Biocatalysis using immobilised enzymes in continuous flow for the

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Review

Biocatalysis using immobilised enzymes in continuous flow for the synthesis of fine chemicals Matthew Thompson, Itziar Penafiel, Sebastian Cronin Cosgrove, and Nicholas J Turner Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00305 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 10, 2018

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Organic Process Research & Development

Biocatalysis using immobilised enzymes in continuous flow for the synthesis of fine chemicals Matthew P. Thompson 1 § , Itziar Pe ñ afiel 1 , Sebastian C. Cosgrove 1 and Nicholas J. Turner 1 * 1 School of Chemistry, University of Manchester, Manchester Institute of Biotechnology, 131 Princess Street, Manchester, M1 7DN, United Kingdom § Current address: EnginZyme AB, Teknikringen 38A, 114 28, Stockholm, Sweden E-mail: [email protected]

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Organic Process Research & Development 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC Graphic

Cross-linking NO2

HO

OH

Encapsulation

O

N

NH2

O

O

Carrier bound

O SH

N

CO2H

H

NH2

O2N

O2N

Immobilised biocatalysts in continuous flow


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Abstract Biocatalysis has emerged as one of the most promising technologies to enable green synthesis of important chemicals, due to the ambient conditions generally applied for these reactions. Nonetheless, a general uptake of enzymatic transformations has been hindered by the perceived high cost of recombinant proteins. Recent interest in continuous flow from the synthetic chemistry community has now begun to spread to biotransformations, with protein immobilisation playing a key part. As a consequence, continuous biotransformations using immobilised enzymes are becoming more accessible to non-experts. This review will discuss several recent examples of continuous biotransformations that use immobilisation, with a focus on examples involving fine chemical synthesis. It will also examine some of the issues that the community has as a whole, most importantly a lack of unified reporting tools to allow comparison and assessment of the different techniques. Keywords: Biocatalysis, immobilisation.

synthetic

chemistry,

continuous

flow,

enzyme

Introduction With growing interest in biocatalysis as a tool for asymmetric synthesis, a number of complementary technologies have come to the fore for the development of intensified and industrially relevant biocatalytic processes.1–3 The discovery and evolution of new and highly efficient enzymes, new retrosynthetic approaches with an emphasis on biocatalysis, reduced cost of recombinant proteins and enzyme immobilization strategies all combine to improve the overall outlook for biocatalysis.4–7 In particular, there is growing interest in performing enzymecatalysed transformations in continuous flow. The concept of "continuous flow" defines a very general range of chemical processes that occur in a continuous flowing stream.8 The application of flow chemistry relies on the continuous pumping of reagents through a “reaction zone”. Performing such transformations in continuous flow is attractive as a way to improve reaction productivity, efficiency of scale-up and downstream processing compared to batch processes. Continuous flow processes offer the following potential benefits:9–12    

In situ product removal (ISPR) and/or recycling of unreacted reagents. Real-time online optimization of reaction conditions to maximize efficiency. Integration with complementary enabling technologies such as microwave, photochemical and supported catalyst reactions. Alignment to downstream processing, reducing the optimisation required in the transfer from discovery to process scale.

Specifically with respect to biocatalytic transformations, continuous flow can also offer the following additional advantages:   

Reduced enzyme inhibition through continuous removal of products. Facile downstream processing when using immobilized biocatalysts (no lixiviation). Improved total turnover numbers (TTNs).



In a recent review by Puglisi and co-workers, advances in the application of continuous flow reactions to organic synthesis in both academic and industrial settings were highlighted.13 Despite being a relatively new technology, continuous flow is highly developed with well-established guidance on the potential advantages and challenges to implementation.8,14,15 There are many examples from the recent literature that highlight the state of the art in terms of manufacturing, safety and multistep synthesis in continuous flow. Two publications that highlight the application of continuous flow in small molecule manufacture are discussed below. Firstly, the continuous synthesis and formulation of several drug molecules including fluoxetine hydrochloride and diazepam to U.S. Pharmacopeia standards by Adamo and co-workers; it is a remarkable example of the use of flow to implement safe, efficient, and sustainable processes for the production of pharmaceuticals.16 The continuous, end-to-end, synthesis of these molecules is achieved in a highly automated fashion with a small temporospatial footprint, and exemplifies the possibilities of on-demand continuous small molecule manufacture.

