A generic platform for the immobilisation of engineered

biocatalysts

Matthew P. Thompson1, Sasha R. Derrington1, Rachel S. Heath1, Joanne L. Porter1,

Juan Mangas-Sanchez1, Paul N. Devine2, Matthew D. Truppo,2 Nicholas J. Turner*1.

Author Affiliations

1) School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester,

131 Princess Street, Manchester, M1 7DN, UK

2) Merck & Co., Inc., 126 East Lincoln Avenue, Rahway, New Jersey 07065, United States

* Corresponding author Prof. Nicholas J. Turner, nicholas.turner@manchester.ac.uk

Abstract

The application of biocatalysis in the pharmaceutical industry is rapidly growing as a

result of increased access to enzymes that meet the demands of industrial processes.

This expansion of activity has led to a corresponding increase in the demand for

immobilised enzymes. EziG™ (EnginZyme AB, Sweden) is marketed as a general

matrix for enzyme immobilisation on controlled porosity glass. In this work we

identified criteria for a “general” enzyme immobilisation technology in the context of

the requirements of the pharmaceutical industry. We subsequently evaluated EziG™

for generality in a series of case studies for the application of immobilised

biocatalysts. In this study we have focussed on the challenges facing both academic

1

and industrial applications such as enzyme stability, multistep reactions and reactions

in continuous flow.

Introduction

The value of biocatalysis lies in the unparalleled specificity with which enzymes

catalyse reactions. This high specificity allows for synthetic chemists to build

molecular complexity by running reactions in tandem, avoiding costly and time

consuming protection and deprotection steps and leading to a simplified supply chain

and lower cost of goods (COG). The increase in enzymatic reactions being utilised

throughout industry has also led to an increase in demand for immobilised enzymes.

Enzyme immobilisation can be a key step to ensuring commercial viability of

industrial biocatalytic processes. Immobilisation can be utilised to facilitate protein

removal from reaction mixtures, which is of tremendous value in the pharmaceutical

industry where it is critical to control purity in drug substances.

Immobilisation can also allow for recycling of enzymes and for reactions to be

conducted in organic solvent, performing reactions in continuous flow, and modular

integration into existing reaction steps thereby enhancing the scope for cascade

biocatalysis.

Enzyme immobilisation strategies can be categorised into three types: cross-linking,

encapsulation, and binding to supports (Figure 1).1 Successful examples of each

immobilisation method have been reported, however it is often difficult to make direct

comparisons between the different strategies.1,2 Perhaps the most well-known

2

immobilised enzyme, CalB (Novozym® 435), is immobilised on an acrylic support.

CLEAs (Cross-linked enzyme aggregates) have also been reported with a number of

impressive examples, in particular combi-CLEAS for multistep reactions and

cascades.3–5

Figure 1 Overview of immobilisation strategies. A) cross-linking B) encapsulation C) binding to a carrier

either by adsorption, affinity or covalent attachment

One significant drawback to many of these technologies is the requirement for

extensive screening and optimization of immobilisation conditions to obtain

acceptable performance. In order to maximise impact and minimise development

time, immobilisation strategies need to be as general as possible. 6,7

Although generality is often an ambiguous term, in the following section we comment

on the meaning of “general” from the perspective of a major pharmaceutical

organization. Considerations are made for a process chemistry implementation of

immobilised biocatalysts and broad guidance for these requirements are detailed in

Figure 2.

3

1.1. Guidelines for a “general” immobilisation strategy

Any immobilisation process should have broadest possible application across

numerous enzyme classes but, as an absolute minimum, generality within the same

enzyme class. The time taken to develop the immobilisation protocol needs to be fast

(i.e. less than one month) thereby allowing for biocatalytic solutions to be

implemented earlier in the development process and allowing for the use of the most

advanced enzyme variant generated at that stage in development. The immobilised

enzyme should be stable in organic solvent with no leachable or extractable material

from either the protein or solid support. Transparency in the manufacturing of the

solid support is also required to ease the regulatory burden by ensuring that all process

related impurities are detected. This issue is essential in controlling the purity profile

of the active pharmaceutical ingredients (APIs). The purity profile should also remain

unchanged over time. The immobilised material must be mechanically stable and have

good flow properties making it amenable to both flow and batch processes. This

4

would allow chemists greater flexibility in developing the biocatalytic process.

