R. WOHLGEMUTH, Molecular and Engineering Perspectives of the Biocatalysis …, Chem. Biochem. Eng. Q.

25 (1) 125–134 (2011) 125

Molecular and Engineering Perspectives of the Biocatalysis Interface

to Chemical Synthesis*
R. Wohlgemuth Review
Research Specialties, Sigma-Aldrich, Industriestrasse 25, Received: January 11, 2011
CH-9470 Buchs, Switzerland Accepted: March 14, 2011

The sustainable use of limited resources by nature to provide target molecules with
biocatalytic reactions continues to be a role model for chemical synthesis. The applica-
tion of biocatalysts to functional group transformations is shaped by the various parallel
influences like e.g. the search for selectivity, the shift from fossil-based to biobased raw
materials and the economy of molecular transformations like atom economy and step
economy. As safety, health and environment issues are key drivers for process impro-
vements in the chemical industry, the development of reactions or pathways replacing
hazardous reagents is another major factor determining the sequence of molecular trans-
formations from raw material to product.
Biocatalyst production technologies and integrated process engineering have been
instrumental in the establishment of biocatalytic reaction steps in chemical synthesis.
The inherent properties of biocatalysts make them the privileged catalysts for highly se-
lective asymmetric molecular transformations like e.g. hydrolysis reactions, oxidation re-
actions, carbon-carbon bond formation reactions as well as molecular unit transfer reac-
tions. The universe of six enzyme classes provides a tremendous goldmine for dis-
covering improved versions of enzymes with known functions as well as for finding
completely novel enzymes. With the growing collection of biocatalytic reactions, the
retrosynthetic thinking from chemical synthesis can be applied to biocatalysis as well.
Once the feasibility of a biocatalytic reaction has been proven, up- and downscaling
experiments have been useful for engineering the most adequate process design. In the
case of the first large-scale biocatalytic Baeyer-Villiger oxidation, the debottlenecking of
the substrate feed and product recovery, final purification and overcoming thermody-
namic limitations have been essential in establishing bioprocesses with high yields of
enantiopure products. These downscaling experiments in conjunction with new analyti-
cal techniques have proven useful also in the case of asymmetric synthesis of natural
compounds. Spatial and temporal organisation of biocatalysts, reactants or products is
another interesting engineering option for biocatalytic process design.
The interdisciplinary character of the dead ends and locks between chemistry, biol-
ogy and engineering requires investigations of the interfaces. Communication across sci-
entific and technological disciplines including the value creation perspective is important
for the development of a better synthesis for the final product-in-the-bottle. Whether the
successful problem solution will come from the engineering of substrates, reaction me-
dia, process conditions or from the search for better and new enzymes, progress in the
understanding of the molecular mechanisms of enzyme action will be key for the further
development of the science of synthesis with its challenges towards the more difficult
and more complex target molecules.
Key words:
Atom economy, step economy, redox economy, enzymatic resolution, asymmetric syn-
thesis, biotransformation, biocatalysts, oxidoreductases, transferases, hydrolases, lyases,
biocatalytic process design, downstream processing, scalability

Introduction creasing number of applications. The growing hu-

man population is requiring larger amounts of prod-
The knowledge in the preparation of natural
and synthetic compounds has led to an enormous ucts and intermediates, which have been and con-
number of different molecular structures for an in- tinue to be supplied by scaling up manual syntheses
from small volumes to industrial large-scale and
*Based
highly automated and controlled productions.
on an Invited Lecture at the XXI Croatian Meeting of Chemists
and Chemical Engineers, Trogir, April 19–22, 2009. While the science of synthesis has contributed spec-
E-mail: roland.wohlgemuth@sial.com tacular advances in the high-yield preparation of the
126 R. WOHLGEMUTH, Molecular and Engineering Perspectives of the Biocatalysis …, Chem. Biochem. Eng. Q. 25 (1) 125–134 (2011)