Figure 1 Formulated drug preparations of diphenhydramine hydrochloride (1), lidocane hydrochloride (2), diazepam (3) and fluoxetine hydrochloride (4) produced by continuous end-to-end synthesis to U.S. Pharmacopeia standards

Continuous manufacture on production scale has several advantages over batch processes including improved safety, improved performance, and reduced isolation steps that in turn reduce handling steps. An illustration of this, in contrast with the small scale of the previous example, describes a multi-kilogram scale production of prexasertib 7 under continuous good manufacturing practice conditions.17 While numerous examples of APIs produced by continuous manufacture are reported, the work by Cole and co-workers represents the first cGMP end-to-end synthesis.13 The impressive overall yield of 60 % over three reactor stages, including in-process filtration and re-dissolution steps, is noteworthy. Finally, the use of online process analytical technologies (PAT) permits further automation and monitoring of the process.


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N HN O

O

O

O CN

6 steps OH

N H

4 steps O

O

6

N •H2O X = L-Lactate or Mesylate NH3X

NH2 5

CN N

O

7

Figure 2 cGMP synthesis of prexasertib from 1-(2-hydroxy-6-methoxyphenyl)ethan-1-one (5). The first reagent in the cGMP campaign nitrile (6) is produced in batch over 6 steps from (5). The four step procedure gives prexasertib (7) as either the lactate or mesylate salt in an entirely continuous procedure.

Previous reviews and scope of this review Recent reviews have included an examination of biocatalysis in continuous flow. Kroutil and co-workers extensively reviewed enzymatic and chemo-enzymatic cascade reactions including some performed in continuous flow.18 Additionally Liese and co-workers have comprehensively reviewed chemo-enzymatic coupled cascades in continuous flow.19 Furthermore, Britton and Weiss delivered a wideranging review that detailed many of the new technologies associated with continuous biotransformations, including immobilisation.20 In both of these reviews reactions performed in flow with soluble enzymes and reactions performed in enzyme membrane reactors (EMRs) or dynamic kinetic resolutions catalysed by immobilized lipases were given particular attention. Tamborini and co-workers recently gave an excellent overview of recent innovations in flow reactor design in addition to comments on appropriate terminology and metrics for biocatalytic reactions in flow.21 This review focuses on the use of immobilised enzymes in continuous flow; biotransformations performed in flow but using soluble enzymes are excluded, including those performed with EMRs. Additionally, there are industrial applications of immobilised enzymes that are not performed in flow reactors. For example, fructose corn syrup is a sweetener that is obtained by the enzymatic isomerisation of glucose to fructose.22 The enzyme, glucose isomerase, is costly so is immobilised to permit reuse. Additionally, penicillin G acylase is used as an immobilised preparation for the mass production of 6-aminopenicillanic acid, a precursor to a host of semi-synthetic antibiotics.23 Both of these are run as multiton scale processes. In this review, particular attention is given to enzymes employed as biocatalysts for the synthesis of target molecules in a continuous fashion. An important note regarding continuous reactions is a lack of unified reporting of results. This makes direct and critical comparison of enzymatic flow reactions difficult: consequently, comparative discussion of the different reactors discussed here will be limited as we could not identify a fair way to do this.

Early biocatalysis and immobilisation Early processes for biocatalysis in continuous flow relied heavily on membrane reactors or flowing soluble enzymes through the reactor system.24–26 The