Figure 2 Requirements for a "general" enzyme immobilisation strategy

In order for a process using immobilised biocatalyst to be viable it must serve to

lower cost of goods (COGs) compared to the alternatives. COGs are comprised of raw

materials and labour and overhead (L&O) costs with L&O being proportional to the

amount of time a reaction spends in a vessel. Recycling the immobilised enzyme can

lower raw material costs. Typically a minimum of 20 recycles is desired for a cost

effective process. L&O costs are managed by ensuring the immobilised process is

productive, with a desired value of 2 Kg product/Kg enzyme/hour. The enzyme

should be inexpensive, easily immobilised and retain at least 50% of its activity

during the immobilisation process. It is also appealing if multiple enzymes can be

supported with high activity, as this would facilitate multistep transformations. A

loading of at least 10 wt.% active proteins with respect to the solid support is also 5

required for a cost effective process. The immobilised enzyme should be stable for

one year, preferably at ambient temperatures, with no loss in activity, in order to

allow for cost effective shipping and storage. This provides flexibility as to where the

API is manufactured and serves to keep costs down and get medications to patients in

an expedient manner. Immobilised enzyme preparation should also allow for good

mass transport of substrates into, and products out of the matrix.

Currently, enzyme immobilisation on a solid support provides the highest probability

of success but is also the most inefficient from a product mass intensity (PMI)

perspective. PMI is a measurement of the environmental impact of the process.

Greener immobilisation processes such as cross-linking and encapsulation could

prove to be more cost effective than carriers. However, the use of toxic cross-linkers

such as glutaraldehyde adds an analytical burden to the regulatory process. Despite

the indisputable success and efficacy of existing immobilisation strategies, most

existing immobilisation techniques fail to meet several of the criteria for industrially

applicable immobilisation strategies but most critically, they lack generality.8

EziG™TM (EnginZyme AB, Sweden), is a general technology for immobilisation of

enzymes possessing His-tags. The EziG™™ material is based on controlled pore

glass, which is coated with an organic polymer and chelated Fe(III) ions, facilitating

the selective binding of His-tagged proteins directly from crude cell lysate. Due to

efficient mass transfer through interconnecting pores, and selective and non-

destructive binding through His-tags, a high enzyme mass loading can be reached

without the loss of activity caused by a high degree of diffusion limitation and

deactivation.9 His-tag binding to Fe(III) gives strong non-covalent attachment 6

resulting in low levels or even no protein leaching. Fe(III) posesses low human

toxicity and is environmentally benign. EziG™ is currently available in three different

versions with varied surface hydrophobicity Opal (Hydrophilic), Coral (Hydrophobic)

and Amber (Semi-hydrophilic). Previously CalA and CalB immobilised on EziG™

have been shown to give enhanced volumetric activity and speed of immobilisation

compared to Accurel resin. An ω-transaminase and Baeyer–Villiger monooxygenase

(BVMO) immobilised on EziG™ were found to be active in the presence of organic

solvents.10,11 The immobilised preparations were shown to catalyse reactions that did

not proceed with the soluble protein. Recently, Mutti and co-workers showed that the

dual enzyme hydrogen-borrowing amination of alcohols could be performed with co-

immobilised amine dehydrogenase and alcohol dehydrogenases.12–14 Finally Höhne

and co-workers have shown that an imine reductase (IRED) could be immobilised

from cell-free extracts and recycled for up to 14 reaction cycles.15

Results and Discussion

In this current study we set out to investigate the generality of EziG™ as an enabling

technology for the immobilisation of enzymes from a diverse range of classes, with

different reaction chemistries, cofactor requirements and reaction conditions. In line

with the industrial requirements for immobilised enzyme systems discussed above, we

have carried out a series of case-studies, examining the ability of EziG™ to solve

problems routinely encountered in biocatalysis.