most complex molecular structures from simple minimization becomes an economic necessity for
building blocks or intermediates, the applications the viability of manufacturing processes worldwide.
on industrial large-scale or in biological systems re- This has led to the proposal of the Environmental
mind us of the many challenges, bottlenecks and Factor (short E Factor) by Roger Sheldon1 in the
dead ends which may be encountered on the route late 1980s for assessing the environmental impact
from educt to product: of manufacturing processes. The E factor concept
1) Protection of the macroenvironment (hu- (E equals kg waste divided by kg product), as an
mans, environment, product) or the microenviron- overall measure for the amount of waste produced
ment (other functional groups of the molecule, in a synthesis compared with the amount of isolated
other molecular components or parts of the cell) product, has become an important parameter driv-
may represent challenging tasks ing new synthetic developments.1 The economy of
2) Available known reactions may not be selec- a synthetic scheme on a molecular scale can be
tive enough viewed from different perspectives, but all aim at
making the best use of materials and minimizing
3) No direct synthetic route from the starting
the waste. This is a strategy that biological cells use
material to the product may be known
with the help of nature’s catalysts in order to avoid
4) Too many steps and detours for the intended the scaling of waste with the increasing amount of
synthetic route may be required or a key reaction product as in stoichiometric reactions in chemical
step may turn out to be a dead-end synthesis. From the single reaction step perspective,
5) Waste growth in relation to the growth of selective transformations with high atom economy
the desired product may create problems due to un- continue to be a main focus of research in the sci-
favourable product/waste-ratios. ence of synthesis.2
Waste accumulation with each reaction step is The attention to the overall number of steps in
a common feature of production processes in chem- a synthesis scheme aims at achieving a high step
ical synthesis, illustrated in Fig. 1 schematically economy by reducing the number of steps and
from starting material to product via one or more replacing protection-transformation-deprotection
intermediates. Therefore, increased attention to schemes by direct transformations.3 Further waste
waste is of prime importance, in addition to the tra- minimizations in synthetic schemes involve the re-
ditional synthetic criteria such as product yield and dox economy4 and reducing the use of auxiliary re-
purity. If the cumulated costs of waste disposal for agents and solvents in product isolations and
each non-sustainable reaction step in Fig. 1 ap- purifications. As waste is still accumulating with
proach the market value of the product, waste many existing technologies at present, searching for
waste degradation to harmless compounds or for
waste conversion into new useful products contin-
ues to be relevant and may help close the path of
carbon. For producing useful compounds as well as
for converting waste into harmless or even useful
materials rather than just waste disposal, catalysis
has been and continues to be the key to economical,
energy-saving and environmentally benign chemi-
cal transformations.5,6
The early history of biocatalysis is closely re-
lated to the preparation of alcoholic beverages like
beer and wine. A scientific milestone in the 19th
century has been the theoretical concept that chemi-
cal transformations of living organisms in all forms
of life, not only microorganisms, could be related
back to the work of specific substances which
would accelerate reactions and function in a similar
way as cellular machines. The experimental discov-
ery of enzymes as these specific substances acting
like molecular machines with the ability to acceler-
ate chemical reactions in living organisms has
sparked tremendous interest until the present day
and the scientific discipline of biochemistry origi-
F i g . 1 – Waste accumulation from starting material to the nated with the goal to isolate enzymes from biolog-
yield of pure product ical cells and determine their functioning on a mo-
R. WOHLGEMUTH, Molecular and Engineering Perspectives of the Biocatalysis …, Chem. Biochem. Eng. Q. 25 (1) 125–134 (2011) 127

lecular level. As enzymes within a biological cell cro- and macroeconomic value creation by natural
perform the required molecular transformations biotransformations of raw materials on our planet
with excellent selectivity, the waste is minimized has been central to life and the knowledge of the
and product yield maximized. This is due to an molecular diversity of natural compounds is far
enormous amount of work that enzymes have from complete. The molecular economy of bio-
achieved today a remarkable success in catalyzing catalytic transformations in nature has its counter-
useful reactions in chemical synthesis.7–10 part in the micro- and macroeconomic aspects of
The importance of both the molecular and en- chemical syntheses in industry, where raw material
gineering aspects of the application of biocatalysis costs, labour, energy and waste costs are related to
in the synthesis of chemicals has already been dem- the atom-, step- and redox-economy of the overall
onstrated by Louis Pasteur in 1858 in the successful process. An area where these aspects have been
microbial resolution of racemic tartaric acid, illus- particularly pronounced, is the manufacturing of
trated in Fig. 2, combining the selective microbial asymmetric molecular architecture. The fascinating
degradation of one enantiomer with the crystalliza- asymmetry of living organisms like snails17 or the
tion of the remaining enantiomer.11,12 The under- importance of chiral molecules in the chemical and
standing of biocatalysis as a key to the chemistry of pharmaceutical industry,18 justify the attention
living systems and the exciting discoveries on the given to clean manufacturing methods for products
molecular nature of enzyme action have paved the with high stereochemical purity. The three basic ap-
way for recognizing the importance of enzymes.13 proaches discovered already in the 19th century by
Louis Pasteur, involve the separation of enantio-
mers from a racemic mixture, asymmetric synthesis
by one or more stoichiometric and/or catalytic steps
and the conversion of chiral compounds from re-
newable natural raw materials. The standardization
of chirality description by the Cahn-Ingold-Prelog
rules19 and the demonstration of the enantioselec-
tive catalytic action of enzymes by John Corn-
forth20 stimulated research into the use of enzymes
for asymmetric synthesis. The inherent chirality of
enzymes due to the chirality of its amino acid con-
stituents has been applied in an ever-increasing
number of reactions for preparing molecular asym-
metry with high efficiency, enantioselectivity and
yield.7–10 The applications of different classes of en-
zymes like oxidoreductases, transferases, hydrolas-
es and lyases have been growing along various di-
F i g . 2 – Resolution of racemic tartaric acid by Louis Pas- rections of synthesizing enantiomerically pure
teur 1858
products by resolutions of racemic mixtures, asym-
metric synthesis or bioconversions from chiral nat-
Scientific and technological progress in the ural raw materials. The high efficiency and selectiv-
molecular and engineering sciences and their inte- ity of enzymes in catalyzing functional group trans-
gration towards viable industrial processes has cre- formations is however not restricted to asymmetric
ated sustainable value by enabling a large range of transformations but has been also useful for many
industrial biotransformations.14–16 Therefore, reac- catalytic chemoselective conversions where no
tion development of selected examples will be dis- chiral center was involved. The molecular perspec-
cussed in the following sections first from a molec- tive towards organic and enzymatic reactions and
ular perspective, then from an engineering perspec- the organic reaction types catalyzed by different
tive, and subsequently from the practical overall classes of enzymes continue to be an inspiring and
perspective. fruitful approach.21–23
Safety, health, energy and environment aspects
of chemical production technologies have become
Molecular perspectives key determinants of synthetic routes and are very
much related to the reaction design. With the in-
The creation of value by converting abundant creasing number of enzymes becoming available,
and easily available raw materials into products of not only can biocatalytic tools and methodologies
use for the quality of life continues to be a common provide important improvements in these aspects,
goal of both nature and human societies. The mi- but can also reduce the amount of waste.24 Even
128 R. WOHLGEMUTH, Molecular and Engineering Perspectives of the Biocatalysis …, Chem. Biochem. Eng. Q. 25 (1) 125–134 (2011)

