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TRANSCRIPT
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, [email protected]
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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