limitation in these cases is that many of the benefits of continuous flow such as high catalyst loading and improved downstream processing are not realised. Moreover, while the cost of recombinant proteins is declining, they still constitute a significant contribution to any large-scale manufacturing process. To some extent therefore, the groundwork for biocatalysis in continuous flow has been the continued development in the area of enzyme immobilisation. Enzyme immobilisation can be broadly categorised into three types: cross-linking, encapsulation, and binding to supports (Figure 3).27 Each immobilisation strategy comes with its own set of advantages and disadvantages, however, it is often difficult to make direct comparisons. In addition, enzyme choice, immobilization strategy and reactor configuration can significantly influence the outcome.27,28 Cross-linked enzymes are prepared by first aggregating the enzyme to precipitate it followed by cross-linking the aggregate with a cross linking agent (typically glutaraldehyde). Despite the appeal of a simple, carrier free enzyme immobilisation, optimisation of the aggregation and cross-linking procedure can be tedious and often results in poor recovery of enzyme activity.27,29 Encapsulated enzymes are soluble or aggregated enzymes trapped inside of a bulk matrix such as a polymer network. The popularity of encapsulation was spurred by a desire to trap enzymes in an “active” state. However problems with leaching in addition to low activity and generality has plagued their wider adoption. 28,30 Finally, enzymes bound to supports are probably the most ubiquitous of all immobilised enzymes, thanks to the breadth of technologies for binding enzymes to solid supports ranging from polymeric beads to porous glass particles. Regardless of the material, the supports can be generally categorised into “covalent” and “non-covalent” immobilisation strategies. In the case of covalent attachment, reactive groups (epoxides, aldehydes etc…) on the surface of the support form covalent bonds to surface lysine groups on the protein. Meanwhile non-covalent supports bind proteins via any number of physiochemical interactions such as hydrophobic, charge-charge and affinity-tag binding. Since binding to a carrier introduces a large amount of “non-catalyst” bulk material, such methods typically result in enormous activity loss. Consequently, the application of carrier-immobilised enzymes can result in a reduction in reaction productivity owing to dilution effects.27,31–33 An important note regarding immobilisation is that most enzymes do not retain their in vitro activity. The use of continuous flow can overcome this, as packed-bed reactors have a high ratio of biocatalyst vs. substrate relative to an equivalent batch reactor, often overcoming the observed loss of activity. One significant draw back to many immobilisation technologies is the amount of screening and optimization required to obtain acceptable performance. In order to maximise appeal and minimise time to operation, immobilisation strategies need to be as general as possible. 1,2


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Figure 3 overview of immobilisation strategies. A) encapsulation B) cross-linking C) binding to a carrier either by adsorption, affinity or covalent attachment.

Early examples of biocatalysis in packed bed reactors Amongst the earliest reports of biocatalysis in continuous flow with immobilised enzymes is the continuous production of uridine diphosphate galactose (UDPgalactose) 9, from galactose and uracil monophosphate (UMP) at the expense of polyphosphate (polyP) used for ATP regeneration.34 Seven enzymes involved in the biosynthetic pathway were immobilized via His-tags to Ni-NTA agarose resin. This resin was packed into a column and reactions performed by recirculation of a reaction mixture through the column over a period of 48 hours with an overall conversion of 50 % from galactose (8) to UDP-galactose. Both the polyphosphate kinase (PpK) and galactose kinase (GalK) are unstable in solution; immobilization of these enzymes afforded an improvement in Vmax (up to 150% for PpK). Additionally, performing the reaction in flow using a packed column showed reduced attrition of enzyme activity compared with using the immobilized preparation in a stirred reactor.



OH HO OH

OH

OH GalK

O OH ATP

8

O

HO

OHOPO 23

OH

O HO HO

O

OHOUDP

PolyPn-1

OHOUDP

OH

HO HO

PpK

OH

OH

GalT

O

HO ADP

PolyPn

OH

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9

OHOPO 23

GalU UMP

UMK

ATP

UDP ADP

ATP

PpK PolyPn

NDK

UTP

PPi

PPA

2Pi

ADP PpK

PolyPn-1

PolyPn

PolyPn-1

Figure 4 Continuous synthesis of UDP-galactose (9) from galactose (8). GalK; galactose kinase. GalT; galactose 1-phosphate uridyltransferase. GalU; UDP-glucose pyrophosphorylase. UMK; UMP kinase. NDK; nucleotide diphosphate kinase. PPA; inorganic pyrophosphatase. PpK; polyphosphate kinase.

Baxendale and co-workers reported the first enantioselective total synthesis of the natural product grossamide 10 in a fully automated flow reactor. The product 10 was synthesized through a three-column flow reactor from 4-hydroxy-3methoxy cinnamic acid 11.35 Activation of the acid on polymer supported hydroxybenzotriazole in the presence of a reaction stream containing the cinnamic acid, PyBrOP and DIPEA followed by coupling with the amine partner gave the amide 12. The coupling reaction stream was then diluted by mixing with a buffer containing hydrogen peroxide urea complex, potassium phosphate buffer (pH 4.5) and acetone-water (1:4). The combined reaction stream was then passed through a column containing horseradish peroxidase (type II) adsorbed onto silica, which mediated the dimerization to afford grossamide 10. This also represented the first total synthesis completed under a fully continuous system.

