View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Repository of the Academy's Library catalysts Article Immobilized Whole-Cell Transaminase Biocatalysts for Continuous-Flow Kinetic Resolution of Amines Zsófia Molnár 1,2,3, Emese Farkas 1, Ágnes Lakó 1 , Balázs Erdélyi 2, Wolfgang Kroutil 4 , Beáta G. Vértessy 3,5, Csaba Paizs 6 and László Poppe 1,6,7,* 1 Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, M˝uegyetemrkp. 3, 1111 Budapest, Hungary; molnar.zsofi[email protected] (Z.M.); [email protected] (E.F.); [email protected] (Á.L.) 2 Fermentia Microbiological Ltd., Berlini út 47–49, 1405 Budapest, Hungary; [email protected] 3 Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Science, Magyar tudósok krt. 2, 1117 Budapest, Hungary; [email protected] 4 Institute of Chemistry, University of Graz, NAWI Graz, BioTechMed Graz, BioHealth, Heinrichstraße 28, 8010 Graz, Austria; [email protected] 5 Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics, M˝uegyetemrkp. 3, 1111 Budapest, Hungary 6 Biocatalysis and Biotransformation Research Centre, Faculty of Chemistry and Chemical Engineering, Babe¸s-BolyaiUniversity of Cluj-Napoca, Arany János Str. 11, RO-400028 Cluj-Napoca, Romania; [email protected] 7 SynBiocat Ltd., Szilasliget u 3, H-1172 Budapest, Hungary * Correspondence: [email protected]; Tel.: +36(1)463-3299 Received: 29 March 2019; Accepted: 25 April 2019; Published: 10 May 2019 Abstract: Immobilization of transaminases creates promising biocatalysts for production of chiral amines in batch or continuous-flow mode reactions. E. coli cells containing overexpressed transaminases of various selectivities and hollow silica microspheres as supporting agent were immobilized by an improved sol-gel process to produce immobilized transaminase biocatalysts with suitable stability and mechanical properties for continuous-flow applications. The immobilized cell-based transaminase biocatalyst proved to be durable and easy-to-use in kinetic resolution of four racemic amines 1a–d. The batch and continuous-flow mode kinetic resolutions with transaminase biocatalyst of opposite stereopreference provided access to both enantiomers of the corresponding amines. By using the most suitable immobilized transaminase biocatalysts, this study describes the first transaminase-based approach for the production of both pure enantiomers of 1-(3,4-dimethoxyphenyl)ethan-1-amine 1d. Keywords: stereoselective biocatalysis; transaminase; kinetic resolution; flow chemistry; sol-gel; whole-cell immobilization 1. Introduction Enantiopure amines are essential chiral building blocks for the synthesis of a wide variety of active pharmaceutical ingredients. Chemical synthesis of these compounds usually employs transition metal catalysts of relatively high toxicity, and may require harsh reaction conditions. In recent years, there has been a growing interest in transaminases (TAs), which offer a sustainable alternative to these synthetic chemical processes [1–3]. Transaminases have been successfully used in the preparation of several pharmaceutically relevant compounds, like 3-aryl-γ-aminobutyric acid derivatives [4], sitagliptin [5] and valinol [6]. Transaminases can be applied for separation of the enantiomers from their racemates in kinetic resolution (KR) with a maximum of 50% yield, or for conversion of a prochiral carbonyl compound to a Catalysts 2019, 9, 438; doi:10.3390/catal9050438 www.mdpi.com/journal/catalysts Catalysts 2019, 9, 438 2 of 11 Catalysts 2019, 9, x FOR PEER REVIEW 2 of 11 tochiral a chiral amine amine in asymmetric in asymmetric synthesis synthesis with a with theoretical a theoretical yield of yield 100%. of Although 100%. Although asymmetric asymmetric synthesis synthesiscould provide could higher provide yields, higher it usually yields, su itff usuallyers from su disfavoredffers from reaction disfavored equilibrium reaction [7equilibrium]. Furthermore, [7]. Furthermore,the costs of enzyme the costs production of enzyme indicates production the need indi forcates biocatalyst the need recovery for biocatalyst in an economically recovery viablein an economicallyprocess [8]. These viable are process the most [8]. significant These are ditheffi cultiesmost significant hindering difficulties the widespread hindering industrial the widespread application industrialof transaminases. application Several of transaminases. strategies have beenSeveral developed strategies to overcomehave been the developed difficulties to arising overcome from the the difficultiesdisfavored arising reaction from equilibrium the disfavored in asymmetric reaction synthesis equilibrium with in