7

Two excellent reviews published separately by Sheldon and Bommarius respectively

highlight several long-standing challenges in the field. These include enzyme

engineering, enzyme stability and availability of ready-to-use biocatalysts.16,17 We

have focussed our case-studies on demonstrating that immobilisation on EziG™ can

be used to expand the operating window of biocatalysis – be that via enzyme

stabilisation or reactions in organic media. We also show that combining enzymes in

“plug and play” type packed bed reactors could provide more ready to use

biocatalysts.

Moreover, from an academic perspective, many of the benefits of immobilisation can

also be exploited. Co-immobilisation of multiple enzymes on single carriers opens the

door to multistep cascades and convergent transformations. On the other hand, the

potential for immobilisation and performing reactions in continuous flow opens a new

area of intensified biocatalytic reactions and even the possibility of combinatorial

synthesis using immobilised biocatalysts.

Initially we selected a broad panel of enzymes with known activities available within

our laboratory. The panel of enzymes examined all have significant value as industrial

biocatalysts (Table 1).

8

Table 1 Panel of enzymes immobilised on EziG™. For details see Supporting Information.

Enzyme EziG™Maximum % wt.

enzyme on carrier

Activity

Recovery (%)

Alcohol oxidase (AOx) Amber 20 > 99

P450 Monooxygenase (P450) Amber < 2 30–40

Alcohol dehydrogenase (ADH) Amber 25 > 99

Amine dehydrogenase (AmDH) Amber 32 > 99

Carboxylic acid reductase (CAR) Opal 26 > 99

Reductive aminase (RedAm) Amber 15 > 99

A major limitation of many immobilisation strategies is the significant loss in activity

coupled to a low maximum weight loading of enzyme on the carrier. This loss of

enzyme activity can increase the amount of enzyme required thereby reducing the

PMI of a process. The success of immobilisation can be quantified using activity

recovery metric reported by Sheldon.1 Activity recovery gives a value to the overall

success of enzyme immobilisation. It is the product of the immobilisation yield

9

(amount of enzyme immobilised) and immobilisation efficiency (measured activity of

immobilisation vs. theoretical activity based on immobilisation yield).

Initially we investigated the levels of retained activity and maximum loading on each

EziG™ carrier (Opal, Coral and Amber). The activity of the immobilisates was

determined with respect to model reactions performed with an equivalent amount of

soluble enzyme(s) (see Supporting information). For the enzymes that require

addition of exogenous cofactors, stoichiometric cofactor was used for the initial study.

Most of the enzymes showed good retention in activity (> 50%) compared to the

soluble preparations (Table 1).

Next, the maximum weight loading of the different enzymes on the carrier of choice

was determined; in each case a purified sample of the His-tagged enzyme was

exposed to the preferred EziG™ carrier at a theoretical maximum weight loading of

80 %. After incubation for 30 minutes, the amount of protein remaining in the

supernatant (as determined spectrophotometrically) was determined to give, by

inference, the weight of immobilised protein (see Supporting Information).

Amongst the enzymes tested, only the P450 monooxygenase showed markedly

reduced activity compared to the soluble protein. This could be partially attributed to

use of cell-free extracts for immobilisation of the P450. Immobilisation of His-tagged

proteins directly from cell free extracts is less efficient than for purified proteins

owing to the presence of non-specifically bound proteins. However, the selective

binding of the His-tagged protein from crude cell extracts has been successful in

several cases when subjected to optimisation.9,11,15 In this case the total P450 bound to 10

the carriers is probably overestimated and hence the actual recovered activity may be

higher than reported (see Supporting Information).

Given the excellent activity recovery, weight loadings and immobilisation yields

obtained with the majority of tested enzymes (RedAm, AmDH, AOx, ADH and

CAR), EziG™ appeared to be an excellent platform for enzyme immobilisation.

However, as discussed in the introduction, there are many criteria that a general

immobilisation technology should meet. In the following sections we present case

studies examining some of the remaining requirements. We chose to focus

specifically on challenges appealing to both academic and industrial applications such

as enzyme stability, multistep reactions and reactions in continuous flow (Figure 3).