Figure 5 Enantioselective total synthesis of grossamide (10) in a fully automated flow reactor

In a similar manner, 2-aminophenoxazin-3-one 16 was produced from nitrobenzene in three-steps.36 The first step was the zinc-catalysed reduction of nitrobenzene 13 to phenylhydroxylamine 14. Next, hydroxylamino benzene mutase (HAB mutase) from Pseudomonas pseudoalcaligenes JS45, adsorbed on silica, converted 14 to 2-aminophenol 15. In the final step soybean peroxidase


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(SBP), also adsorbed on silica, catalysed the oxidation of 15 to give the dimer 16. However, substrate concentration (1 mM), the overall yield (19 %) and productivity require further improvement.

Figure 6 Continuous synthesis of 2-aminophenoxazin-3-one (16)

Wever and co-workers reported the production of chiral carbohydrate analogues from dihydroxyacetone 17 and aldehydes using three separate packed bed reactor systems.37 Phosphorylation of the dihydroxyacetone by acid phosphatase (PhoN-Sf) covalently bound to epoxy functionalised Immobeads (ChiralVision, the Netherlands) gave the activated intermediate 18. Aldol condensation of 18 catalysed by either fructose-1,6-aldolase from rabbit muscle (RAMA) or rhamnulose-1-phosphate aldolase from Thermotoga maritima (RhuA) and finally dephosphorylation of the aldol adduct by the PhoN-Sf yielded the product. Despite screening of several immobilization resins for the RAMA and RhuA enzymes, the selected matrix, Relizyme EP-403 (Resindion, Italy), afforded only 30 % recovery of activity compared to the soluble protein. However, immobilisation had the advantage of suppressing a retro-aldol cleavage reaction, hence the immobilized enzyme operating in continuous flow showed an equilibrium favouring the formation of product. Overall the reaction gave an impressive space-time yield (STY) of up to 207 g L-1 day-1 from a residence time of approximately 120 minutes.

Figure 7 continuous syntheses of complex carbohydrates from dihydroxyacetone (17)

Immobilised ω-transaminases in continuous flow ω-Transaminases are amongst the most widely used biocatalysts for the synthesis of chiral amines from the corresponding carbonyl compound. Immobilised preparations of transaminase have been implemented within industrial processes, including the landmark Merck process for production of sitagliptin.38,39 In addition to a number of examples of transaminases immobilised on supports or by cross-linking, several groups have recently described the operation of immobilised transaminases under continuous flow. 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 %). The use of porous materials with good fluid properties is attractive, however the low yield and volumetric intensity with respect to wt. enzyme/wt. support severely limits this approach. Jamison and co-workers reported the first example of an immobilised transaminase operating in continuous flow for the efficient synthesis of chiral amines 20 at the expense of isopropylamine 19.41 Whole cells of E. coli expressing the (R)-selective ω-transaminase from Arthrobacter sp. were immobilised directly onto a methylacrylate polymeric resin and packed into a steel column. Water saturated MTBE containing substrate (50 mM) and isopropylamine 19 (200 mM) was flowed through the column. Conversions up to 85 % were obtained with a residence time of 30 min giving a STY of 349 g L-1 day-1. This system benefits from the use of immobilised whole cells: not only does this permit the use of cofactor dependent enzymes in an organic bulk phase, but whole cell preparations are the cheapest option for a large-scale process. Additionally, it was possible to install a silica column after the reactor column in order to trap the amine product that was subsequently eluted with methanol. Nevertheless, the use of whole cells could result in overall lower productivity compared to isolated proteins owing to the need for mass transfer across cell membranes.

Figure 8 Synthesis of amino esters with immobilised E. coli cells expressing (R)-selective ω-transaminase from Arthrobacter sp.