11a-hydroxylation of progesterone catalyzed by

Rhizopus arrhizus in the synthesis of steroid hor-
mones,28 the use of biocatalysts in oxidation reac-
tions has been growing.25,26
The synthetic applications of oxidases are sim-
ple and therefore a variety of alcohols, amino acids,
amines, carbohydrates, metabolites, nucleosides
and other building blocks have been oxidized
enantioselectively by suitable oxidases. Among the
variety of enzymatic routes for the production of
chiral amino acids from the racemic aminoacid sub-
strates,29 the use of D- and L-specific amino-acid
oxidases has been widespread.8 The peroxides,
which are formed naturally as reaction byproducts
of the enzymatic oxidation of amino acids and often
negatively influence enzyme stability, can be easily
removed selectively by enzymatic oxidation with
catalase. The resolution of racemic m-DL-tyrosine
by immobilized D-aminoacid oxidase from Trigo-
nopsis variabilis and a subsequent Pictet-Spengler
reaction has given rapid access to enantiopure
(S)-3-carboxy-6-hydroxy-1,2,3,4-tetrahydroisoquino-
line.30 The yield can be further enhanced by
non-enantiospecific chemical reduction of the oxi-
dation product.31
With oxygen being available as abundant natu-
F i g . 3 – Sustainable Functional Group Conversions from
ral oxidant and electron acceptor, the reactions,
starting material to product where one or both of the two oxygen atoms of mo-
lecular oxygen are introduced selectively into an or-
ganic substrate, are of great synthetic interest. One-
more decisive reasons for the choice of a particular -oxygen insertion reactions into organic substrates
biocatalytic reaction is the non-availability of the like hydroxylations, epoxidations, Baeyer-Villiger
corresponding organic reaction type, avoiding harsh oxidations, heteroatom oxidations have catalyzed a
reaction conditions, which would lead to side reac- variety of monooxygenases, with the other oxygen
tions, or the selective and protecting-group-free ap- atom ending up in a molecule of water.
proach schematically illustrated in Fig. 3. The Baeyer-Villiger reaction has been a key
Biocatalytic oxidations combine the utilization synthetic transformation for more than a century.
of mild oxidants like molecular oxygen, environ- Since the selective oxidation of one out of several
mentally friendly solvents like water and highly se- ketone functions in an enantioselective way is a
lective and orthogonal non-stoichiometric oxidation competence of biocatalytic systems, asymmetric
reactions in an ideal way with the advantages of Baeyer-Villiger oxidations and asymmetric hetero-
natural or recombinant oxidizing biocatalysts.25,26 atom oxidations are best catalyzed by biocatalysts.
This is an attractive alternative to classical chemical Baeyer-Villiger monooxygenases have been a very
oxidation reactions, where the simultaneous pres- useful group of enzymes due to their highly
ence of reactive oxidants, flammable organic sol- enantioselective oxidation of linear ketones to es-
vents and toxic auxiliary reagents requires special ters, cyclic ketones to lactones, N-and S-heteroatom
safety, health and environment precautions. The oxidations.32,33 The increasing number of recombi-
prime importance of biocatalytic oxidations goes nant Baeyer-Villiger monooxygenases, explorations
however far beyond improvements in safety, health and systematic extensions of the substrate range of
and environment issues. Biocatalytic oxidation can these enzymes as well as progress in the bio-
follow asymmetric reaction paths with remarkable catalytic process design has significantly broadened
selectivity and versatility as well as improved com- the range of applications for enzymatic Baeyer-Vil-
patibility with labile functional groups.26 Since the liger oxidations in organic synthesis.34–38
early examples of the C5-oxidation of D-(-)-sorbi- The selective introduction of two atoms of mo-
tol to L-(-)-sorbose catalyzed by Acetobacter sub- lecular oxygen into an organic substrate can be
oxydans in the synthesis of vitamin C27 and the achieved by a number of different reactions.
R. WOHLGEMUTH, Molecular and Engineering Perspectives of the Biocatalysis …, Chem. Biochem. Eng. Q. 25 (1) 125–134 (2011) 129