Figure 3 Challenges for biocatalysis investigated in the following case studies. 1) General enzyme

immobilisation and operational stability 2) Biocatalysis in continuous flow 3) Enzyme cascades with

immobilised enzymes 4) Enzymes in organic media

11

1 2

3 4

1.2. Reductive Aminase (RedAm) – expanding the operational window

Recently our group reported the discovery, characterisation and application of a

reductive aminase from Aspergillus oryzae (AspRedAm) which catalyses the

NADPH-dependent reductive coupling of a wide range of carbonyl compounds and

amines, in some cases at near-stoichiometric equivalents of the amine partner, to give

a variety of secondary amines.18 However, despite the attractive reaction scope,

AspRedAm suffers from a lack of operational stability. At temperatures above 30 °C

the enzyme rapidly denatures and precipitates within a matter of hours. Developing

stable, immobilised enzymes is attractive for process development applications. We

investigated whether co-immobilisation of AspRedAm and glucose dehydrogenase

(for cofactor recycling) on EziG™ could offer a facile way to stabilise AspRedAm.

AspRedAm and glucose dehydrogenase (GDH-II) from Bacillus megaterium were co-

immobilised on all three EziG™ carriers (10 % wt. each)19 (see Supporting

Information). The activity was compared to reactions containing an equivalent

amount of soluble protein. When the reductive amination of cyclohexanone 2 with

allylamine 1 was performed under the previously reported conditions for 1 hour,

(Figure 4a) all the immobilised preparations showed good retention of activity

compared to the soluble preparation. The best activity was obtained with EziG™

Amber showing better conversions than even the soluble preparation under the

conditions examined.

12

Figure 4 Top: Reductive amination of cyclohexanone 2 with allylamine 1 by AspRedAm to give amine 3

Bottom: A) Comparison of conversion obtained by soluble and immobilised AspRedAm catalysed reductive

amination performed at 37 °C B) Conversions obtained from recovery and recycling of co-immobilised

AspRedAm and GDH

The co-immobilised AspRedAm and GDH on EziG™ Amber was further investigated

with respect to temperature and total turnover number (TTN). When the reductive

coupling of cyclohexanone 2 (5 mM) and allyamine 1 (10 mM) was performed

overnight at 37 °C, the reaction with soluble enzyme reached a maximum conversion

to 3 of 49 % with visible precipitation of protein inside the reaction vessel. By

comparison, the co-immobilised preparation reached a conversion of > 90 %

demonstrating significant stabilisation of the immobilised AspRedAm preparation. To

further examine this stabilisation effect, the immobilised preparation was recovered

from the reaction mixture, and recycled an additional four times. Over the course of

five reaction cycles the immobilised enzyme retained partial activity, albeit with a

13

lower final conversion of 26% (Figure 4b). Moreover, immobilisation of AspRedAm

increased the TTN to 4105 compared to 666 for the soluble enzyme.

1.3. Amine Dehydrogenase – catalysis in continuous flow

Amine dehydrogenases (AmDHs) have been engineered to perform the reductive

coupling of ammonia to aldehydes and ketones.20–22 Biocatalytic reductive amination

reactions employing AmDHs are very attractive, as, unlike amine transaminases

(ATAs) that perform the reaction at the expense of an excess of amine donors such as

isopropylamine and alanine, AmDHs use inexpensive ammonia salts as the amine

source. AmDHs have also been utilised in dual-enzyme cascades for the direct,

asymmetric amination of alcohols including with immobilised enzymes.12–14,23

Recently Mutti et al. coupled AmDHs to formate dehydrogenase from Candida

boidinii (cbFDH), using ammonium formate as a source of ammonia and formate as

the reductant for the cofactor regeneration.24 Pushpanath and co-workers showed

impressive intensification of an engineered AmDH from Caldalkalibacillus

termarium, with [S] up to 400 mM and a space-time-yield (STY) of 40 g L-1 day-1.25

Despite their appeal, AmDHs suffer from product and substrate inhibition, and low

productivity even under intensified conditions.

