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In this tutorial review we describe a holistic approach to the invention, development and optimisation of
biotransformations utilising isolated enzymes. Increasing attention to applied biocatalysis is motivated by
its numerous economic and environmental benefits. Biocatalysis engineering concerns the development
Received 28th November 2016 of enzymatic systems as a whole, which entails engineering its dierent components: substrate
DOI: 10.1039/c6cs00854b engineering, medium engineering, protein (enzyme) engineering, biocatalyst (formulation) engineering,
biocatalytic cascade engineering and reactor engineering.
rsc.li/chem-soc-rev
1. Introduction Today almost all industrial enzymes are recombinant, with the
exception of those used in food processing.
The use of enzymes, before their existence was even known, Biocatalysis generally refers to the use of enzymes, or
dates back thousands of years to bread- and cheese-making, enzyme containing cells, in chemical transformations. There
beer brewing and wine-making. Food and beverage processing are basically two types of biotransformations: those involving
together with animal feed and detergents still account for more growing (microbial) cells and resting cells, respectively. The
than 60% of the total enzyme market, the remainder consisting former are fermentations, the main goal of which, in vivo, is
mainly of starch processing, leather, and pulp and paper, and to produce more microbial cells but in vitro the objective
this is reflected in the three highest volume industrial enzymes is generally the production of chemical products. Buchner
being proteases, amylases, and glucose isomerase.1 The first showed in 1897 that the degradation of glucose into ethanol
commercial enzyme preparations were produced in the late by living yeast, found by Pasteur in 1860, didnt actually require
19th and early 20th centuries and include the use of dried living yeast but only the catalytic components he extracted from
calves stomachs in cheese manufacture and pancreatic extracts the cells. Nonetheless, the term fermentation continued
in laundry cleaning. It was the development of recombinant to be used for transformations with growing microorganisms.
DNA technology in the 1970s, however, which provided the The catalytically active substances present in cell extracts,
basis for the shift from the traditional animal and plant sources later shown to be proteins, were given the name en-zymes,
to much more ecient production with recombinant micro- meaning in-yeast. While acknowledging that advances in
organisms, thus aording cheaper and higher purity enzymes. metabolic pathway engineering and the underpinning synthetic
biology2 have revolutionised the microbial production of chemical
a
Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, and pharmaceutical products, in this review we will focus on
Johannesburg 2050, South Africa. E-mail: roger.sheldon@wits.ac.za; the use of isolated enzymes as biocatalysts.
Tel: +27 11-7176727
b
Organic synthesis with the aid of enzymes has been known
Department of Biotechnology, Delft University of Technology, Section BOC, van der
Maasweg 9, 2629 HZ, Delft, The Netherlands. E-mail: r.a.sheldon@tudelft.nl;
since the early 20th century but until fairly recently, with
Fax: +31 (0)15 2781415; Tel: +31 (0)15 2782675 some notable exceptions, such as the synthesis of beta lactam
Electronic supplementary information (ESI) available. See DOI: 10.1039/c6cs00854b antibiotics,3 was not widely used in industrial processes.
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This situation changed in the mid-1980s following two low cost. Consequently, more enzymes are available and they
coincidental developments. First, the landmark publication can be produced for commercially acceptable prices, making
by Zaks and Klibanov4 in 1984, describing enzymatic catalysis the notion that enzymes are expensive obsolete.
at 100 1C in organic media, demonstrated that many enzymes Furthermore, protein engineering techniques, such as directed
were actually more thermally stable in organic solvents, such as (in vitro) evolution,6 have enabled the engineering of enzymes
toluene, than in water. This was a revelation for most synthetic such that they exhibit pre-defined properties with regard to,
organic chemists since conventional wisdom held that enzymes inter alia, substrate specificity, activity, selectivity, stability and
work well only in water. It led to the realisation among organic pH optimum. Lastly, the development of eective immobilisation
chemists that biocatalysis had a much broader scope in organic techniques has improved their operational stability and cost-
synthesis than was previously imagined, totally and lastingly eectiveness by facilitating recovery and recycling.7
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changing the biocatalysis landscape. Second, at about the same Consequently, in the last decade biocatalysis has been
time, recognition of the importance of stereoisomerism in drug integrated into mainstream organic synthesis, particularly in the
action signaled the advent of regulations by, for example, pharmaceutical industry.8 Indeed, Turner and OReilly9 proposed the
the FDA which required that producers separate and test both introduction of guidelines and rules for biocatalytic retrosynthesis
enantiomers of chiral drugs. In commercial practice this to aid synthetic chemists in identifying where biocatalysts could be
generally led to the decision to produce and market the drug applied to the synthesis of target molecules.