In contrast to the immobilised whole-cell reactor, an undisclosed ω-transaminase, provided by c-LEcta (Leipzig, Germany), was encapsulated within a PVA-gel matrix, LentiKats (LentiKats, Czech Republic). The LentiKat preparation was applied to a miniaturised packed bed reactor for the synthesis of enantiopure amino acids 21. 42 The reactor was systematically optimised towards a number of operational parameters including the width of the channels and distribution of LentiKats. The system was shown to be stable for at least 21 days however the continuous addition of exogenous PLP could be a limitation of this reactor system without the ability to efficiently recycle an aqueous stream containing the cofactor.


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Figure 9 Deracemisations of MBA with transaminase entrapped within LentiKat

In a related fashion, Paradisi and co-workers recently reported a transaminase from Halmonas elongata (HEWT) displaying broad substrate scope, thermal and pH tolerance in addition to the presence of high concentrations of salt and/or organic solvents.43 In a recent publication this transaminase has been operated for the production of a panel of amines in continuous flow. The enzyme was immobilized using a method developed by Guisan44 to selectively immobilize Histagged proteins to a metal derivatised epoxy resin. Despite the selective enrichment of the His-tagged protein, covalent attachment via the epoxy group resulted in the loss of up to 70 % of the total enzyme activity. An elegant in-line purification protocol was used, in which basification of the reaction stream and extraction with EtOAc followed by passing through a benzylamine functionalized resin, effectively scrubbed any unreacted aldehyde 22 and afforded the desired amine product 23. With excellent conversions and total reaction times reduced to minutes compared to hours in batch, this system represents one of the most efficient examples of biocatalysis in continuous flow reported to date.

Figure 10 Synthesis of enantiopure amines by immobilised TA, integrated with two-step in-situ product removal process.

More recently, the Paradisi group reported the conversion of primary amines to aldehydes catalysed by the immobilized HEWT system used previously.45 In-line acidification of the reaction stream followed by liquid-liquid extraction using a Zaiput separator allowed facile separation of the aldehyde product with residence times of 3–15 minutes affording up to five-fold improvements in productivity compared with equivalent batch processes. Sans and co-workers recently showed that following functionalization of a 3Dprinted nylon microreactor with glutaraldehyde, the commercially available ωtransaminase ATA117 (Codexis, USA) can be efficiently immobilised.46 3D printed reactors are attractive owing to their ease of production, modification and use.47 However, the use of glutaraldehyde for covalent attachment of ATA-117 resulted in recovery of only 25 % of the total enzyme activity. The immobilised ATA117 was applied to the kinetic resolution of rac-methylbenzylamine under a continuous flow regime. The immobilised reactor reached a steady state productivity of ca. 8 µmol h-1 mg-1 of immobilised ATA117. The reactor afforded (s)-MBA (> 99 % ee) with a residence time of ca. 50 minutes and was shown to be stable for ca. 100 hours.

Immobilised oxidoreductases in continuous flow The Vincent group recently described a series of enzymatic hydrogenations in continuous flow. They combined an alcohol dehydrogenase with a hydrogenase from E. coli and a NAD+ reductase variant from Ralstonia eutropha immobilized



by adsorption on carbon nanotubes.48 Solutions saturated with H2 provided the hydrogenase with an electron source, coupled to NAD+ reductase resulted in an efficient system for the regeneration of NADH (Fig. 11). One drawback of this approach is the requirement for anaerobic conditions and rate limitations caused by low availability of H2 to drive the reaction system. The authors noted that devices to improve H2 availability are known and could be adapted for their process accordingly.

Figure 11 Asymmetric reduction of ketones using hydrogen in continuous flow.

Serra and co-workers immobilised a ketoreductase from Pichia glucozyma and glucose dehydrogenase from Bacillus megaterium to aldehyde activated agarose with loss of up to 60 % of the total enzyme activity.49 However, the immobilised protein only constituted 1.4 wt. % of the matrix and hence displayed poor volumetric activity. Using the immobilised agarose matrix, it was possible to perform stereoselective reduction of various ketones with residence times between 7 and 180 minutes. In particular the stereoselective reduction of bulky substrate ethyl secodione 24 gave a recovered yield of 60 mg of hydroxy ketone 25 over 15 days of continuous operation (STY = 4.8 g L-1 day-1). This low volumetric productivity can be attributed to the overall low activity of the immobilised enzyme compared to the free enzyme.