One strategy for overcoming both of these limitations is to perform reactions in

continuous flow wherein substrate and product are continuously removed as they pass

through the system. The application of continuous flow to chemo- and photocatalysis

is well established.26 However, there are few examples of transformations performed

14

using enzymes in continuous flow as detailed in an excellent review by Kroutil and

co-workers.27,28

Considering the excellent atom efficiency of the AmDH/CbFDH process reported by

Mutti et al., we investigated the translation of this process to continuous flow. The co-

immobilisation of chimeric amine dehydrogenase and formate dehydrogenase

(cbFDH) was optimised with respect to EziG™ carrier type and % relative weight

loading. Despite the higher activity of cbFDH compared to ChiAmDH the best

activity was obtained with a 1:1 ratio of ChiAmDH and cbFDH on EziG™ Amber

(see Supporting Information).

To perform the reaction in continuous flow, a steel column was slurry packed with

EziG™ Amber (150 mg) and the solution of AmDH and cbFDH was applied to the

column, 15 wt. % of each enzyme. Activity assays performed with the eluent showed

complete binding of the target proteins to the EziG™ carrier. The column was

attached to a HPLC pump as shown in Figure 5.

Figure 5 schematic of packed bed reactor (PBR) containing EziG™ with pump and fraction collector and

continuous production of (R)-4-fluoroamphetamine by co-immobilised ChiAmDH and cbFDH.

15

With a flow rate of 0.2 ml min-1 of ammonium formate (1 M, pH 9) containing 4-

fluorophenyl acetone 4 (10 mM) and NAD+ (0.5 mM) conversions to 5 of up to 68 %

were achieved in continuous flow mode for more than 3 hours with no apparent loss

in productivity. The productivity of the reactor equates to a STY of more than 300 g

L-1 day-1. Rapid deterioration in productivity occurred after 6 hours, this is likely due

to inactivation of the cbFDH via oxidation of exposed cysteine residues as previously

reported.29 In contrast, addition of NADH (1 mM) to the reaction buffer showed the

AmDH remained active for at least 6 more hours (data not shown).

Next we applied our recently developed hydrogen-borrowing cascade for the

asymmetric animation of racemic alcohols. Mutti and co-workers have shown that a

similar transformation can be performed for the amination of enantiopure alcohols

immobilised on EziG™ carriers.14 However this type of enzyme cascade has not been

reported under continuous flow conditions.

An engineered variant of secondary alcohol dehydrogenase from Thermoanerobacter

ethanolicus (TeSADH W110A/G198D) and ChiAmDH were co-immobilised on

EziG™ Amber. The efficient amination of 1-(4-fluorophenyl)propan-2-ol 6 to give 5

could be achieved using the immobilised biocatalyst under previously optimised

conditions (Figure 6).13

16

Figure 6 Hydrogen-borrowing amination of 1-(4-fluorophenyl)propan-2-ol in continuous flow

The co-immobilised TeSADH W110A/G198D and ChiAmDH preparation was slurry

packed into a steel column as described for the AmDH/FDH system. A reaction buffer

containing ammonium formate (1 M, pH 9), 1-(4-fluorophenyl)propan-2-ol 6 (10

mM) and NAD+ (1 mM) was passed continuously through the column at a flow rate

of 0.02 mL min-1. The reaction reached steady state within 10 column volumes and

the product 4-fluoroamphetamine 5 could be obtained in the effluent with a STY of 13

g L-1 day-1. This steady state conversion (ca. 30 %) was maintained for at least 20

hours. (see Supporting Information).

One limitation of the current system is a single pass of the aqueous phase containing

substrates and cofactor; this could be improved by the addition of an in situ product

removal such that a small volume of the aqueous buffer containing ammonia and the

cofactor could be continuously recycled and mixed with a substrate stream. However,

this system provides proof-of concept that simple plug-and-play reactor set ups are

possible using immobilised enzymes and simple off the shelf equipment. Moreover,

17

this represents the first example of the hydrogen-borrowing amination of alcohols

performed in continuous flow.

1.4. Carboxylic acid reductase – cofactor recycling (ATP, NADPH)

Carboxylic acid reductase (CAR) catalyses the reduction of a broad range of

carboxylic acids to the corresponding aldehydes at the expense of ATP and

NADPH.30–33 The enzyme is related to the nonribosomal peptide synthetases and

consists of an adenylation domain fused via a peptidyl carrier protein (PCP) to a

reductase termination domain.34,35 Reactions with CAR are almost exclusively

performed in whole cells owing to the need to regenerate both NAD(P)H and ATP.36–

38 This approach has the advantage of exploiting cellular machinery for cofactor

regeneration but suffers from the possibility of unwanted side reactions and/or mass

transfer limitations compared to combining several purified proteins in vitro.