as a single, active enantiomer. It created a need for cost- The broad application of biocatalysis can also be attributed to its
eective methods for enantioselective synthesis and biocatalytic numerous environmental and economic benefits. Enzymes are
methods were obvious candidates since enzymes are usually natures sustainable catalysts. They are derived from renewable
highly enantioselective. resources and are biocompatible, biodegradable, essentially non-
Hence, biocatalysis evolved from an academic curiosity to an hazardous and non-toxic. Biocatalysis avoids the use of scarce
industrially attractive technology for the enantioselective synth- precious metals and the associated, often prohibitive, costs of
esis of APIs.5 However, in the early 1990s a major obstacle to removing traces of noble metals, to an acceptable ppm level, from
their widespread use was the number of commercially available end products. Enzymatic reactions are performed under mild
enzymes. This was largely limited to those already in use in the conditions (physiological pH and ambient temperature and
food and beverages and detergent industries: mainly proteases, pressure) in water, often without the need for functional-group
lipases, esterases and glycosidases, often from animal origin. activation and protection and deprotection steps required
Over the last two decades this situation has changed dramati- in conventional organic syntheses. This translates to shorter
cally thanks to modern biotechnology. As a result of high syntheses, higher selectivities and purer products in processes
throughput DNA sequencing, more than 20 000 whole genome that are more resource and energy ecient and generate less
sequences have become publicly available. Thanks to advances waste than conventional routes. Moreover, enzymatic processes
in recombinant DNA technology it is now possible to identify a can be conducted in standard multi-purpose batch reactors and,
target gene, by in silico analysis of a genome sequence data hence, do not require any extra investment, e.g. for high-pressure
base, have the gene synthesised chemically within weeks, ready equipment. Lastly, most enzymatic reactions are conducted
for cloning into a host production organism and at relatively under roughly the same conditions of temperature and pressure
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and, hence, it is relatively easy to integrate multiple reactions biocatalyst is merely one part. In this tutorial we shall describe
into eco-ecient catalytic cascade processes (see later). dierent strategies for biocatalysis engineering aimed at
Wild-type enzymes have evolved over millions of years to be improving existing biotransformations or creating new ones.
able to convert their natural substrates in high rates in vivo. It These include engineering of the substrate, the reaction
is not surprising therefore, that they have a problem with medium, the protein, and post-translational modification of
sustaining a high activity and productivity with non-natural the enzyme. Finally, reactor engineering in combination with
substrates under more challenging conditions in vitro, such downstream processing facilitates product recovery and re-use
as high substrate concentrations and non-aqueous media. of the biocatalyst. Hence, the holistic approach of biocatalysis
Nonetheless, spectacular results have been obtained in some engineering can aord an environmentally and economically
cases with wild-type enzymes. An early example is the enzymatic viable biotransformation by optimising all the variables.
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3. Medium engineering
Fig. 4 Epoxide ring opening reactions catalysed by HHDH. As we already noted (see above), the 1984 paper of Zaks
and Klibanov served as a wake-up call to organic chemists.
Enzymes generally function optimally in water but they are also
react with the epoxide. Indeed, HHDHs are highly promiscuous active in organic solvents. Indeed, biocatalysis in organic media
enzymes that can accept at least nine other anions as nucleo- has several benefits: many organic substrates are at best
philes in the enantioselective ring opening of epoxides,15 only sparingly soluble in water, and some reactions e.g. (trans)-
forming the basis for the highly enantioselective synthesis of esterifications and amidations, cannot be conducted in water
a broad range of b-substituted alcohols. The use of cyanide ion owing to equilibrium limitations and/or competing product
as the nucleophile, for example, is particularly interesting as it hydrolysis. Additional benefits of non-aqueous biocatalysis are
generates a new CC bond. It has been applied in a process, easier product recovery from volatile organic solvents and
commercialised by Codexis, for the synthesis of a key inter- elimination of microbial contamination. In short, engineering
mediate in the manufacture of atorvastatin,16 the active ingre- of the reaction medium can be used to optimise the synthetic
dient of Pfizers cholesterol lowering agent Lipitor (Fig. 4). potential of biocatalytic transformations.