TAs, asymmetric such as the synthesis removal with of products TAs, such by asextraction the removal [9], by of evaporation products by [10 extraction] or by coupled [9], by cascade evaporation reactions [10] [11 or,12 by]. Thecoupled overall cascade efficiency reactions of the [11,12].KR process The mayoverall be enhancedefficiency byof the recycling KR process the formed may be ketone enhanced to racemic by recycling amine bythe a formed proper ketone reductive to racemicamination amine method by a [proper13]. Although reductive these amination solutions method work [13]. well Although in batch reactions, these solutions for the work sustainable, well in batchindustrial reactions, production for the of sustainable, enantiopure industrial amines the pr intensificationoduction of enantiopure possibilities oaminesffered bythe immobilized intensification TA possibilitiesbiocatalysts andoffered by theby continuous-modeimmobilized TA biocatalysts operations areand needed by the [1continuous-mode]. This trend is indicated operations by are the neededsuccessful [1]. applicationsThis trend is ofindicated TAs in continuous-flowby the successful reactors applications which of haveTAs in been continuous-flow developed in reactors the past whichyears [14have–23 been]. developed in the past years [14–23]. A wide range of immobilization methods for transaminases have have been been reported over the last couple of of years. years. Transaminases Transaminases have have been been successfully successfully immobilized immobilized by byusing using different different kinds kinds of carriers,of carriers, such such as polymeric as polymeric resins [7,16,23], resins [7 ,functionalized16,23], functionalized cellulose cellulose[21], chitosan [21], [24–26], chitosan inorganic- [24–26], basedinorganic-based nanoflowers nanoflowers [27,28], macrocellular [27,28], macrocellular silica monoliths silica [19], monoliths MnO2 nanorods [19], MnO [29]2 nanorods or porous [29 glass] or metalporous affinity glass metalsupports affi nity[22]. supports In addition [22]. to Inimmobilization addition to immobilization of TAs in their of isolated TAs in theirand fully isolated or partiallyand fully purified or partially form, purified the entrapment form, the of entrapmentcell-free extracts, of cell-free or even extracts, whole cells or even with whole comprising cells with TA activitycomprising in different TA activity sol-gel in dimatricesfferent sol-gel[14,30,31] matrices proved [ 14to, 30be, 31an] provedexcellent to approach be an excellent to prepare approach high- performanceto prepare high-performance and stable immobilized and stable biocatalysts. immobilized In many biocatalysts. cases, utilization In many of cases, whole utilization cells over of enzymewhole cells solutions over enzymeis advantageous solutions due is advantageousto the lower production due to the costs, lower increased production stability costs, and increased easier handlingstability and[32]. easier Immobilize handlingd transaminases [32]. Immobilized have been transaminases successfully have employed been successfully in the preparation employed of severalin the preparationenantiopure ofcompounds, several enantiopure such as 1-phenox compounds,ypropane-2-amine such as 1-phenoxypropane-2-amine [33], 3-amino-1-Boc-piperidine [33], [34],3-amino-1-Boc-piperidine 1-methyl-3-phenylpropylamine [34], 1-methyl-3-phenylpropylamine [23,26] and mexiletine [16]. [23 ,26] and mexiletine [16]. Scheme 1. Continuous-flowContinuous-flow kinetickinetic resolutionresolution of of primary primary amines aminesrac rac-1a-1a–d–withd with immobilized immobilized whole-cell whole- celltransaminase transaminase biocatalysts. biocatalysts. This study aimed at the preparation of robust, stereoselective TA biocatalysts capable of operating This study aimed at the preparation of robust, stereoselective TA biocatalysts capable of under continuous-flow conditions to produce enantiopure amines. A promising sol-gel-based operating under continuous-flow conditions to produce enantiopure amines. A promising sol-gel- whole-cell immobilization method has already been applied in a previous work of our group for based 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 Catalysts 2019, 9, 438 3 of 11 Catalysts 2019, 9, x FOR PEER REVIEW 3 of 11 immobilizationthe transaminase of fromfurtherChromobacterium transaminases with violaceum differentexpressed enantioselectivity in E. coli [14 and]. Hereby substrate we specificity. report the Theimmobilization immobilized of cells further proved transaminases to be easy-to with store, di ffcheaperent and enantioselectivity