Previously, in vitro reactions with CAR have been performed with

suprastoichiometric ATP and NADPH, although this is uneconomical for large-scale

transformations.

A two-enzyme system for the regeneration of ATP from polyphosphate was reported

by Resnick using cell extract or partially purified enzymes.39 We envisaged that, by

exploiting the generality of EziG™ carriers, CAR in combination with the AMP

phosphotransferase (PAP) and adenylate kinase (AdK) from Acinetobacter johnsonii

could be immobilised onto a single carrier. (Figure 7a).

18

Figure 7 A) In vitro recycling of ATP drives the CAR mediated reduction of benzoic acid to benzaldehdye B)

Conversion of benzoic acid (5 mM) to benzaldehdye with in vitro recycling of ATP.

In order to apply this two-enzyme system for the regeneration of ATP whilst

immobilised on a single carrier with CAR we needed to express and purify PAP and

AdK from A. johnsonii. Recombinant expression in E. coli and purification was

achieved, introduction of an N-terminal His-tag to enable immobilisation onto the

EziG™ carrier (see Supporting Information). Prior to immobilisation we

demonstrated that the two-enzyme system was sufficient for regeneration of ATP in

solution, facilitating the reduction of benzoic acid to benzaldehyde using sub-19

A

B

stoichiometric concentrations of ATP with polyphosphate as the phosphate donor (see

Supporting Information).

Co-immobilisation of CAR and the polyphosphate:AMP phosphotransferase (PPT)

and adenylate kinase (AdK) was performed on all the EziG™ carriers. The loading

with respect to all three enzymes was 10, 5 and 5 % by weight respectively.

Regeneration of NADPH was performed using soluble glucose dehydrogenase (CDX-

901) devoid of a His-tag. Using the reduction of benzoic acid 7 to benzaldehyde 8 as a

model reaction, only EziG™ Opal afforded any detectable activity.

In order to demonstrate that the immobilised system is catalytic we varied the

concentration of ATP from 20 to 0.02 mol% (Figure 7b). The soluble protein and

immobilised cascade system achieved excellent conversions as low as 0.2 mol% ATP

(98 %). However, at very low concentrations of ATP the soluble enzymes reach

higher conversion (98 % vs. 69 % with 0.02 mol% ATP). We attribute this effect to

adsorption of the cofactor onto the matrix resulting in less ATP being available to

participation in the reaction.

We have shown it is possible to perform the in vitro reduction of carboxylic acids at

the expense of catalytic quantities of both NADPH and ATP by co-immobilisation of

a parallel cofactor regeneration cascade. The ATP concentrations required were as

low as 0.02 mol% with a TTN of 3400 with respect to ATP. In addition, the ability to

recover and reuse this complex network of enzymes should broaden the appeal of this

versatile biocatalyst formulation.

20

1.5. Alcohol Oxidase – reactions in organic media

The ability to perform selective oxidation of primary alcohols to the corresponding

aldehyde under mild conditions at the expense of molecular oxygen is an attractive

alternative to classical methods such as Collins, Swern, Dess-Martin, Pfitzner-Moffat

and Ley reactions that often suffer from over oxidation to the corresponding

carboxylic acid.40 Recently, choline oxidase from Arthrobacter chlorophenolicus has

been engineered yielding a four amino acid variant with much broader substrate

specificity towards primary alcohols, e.g. hexanol. A further two mutations gave

increased temperature stability and solvent tolerance.41 Compared to NAD(P)H-

dependent alcohol dehydrogenases (ADHs), which require external cofactor

regeneration, alcohol oxidases (AOx) are highly atom efficient. Enzymatic

transformations performed in organic solvents are attractive because they allow

solubilisation of hydrophobic substrates. In the context of alcohol oxidations a low-

water environment also reduces the possibility of over oxidation to the carboxylic acid

via oxidation of the geminal diol (Figure 8a).