A more recent, remarkable example of enzyme promiscuity Although enzymes are able to perform as suspensions in
designed by intuitive, mechanism-based substrate engineering organic solvents, their catalytic eciencies are generally two
is cyclopropanation via carbene transfer catalysed by an engi- orders of magnitude lower than those observed in water. It
neered cytochrome P450-dependent monooxygenase.17 In vivo should be noted, however, that it is dicult to meaningfully
these enzymes catalyse the aerobic oxidation of a wide variety of compare the rates of reactions conducted in a homogeneous
organic substrates via a high-valent oxo-iron species as the enzyme solution in water with reactions catalysed by the same
active oxidant. Reaction of the latter with an olefin leads to enzyme as a heterogeneous suspension of a solid in an organic
transfer of the oxygen atom (oxene transfer) to the double bond, solvent. Moreover, the environmental issues associated with
aording an epoxide (Fig. 5). By analogy, the formation of a high- the use of volatile organic solvents (VOCs) represent another
valent iron-carbenoid species from a diazo ester as a co-substrate drawback of reactions in organic media. In this context it
was envisaged. Reaction of this putative intermediate with an should also be mentioned that many biocatalytic reactions
olefin would result in carbene transfer to the double bond, are performed, both on laboratory and industrial scale, in an
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aqueous biphasic system, consisting of a, preferably environ- suspension of CaLB in anhydrous [bmim][PF6] and [bmim][BF4].20
mentally attractive, organic solvent and water. The reaction To ensure that they were anhydrous, both the IL and the enzyme
takes place in the aqueous phase and the substrate and product were dried over phosphorus pentoxide prior to use. Although the
are dissolved primarily in the organic phase. A good example is rates were only marginally higher than those observed in the best
the three enzyme process for atorvastatin intermediate referred organic solvents these results demonstrated the compatibility
to earlier which is conducted in aqueous ethyl acetate. of enzymes with ILs and marked the advent of prolific studies
The best solvent is no solvent but if a solvent (diluent) is of biocatalysis in ILs in the next 15 years.
needed supercritical carbon dioxide, scCO2, is a potentially The use of ILs as reaction media can also result in increased
attractive alternative to VOCs. It is non-flammable, non-toxic enantioselectivity or stability resulting from conformational
and is the only readily available, inexpensive solvent that is changes of enzymes in IL media. Based on their ability to dissolve
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supercritical under conditions (31 1C and 7.4 MPa) which are large amounts of highly polar substrates they are potentially
conducive to biocatalysis. In short, a natural catalyst in a interesting media for biotransformations of carbohydrates,
natural solvent. Furthermore, supercritical fluids combine the nucleosides and polysaccharides which are, at best, only
solubilising capacity of a liquid with the low viscosity of a gas, sparingly soluble in common organic solvents.
resulting in high rates of mass transfer, and the product can be An important driver for using ILs is the possibility of
recovered by simply reducing the pressure. Enzymes generally replacing VOCs with non-volatile ILs, thereby circumventing
exhibit good activity and stability in scCO2 but there are two the risk of air pollution. However, ILs have significant solubility
properties which can have a detrimental eect: (i) amino groups in water and first generation ILs, such as dialkylimidazolium
in lysine residues can react with CO2 to aord carbamates, and and tetraalkylammonium salts exhibit aquatic ecotoxicity and
(ii) CO2 can react with water to give carbonic acid resulting in a poor biodegradability. Moreover, their preparation often involves
drop in pH. circuitous processes, making them prohibitively expensive.
