Co‐immobilized Whole Cells with ω‐Transaminase and Ketoreductase Activities for Continuous‐Flow Cascade Reactions

Abstract

An improved sol-gel process involving the use of hollow silica microspheres as a supporting additive was applied for the co-immobilization of whole cells of Escherichia coli with Chromobacterium violaceum ω-transaminase activity and Lodderomyces elongisporus with ketoreductase activity. The co-immobilized cells with two different biocatalytic activities could perform a cascade of reactions to convert racemic 4-phenylbutan-2-amine or heptan-2-amine into a nearly equimolar mixture of the corresponding enantiomerically pure R amine and S alcohol even in continuous-flow mode. The novel co-immobilized whole-cell system proved to be an easy-to-store and durable biocatalyst.

Immobilized Whole-Cell Transaminase Biocatalysts for Continuous-Flow Kinetic Resolution of Amines

“…The immobilized The immobilization method applied in this study combines the advantages of cell-adsorption and sol-gel entrapment. The silica microspheres provide the good mechanical properties of the biocatalyst, while the entrapping silica matrix afford the high immobilization yield (~100% of the cells were retained [14];~0.9 g of dry TA biocatalyst could be produced from 1 g of wet cells). The immobilization could be scaled up from g scale to 10 g scale without any noticeable problem.…”

“…Although these solutions work well in batch reactions, for the sustainable, industrial production of enantiopure amines the intensification possibilities offered by immobilized TA biocatalysts and by the continuous-mode operations are needed [1]. This trend is indicated by the successful applications of TAs in continuous-flow reactors which have been developed in the past years [14][15][16][17][18][19][20][21][22][23].…”

“…Transaminases have been successfully immobilized by using different kinds of carriers, such as polymeric resins [7,16,23], functionalized cellulose [21], chitosan [24][25][26], inorganic-based nanoflowers [27,28], macrocellular silica monoliths [19], MnO 2 nanorods [29] or porous glass metal affinity supports [22]. In addition to immobilization of TAs in their isolated and fully or partially purified form, the entrapment of cell-free extracts, or even whole cells with comprising TA activity in different sol-gel matrices [14,30,31] proved to be an excellent approach to prepare high-performance and stable immobilized biocatalysts. In many cases, utilization of whole cells over enzyme solutions is advantageous due to the lower production costs, increased stability and easier handling [32].…”

“…This study aimed at the preparation of robust, stereoselective TA biocatalysts capable of operating under continuous-flow conditions to produce enantiopure amines. A promising sol-gelbased whole-cell immobilization method has already been applied in a previous work of our group for the transaminase from Chromobacterium violaceum expressed in E. coli [14]. Hereby we report the This study aimed at the preparation of robust, stereoselective TA biocatalysts capable of operating under continuous-flow conditions to produce enantiopure amines.…”

Parallel Interconnected Kinetic Asymmetric Transformation (PIKAT) with an Immobilized ω-Transaminase in Neat Organic Solvent

“…This improvement has been achieved through protein engineering (i.e., structurally-guided or directed evolution) of novel ω-TAs possessing a broader substrate scope [9,10], as well as the development of displacement strategies for the shift of reaction equilibria [11][12][13][14][15]. Therefore, ω-TA-catalyzed reactions have been implemented in industry, and current prominent applications entail (chemo)enzymatic cascades and processes with immobilized biocatalysts in batch or flow reactors [3,6,9,[16][17][18][19][20][21][22][23][24].…”