Figure 12 ADH/GDH coupled reduction of ethyl secodione in continuous flow

Tamborini and co-workers recently reported the enantioselective, chemoenzymatic synthesis of captopril 26 in continuous flow.50 Starting from 2-methyl1,3-propandiol 27, using alginate entrapped whole cells of Acetobacter aceti MIM 2000/28 gave regio- and stereo-selective oxidation to the carboxylic acid 28 with Tres = 10 min and STY of 144 g L-1 day-1 (Fig. 13).

Figure 13 Enzymatic oxidation of diol 27 to produce key fragment 28 in captopril 26 synthesis

Oxygen supply is critical to the cofactor recycling in bacterial whole cells. This was accomplished using a segmented air-liquid flow stream. The carboxylic acid 28 in the effluent from the biocatalytic step was trapped using an Ambersep 900OH resin followed by release upon acidification. 28 then underwent chlorination and


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amide coupling, followed by phase separation and formation of the thiol to furnish enantiopure captopril 26 in 50 % isolated yield, which was equivalent to 160 mg L-1. A smart strategy for the immobilization and recycling of both enzyme and cofactor has been recently reported by López-Gallego and co-workers.51 A commercially available KRED (P1-A04, Codexis) was immobilised on a commercial agarosebased cationic carrier based on monolayer of tertiary amine groups (Ag-DEAE). The immobilized enzyme displays 98% conversion of 2,2,2Trifluoroacetophenone 29 (20 mM) into the corresponding chiral secondary alcohol 30 after 14 hours. Further, it was co-immobilized with NADPH to catalyse asymmetric reductions without the addition of an exogenous cofactor. The enzyme was strongly bound to the carrier, whereas the cofactor established an association/dissociation equilibrium with the solid surface through ionic absorbance, then the catalytic system was accessible and recycled inside the porous structure. Using in operando fluorescence studies, the authors showed that lixiviation of NADPH and KRED was negligible. The immobilized catalytic system (0.28 g, 6.5 mg of KRED and 10 µmol of NADPH per gram of carrier) was loaded into a packed-bed flow reactor for the continuous synthesis of (S)(trifluoromethyl)benzyl alcohol 30 in the presence of MgCl2 and 17% IPA. A spacetime yield of 104 g L-1day-1 and a total turnover number of 1076 over 120 h were achieved with a flow rate of 50 µL min-1 (Fig. 14)

Figure 14 KRED/NADPH simultaneously immobilised on agarose-based cationic carrier

The use of self-immobilizing fusion enzymes for the modular configuration of microfluidic packed-bed reactors has been described by Niemeyer and co-workers.52 Specifically, three different enzymes, the (R)-selective alcohol dehydrogenase LbADH (from Lactobacillus brevis), the (S)-selective methylglyoxal reductase Gre2p (form Saccharomyces cerevisiae) and the NADP(H) regeneration enzyme glucose 1-dehydrogenase GDH (from Bacillus subtilis subsp. natto), were genetically fused with streptavidin binding peptide (SPB/STV), Spy and Halobased tags, enabling selective immobilization on magnetic microbeads coated with complementary receptors. Although the HaloTag system has not been widely employed to date, it is becoming a more commonly used immobilisation strategy. It consists of two covalently-bound fragments: a haloalkane dehalogenase and a complimentary synthetic ligand. The ligands are composed of a reactive chloroalkane linker bound to a functional group which can be used as an affinity tag. The microreactors are prepared by addition of the immobilised enzymes as a suspension. The use of this non-destructive immobilisation strategy avoids any significant activity loss compared to the free enzymes. The stereoselective desymmetrisation of the prochiral symmetrical substrate 5nitrononane-2,8-dione 31 takes place in compartments made by four-channel microfluidic chips containing the enzyme-modified beads. The authors carried out



an extensive study into the effect of enzyme loading and order of reaction on the overall stereochemical outcome. Remarkably, fine-tuning of compartment size and enzyme loading leads to the continuous production of the single meso diol 32 with nearly quantitative conversion (>95%) and excellent stereoselectivity (d.r. > 99:1). Additionally, the reactor showed impressive robustness with an operating time of up to 14 days (Fig. 15).