Immobilisation of the enzyme using the CLEA methodology Hence, the current trend is towards the rational design of task
(see later) suppresses inactivation by converting free amino specific, biocompatible ILs that combine cost-eectiveness with
groups on the surface. In the kinetic resolution of 1-phenyl- a low environmental footprint. Cholinium ILs, for example, are
ethanol by CaLB-CLEA catalysed transesterification in scCO2 in prepared by reaction of inexpensive choline hydroxide with a
continuous mode, reaction rates were higher than those wide variety of carboxylic acids or amino acids.21
observed in n-hexane.18 A benefit of using liquid CO2 is that Another class of IL comprises the protic ionic liquids (PILs).
it can be maintained under relatively modest pressure (4.5 MPa The latter are easily prepared, by simply mixing a tertiary amine
at 10 1C). In another recent development transesterification of with a (carboxylic) acid, and exhibit better biodegradability and
triglycerides in scCO2 catalysed by lipases immobilised in lower toxicity than the corresponding quaternary ammonium
monoliths in packed bed reactors was used for continuous salts. Moreover, they have suitable H-bond donating properties
biodiesel production.19 for interaction with and stabilisation of enzymes and, when
The activity of enzymes in organic solvents can be increased combined with carboxylate anions, they are self-buering. PILs
by lyophilisation in the presence of relatively large amounts of derived from a variety of tertiary amines and carboxylic acids
salts such as potassium chloride. This led to the idea that were successfully used, for example, as the reaction medium for
suspension of an enzyme in a room temperature ionic liquid enantioselective transesterification of a-methyl benzyl alcohol
(IL), with its salt- and water-like character, could aord signifi- (Fig. 7) catalysed by CaLB-CLEA (see Section 5).22
cant rate enhancements compared to organic solvents. ILs are An important issue regarding reactions in ILs is: how can the
composed entirely of ions and are liquid at or close to ambient product be separated from the ionic liquid? An attractive option
temperature. They have been widely advocated as potentially is to use supercritical carbon dioxide, scCO2, to extract the
attractive alternatives to volatile organic solvents (VOCs). product. The IL has no measurable solubility in scCO2 but
The first examples of biocatalysis in a water-free IL are the latter is highly soluble in the IL phase and can extract
the transesterifications and amidations (Fig. 6) catalysed by a hydrophobic molecules. This provides the basis for biphasic
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Table 2 Evolution of a HHDH biocatalyst by DNA shuing required and were subject to acetone inhibition. Subsequent
directed evolution using DNA shuffling was focused on improv-
Parameter Process design Wild-type Best variant
1
ing the activity and stability. After an initial three rounds
[Substrate] (g L ) 120 20 140 of evolution the product/enzyme mass ratio was improved 400
[Enzyme] (g L 1) 1.5 30 1.2
Catalyst productivity (g g 1) 80 0.7 117 fold from 1 : 50 to 8 : 1. Further improvements were obtained
STY (g L 1 day 1) 4360 7 672 by optimising the reaction medium to a toluene/water mixture
Isolated yield (%) 490 67 92 and raising the reaction temperature to 40 1C. The final variant
Chemical purity (%) 498 498 498
ee (%) 499.5 499.5 499.5 exhibited a 3000-fold improvement and the process could be
Reaction time (h) 8 72 5 performed in a slurry-to-slurry mode at 100 g L 1 substrate
Phase separation (min) o10 460 o1
loading and 3 g L 1 enzyme concentration in 24 h at 45 1C,
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5. Biocatalyst engineering
Having identified an enzyme for the targeted transformation,
optimised its properties using substrate, medium and protein
Fig. 11 Montelukast intermediate by KRED reduction. engineering techniques, the enzyme is expressed in a microbial
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production host with a GRAS (Generally Regarded as Safe) status to to be remarkably robust in dry isopropyl acetate at 50 1C, with a
enable its overproduction in large amounts at relatively low cost. deactivation rate of 0.5% h 1 over 6 days. When water-saturated
The next step is to identify an eective formulation of the enzyme. isopropyl acetate was used no deactivation was observed over the
Enzymes are soluble in water and it is dicult and costly to recover same time period and 10 consecutive cycles were obtained, with
them from aqueous euents, although this can be achieved using no detectable loss of activity over a 200 h period. In contrast, the
ultrafiltration. Hence, many enzymes are used on a single use, soluble TA was completely denatured and showed no activity in
throw-away basis. The enzyme costs per kg of product can be the organic solvent.