21

Figure 8 A) AOX catalysed oxidation of 1-hexanol with proposed route to formation of carboxylic acid B)

Oxidation of 1-hexanol to hexanal performed by EziG™ immobilised AOx in a panel of organic solvents.

AOx was immobilised onto EziG™ Amber (final 10 % wt. loading), the buffer was

removed by pipetting, spread on a filter paper and allowed to dry affording free

flowing carrier. The immobilisates were used to perform the oxidation of 1-hexanol 9

in a panel of common solvents. After each reaction the carriers were recovered,

washed in the appropriate solvent and the reactions repeated for at least three cycles

(Figure 8b). The AOx showed good activity in all of the solvents: initially the best

activity was found for the solvents that are most miscible with water (EtOAc and

MTBE), however after repeated reaction cycles the activity in these solvents falls

significantly. The reduction in activity suggests that after the immobilisation and

washing, some water important for catalysis remains either within the enzyme or

support. When reactions are repeatedly performed in sparingly miscible solvents this

22

A

B

water may be removed resulting in a loss of activity. Further evidence for the

importance of water is the complete inactivation of the enzyme upon freeze-drying.

Despite the lower observed activity in the remaining solvents, the activity appeared to

be stable for at least three reaction cycles. No over-oxidation to the acid 11 was

observed in any of the biotransformations.

Next we investigated the translation of the reaction in organic media to continuous

flow. The immobilised AOx was slurry packed into a steel column as described for

the earlier. The reaction stream containing 1-hexanol 9 (10 mM) was passed

continuously through the column at a flow rate of 0.1 mL min-1. To our surprise, when

buffer (20 mM Tris-HCl, pH 8) was used as the solvent, neither starting material 9 or

hexanal 10 could be detected in the effluent stream over at least 30 column volumes.

Upon washing the column with DCM, only hexanoic acid 11 could be isolated (100 %

conversion >90 % mass recovery). We propose that the high catalyst loading in

combination with formation of the geminal diol in the flow conditions results in this

over-oxidation.

When the reaction stream was changed to cyclohexane, containing 1-hexanol 9 (10

mM) and the reaction performed under identical conditions only conversion to

hexanal 10 was observed. (> 95 % mass recovery). With a continuous flow rate of 0.5

mL min-1 the oxidation of 1-hexanol quickly reaches a steady state of ca. 30 %

conversion to hexanal (see Supporting Information). This steady state was maintained

for at least 120 column volumes showing excellent tolerance to the operating

conditions.

23

We have shown that immobilised AOx can perform alcohol oxidations even in the

presence of pure organic solvent ensuring ease of product recovery. The reaction was

translated to continuous flow in a packed bed reactor and steady state operation was

found for at least 120 column volumes.

Conclusions

Immobilisation of biocatalysts is a useful, necessary and under-utilised strategy for

developing a viable industrial processes. We have identified a number of conditions

and requirements for a “general” and desirable immobilisation strategy. However,

many existing technologies are not general enough to have this broad appeal. One

such more “general” technology, EziG™ from EnginZyme AB has been evaluated for

performance against a number of these requirements. We have demonstrated that it is

possible to use EziG™ to expand operational windows by improving stability of a

reductive aminase from Aspergillus oryzae, performed reactions with an engineered

alcohol oxidase in organic solvent and shown enzyme cascades in continuous flow

reactions. We have also demonstrated the direct translation from batch to flow of

AmDH catalysed reductive amination and hydrogen-borrowing amination of alcohols,

in addition to in vitro regeneration of ATP in a cascade manner on a single

immobilisate. EziG™ has been demonstrated as a general technology with great

potential. Although we have found limited evidence of enzyme leaching from the

supports, loss of enzyme activity has been observed after a few reaction cycles (the

manufacturer notes that recyclability can be optimised. In addition, EziG™ carriers

can be recycled following elution of the immobilised protein).9 In addition, occasional

difficulty in immobilisation from cell-free extracts was noted. Hence while the current

24

EziG™ carriers can expand some operational windows, protein engineering may still

be necessary to optimise a biocatalyst for operational stability. It remains to be seen if

for an industrial context, regulatory, process and reduced COG needs can be met.

25

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