Figure 15 Three-enzyme, two-step reaction for diastereoselective reduction of ketones

Very recently, Ley and co-workers described the use of HaloTag immobilised alcohol dehydrogenase from Lactobacillus brevis (HaloTag-LbADH), in the continuous asymmetric reduction of a range of ketones to the corresponding chiral alcohols.53 However, the fusion enzyme HaloTag-LbADH that was generated exhibited a residual activity of 35% compared to the reference. Halo TaggedLbADH established a covalent ester bond with the terminal chloroalkane ligands to the complimentary HaloTag in the column, resulting in a HaloLink_HaloTag immobilized protein with a low loading of protein (ca. 12 mg/gram). This low loading along with a low flow rate (30 µL min-1) led to low volumetric activities. However, a good range of ketones were asymmetrically reduced with residence times of 19 min and conversions and ee up to 99%. Additionally, the authors extended the application of this bioreactor to a two-step chemo-enzymatic transformation of 2-bromoacetophenone 33 into (S)-2-phenyloxirane 34 with 98% conversion and 98% ee (Fig. 16).

Figure 16 Stereoselective ketone reduction coupled with hydroxide-mediated ring closure

In a similar approach, Pohl and co-workers employed the same (type) resin and direct immobilization methodology in a 2-step enzyme cascade for the continuous production of (1S,2S)-1-phenylpropane-1,2-diol 35.54 The authors report a similar low loading of protein, with 4 mg of HaloTag-PpBFD L476Q (from Pseudomonas putida) and 4 mg of HaloTag-LbADH bound to 360 mg of wet HaloLink resin along with 65% and 35% residual activity respectively. An elegant strategy was adopted: in the first step, benzoylformate 36 was decarboxylated in situ to benzaldehyde 37 by HaloTag-PpBFD L476Q which subsequently displayed carboligase activity carrying out the acetylation of the resulting benzaldehyde with the excess acetaldehyde 38 present in the reaction media. Afterwards, residual acetaldehyde 38 was removed by membrane-supported stripping using a hollow fibre module and a reversed nitrogen flow. Finally, a pH-stat was employed to automatically adjust pH to 7.0, which was required for the second step (Fig. 17).


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Figure 17 Two-enzyme system for synthesis of chiral diastereomeric 1,2-diols 35

In the second step, 16 mg of immobilized HaloTag-LbADH in a 20 cm column selectively reduced (S)-2-hydroxy-1-phenylpropane-1-one 39 to the desired diol with a residence time of 4.5 h and a conversion >97%. NADPH was regenerated from the combination of NADP+ with 10 vol% 2-propanol as the co-substrate. The system displayed an overall space-time yield of 38 g L-1 d-1. Both enzymes in the two-step reaction remained active for at least 3 days after which a significant drop in activity was observed. This is attributed to deactivation of the PpBFD module, whereas LbADH remained active for at least 14 days. Rabe et al. have also demonstrated 3D printed reactors, with the protein of interest contained within a biodegradable agarose gel, that is melted and printed directly to generate reactor discs containing immobilised protein. The gels were printed at 60 C, meaning the enzymes had to be stable at these temperatures. The authors described two approaches to achieve this: use of naturally occurring enzymes with an inherent stability of >60 C, or by engineering thermostability through directed evolution. The technology was exemplified through the combination of an engineered ketoisovalerate decarboxylase (KVID) with a wt thermostable ADH in a continuous reactor to produce isobutanol from ketoisovalerate, with isobutanol obtained at a rate of 0.8 mM at steady state. Nevertheless, there was a low flow rate of only 25 L min-1 with activity also beginning to decrease after 4 hours.55 Very recently, Bornscheuer et al. demonstrated the use of EziG, a porous-glass resin that contains a metal centre which exploits the histidine tags used for protein purification.33,56 An engineered transaminase from Aspergillus fumigatus (4CHITA) was immobilised and used to continuously convert bi-aryl ketones, obtained from a preceding Suzuki reaction, into benzylamines. The proof of principle study led to 43% conversion to the amine, with a residence time of 3.5 h. One important point to note is the tolerance to up to 30% DMF in the reaction mixture, a high boiling solvent which did not result in any enzyme leaching from the support. It was not clear what flow rate was used, and no comment was made on the length of time the reaction was run for nor how much product was actually obtained. The authors commented on the ease with which the protein was immobilised using the resin, as no pre-treatment was required as is often necessary with many other supports.



