous supports ensured excellent mass transfer and fluid properties. However, the use of covalent attachment via glutaraldehyde grafting resulted in a significant loss in activity (>84%) and correspondingly low yields (6.2%).…”

Self-Sufficient Flow-Biocatalysis by Coimmobilization of Pyridoxal 5′-Phosphate and ω-Transaminases onto Porous Carriers

“…Therefore, this PBR successfully operated at 1.45 mL × min −1 , the highest flow rate ever reported using coimmobilized enzyme and cofactor 16−18 and one of the highest (0.1−1.7 mL × min −1 ) using immobilized enzymes supplied with exogenous cofactor. [11][12][13]25 In principle, this promising approach may be expanded to other ωTAs in order to fabricate a battery of self-sufficient heterogeneous biocatalysts with different catalytic properties (stability, selectivity, kinetics). On the basis of the same architecture, two additional N-terminus His-tagged ωTAs (Cv-ωTA and Pf-ωTA) were immobilized onto EC-Co 2+ and further coated with PEI to finally absorb PLP as described above.…”

Strategic single point mutation yields a solvent- and salt-stable transaminase from Virgibacillus sp. in soluble form

“…This endeavor was made possible by the availability of new TA crystal structures that have provided a better insight into the functional mechanisms of these enzymes 1 , 11 . In addition, immobilisation of TAs has been shown to enable catalysis under practical conditions (in the presence of co-solvents, stoichiometric amounts of the amine donor) in continuous reactors, where the reaction can be performed with better productivity and long-term stability of the biocatalyst 12 – 14 .…”