substantially reduced by immobilisation of the enzyme to form a The combination of increased stability and reuse of the
free-flowing powder, i.e. by creating a heterogeneous catalyst to immobilised TA aorded a 490% reduction in the enzyme
enable catalyst recovery and reuse,7 thereby resulting in process loading compared to the soluble enzyme. Interestingly, the
simplification combined with a higher product quality and a immobilised TA was used successfully in the synthesis of chiral
smaller environmental footprint. It constitutes post-translational amines, in high enantiomeric purity, from the corresponding
biocatalyst engineering, as opposed to (pre-translational) protein ketone precursors. The use of an organic solvent represents a
engineering. Additionally, enzyme immobilisation generally significant advantage compared to the aqueous protocol which
results in increased stability, by suppressing the unfolding requires the use of buer and continuous pH control during
and, hence, denaturation of the enzyme. This enables, in turn, the reaction. Organic solvent is then used to extract the product
the use of a much broader choice of solvents. and the mixture filtered to remove the denatured enzyme. The
A case in point is the aforementioned (R)-selective trans- remaining aqueous euent constitutes a solvent contaminated
aminase (TA) developed, using a combination of computer-aided waste stream. In contrast, the use of an immobilised enzyme in
modelling and directed evolution, for the synthesis of sitagliptin. an environmentally attractive organic solvent obviates the need
The optimum protocol involved the use of aqueous DMSO as the for buer, continuous pH control and laborious removal of the
reaction medium. Subsequently, Merck scientists36 investigated denatured enzyme. This substantially simplifies the work-up
the immobilisation of the TA on a variety of polymer-based and reduces the processing time and the amount of waste
resins and compared the immobilisates with the lyophilised free generated. Moreover, the enzyme can be reused over and over
TA. The best results were obtained using the TA adsorbed on a again and the bottom line is a more commercially attractive
highly hydrophobic octadecyl functionalised polymethacrylate process than the rhodium catalysed asymmetric hydrogenation
resin with 4% loading of the TA, resulting in 45% activity of an enamine which it replaced.
recovery. The immobilised TA was shown to be active in a variety Enzyme immobilisation usually results in some loss of activity,
of organic solvents, the best results being obtained in the compared with the soluble enzyme, but this is more than
environmentally attractive, isopropyl acetate which combined a compensated by the increase in stability coupled with reusability
favourable solubility of the ketone substrate with increased which can aord dramatic cost reductions.1 Two important
stability of the TA compared to other solvents. The TA was found landmarks were the immobilisation of glucose isomerase and
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b-glucosidase, for aroma enhancement in wine.41 Similarly, a Manihot esculenta, a non-selective nitrilase from Pseudomonas
trienzyme magnetic combi-CLEA of an a-amylase, pectinase fluotrescens EBC 191 and an amidase from Rhodococcus
and cellulase was used for clarification of fruit juices.42 erythropolis, which catalysed the enantioselective conversion of
benzaldehyde to (S)-mandelic acid in 90% yield and 499% ee
(Fig. 14).44 It was originally developed as a hydroxynitrile lyase/
6. Biocatalytic cascade processes: nitrilase bienzyme combi-CLEA but large amounts of the corres-
cell-free synthetic biology ponding amide were formed as a byproduct of the nitrilase-
catalysed hydrolysis and an amidase was added to convert the
Brevity is the soul of synthesis and the ultimate in green
amide side product to the acid. Rates were higher than with
catalytic methodologies is to telescope multi-step syntheses
mixtures of the respective CLEAs.
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tea bags in the reaction solution. The so-called perfusion basket lignocellulose, in the production of first and second generation
reactor (BR) constitutes a refinement of the tea bag concept. biofuels, respectively. In the production of bioethanol, for
It consists of a metallic filtration membrane-like module that example, the hydrolysis step and the subsequent fermentation
retains the immobilised biocatalyst. The BR was used success- can be integrated in a one-pot simultaneous saccharification
fully, for example, in the degradation of endocrine-disrupting and fermentation (SSF) process. As already discussed in
chemicals in aqueous euents catalysed by laccase-CLEAs.47 Section 5, in this case smart magnetic CLEAs of the hydrolytic
Another variation on this theme is the spinning basket enzymes could be used to enable their facile recovery and reuse,
reactor which was used in the enzymatic degumming of rice with obvious cost benefits.
bran oil catalysed by an immobilised lecitase.48 In yet another
elaboration, a rotating flow cell reactor, consisting of a catalyst-
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