Economic impact of different carriers The decision of which carrier is appropriate for a given biocatalyst is obviously impacted by the economics, especially when considered in an industrial context and a need for scalability. In a batch-manufacturing context, enzyme immobilisation enables recycling of the preparation, in successful cases offsetting the additional cost of the supporting material compared to the free enzyme only. In the context of continuous flow however, with no intermediate workup/recycling needed, the immobilised preparation can be used without pause until the enzyme activity has decreased below acceptable specifications – even modulating residence time to prolong the useful lifetime of the supported enzyme. Several other advantages can be realised such as in situ immobilisation and preparation of the enzyme onto a packed bed and in some cases in situ recycling of the immobilisation, which can further reduce cost contributions of immobilised enzymes in continuous flow. All immobilisation methods vary in cost and afford differing levels of activity retention. Consequently, a whole process must be considered in the technoeconomic analysis to decide which carrier is the correct choice. For example, glutaraldehyde as a crosslink offers a cheap, commercially available immobilisation source, however the activity retention is often very poor resulting in significantly increased enzyme (raw material) requirements to match the productivity of the free enzyme. The trade-off with activity should therefore have to be considered against the overall cost of a process to determine whether the loss in activity (and hence, increase in enzyme requirement) is rational compared with other strategies. HaloTag is also a popular option amongst academic circles for efficient enzyme immobilisation using selective affinity binding. Nevertheless, the HaloLink resin costs from £20/€23 mL-1 and binds only >7mg/mL of the HaloTagged protein. Combining the cost of the resin with the necessity to clone into a vector that contains the HaloTag construct, the price would most likely be prohibitive for industrial applications. As noted, the choice of immobilisation support has to be weighed against a number of factors including recyclability of the preparation, and the cost of formulating the immobilised enzyme. Both these factors should be considered as key during early-stage techno-economic assessment but in general efficient and selective immobilisation, and stable immobilised enzyme preparations are key to driving down cost contributions.

Conclusions and outlook Effecting biocatalytic transformations in continuous flow has the potential to assist in bioprocess intensification to industrially attractive levels. The number of examples of biocatalysis in continuous flow is growing rapidly. However, the scope and productivity of current examples leaves room for significant improvements in particular with respect to productivity, ISPR and multistep reactions.


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Strategies for efficient enzyme immobilisation continue to be developed, alongside the expanding number of available biocatalysts. Moreover, the use of immobilised enzymes in packed bed reactors further improves the prospect for biocatalysis by opening the possibility for “plug-and-play” enzyme systems that can be used to assemble in vitro cascade reactions or be integrated with other enabling technologies. An area that must be addressed for this field to move forward productively, is the lack of a common method for reporting results. Many different units are used, including space time yield (STY), productivity, product concentration, and all have their own merits as to which is most effective. However, these is no consistency between reports which makes it difficult to draw true comparisons between different reactors. Furthermore, many reports fail to include essential pieces of information such as reactor size, flow rates and substrate concentrations. In our opinion, the minimum set of reported information should include: 

   

Productivity: the report of productivity gives an exact idea of how well a specific reactor performs. With respect to packed-bed reactors, units of moles/unit time/weight carrier give an accurate picture of enzyme efficiency within the reactor (ideally µmol/minute/gramsupport). Reactor volume: alongside productivity, the user can calculate the size of the reactor and therefore make a judgement as to the efficiency. Residence time: As with reactor volume, this is important with respect to the efficiency of the reactor. Substrate/Product concentration: this metric is perhaps the simplest way to give a quick idea of the efficiency of a reactor and the potential for scalability. Other relevant metrics: such as pressure drop across a bed, leaching and time to inactivation data all further the ability to assess and compare continuous biocatalytic systems.

Additional information such as space time yield and flow rate could also be listed, but with the metrics listed above comparisons between different reactors will become more straightforward. This will simplify the process for deciding the best continuous strategy, and will allow continuous biotransformations to become a central technology for enzymatic reactions.

Funding Thanks go to CoEBio3 (M.P.T) and the EPSRC Catalysis Hub (I.P, S.C.C and N.J.T) for funding.

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Biocatalysis using immobilised enzymes in continuous flow for the

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