Sharpless asymmetric cis-vicinal dihydroxylation est in their utilization for a diverse range of syn-
has rapidly become a most important reaction in or- thetic reactions. Transfer reactions of the hydroxy-
ganic chemistry. Dioxygenases as biocatalysts rep- acetyl-group from a variety of ketol donors to a
resent an interesting alternative26 and have been broad range of (2R)-hydroxyaldehydes can be cata-
used for the preparation of more than 300 cis-vici- lyzed efficiently by transketolases with full stereo-
nal diols.39 The tolerance of dioxygenases like the control and high conversion yields. Transketo-
recombinant toluene dioxygenase and chloroben- lase-catalyzed two-carbon chain extensions using
zene dioxygenase to other functionalities like the the irreversible ketol donor b-hydroxypyruvate
nitrile group40 is of interest for the synthesis of have been important for the stereoselective synthe-
nitriledihydrodiols and their corresponding carb- sis of 2-keto-(3S,4R)-diols.59–62
oxylic acids.41
One area where selective biocatalytic transfer
The early investigations on the product stereo- reactions have a tremendous influence on the
chemistry of D-sorbitol oxidation using microbes E factor is the synthesis of carbohydrates and
by Tadeus Reichstein27 and ketone reduction using glycocojugates, because classical chemical synthe-
dehydrogenases by Vladimir Prelog42 have inspired sis using protection-deprotection schemes for each
the still ongoing development of numerous bio- glycosidic bond generates waste in stoichiometric
catalytic reactions. Dehydrogenases and ketoreduc- amounts. The large-scale preparation of highly spe-
tases have therefore become excellent tools for syn- cific natural and recombinant glycosyltransferases
thesizing chiral alcohols,43 hydroxy aldehydes,44,45 for synthetic applications and the corresponding
hydroxy acids and amino acids, both in the oxida- NDP-sugar donors have been instrumental for es-
tion and the reduction direction.46,47 tablishing highly efficient biocatalytic glycosyla-
The simple availability, preparation and appli- tions. Rapid and complete regio- and stereo-
cation of hydrolases like acylases, proteases, ester- selective glycosylations can be achieved in water
ases and lipases has been instrumental in the early without the detour of protecting other functional
introduction of enzymatic methods in organic groups and solving solubility problems.63,64
chemistry. The growing number of applications for
Transfer reactions involving the formal transfer
pig liver hydrolases as practical biocatalyst for
of the amino group from a donor to a ketone or al-
asymmetric hydrolysis reactions in more than 100
dehyde acceptor have been known for more than 60
years48,49 are an illustration of the straightforward
years and the excellent selectivity of recombinant
integration of this biocatalytic methodology into
aminotransferases is finding practical applications
standard chemical synthesis. In addition to hydro-
lytic kinetic resolutions of racemic esters, hydrolas- for the synthesis of both chiral and non-chiral
es have been excellent chiral catalysts for enantio- amines.65,66
selective desymmetrization of meso- and prochiral The admirable molecular diversity of nature’s
esters by elimination of symmetry elements pre- diverse donors to be transferred to various accep-
cluding chirality.50 Acids, alcohols, amines, lac- tors in reactions catalyzed by transferases as the
tones, amino acids and other chiral building blocks ones mentioned above and others like carboxylases,
are among the products most often prepared. methyl- and prenyltransferases, sulfo- and phospho-
Epoxide hydrolases51–53 have been used for transferases is of both fundamental and practical in-
the preparation of chiral diols and epoxides54,55 terest.
by hydrolytic kinetic resolution of racemic epoxi- The use of lyases in the formation of new car-
des or by enantioselective desymmetrization of bon-carbon bonds for constructing complex struc-
meso-epoxides. tures from simple building blocks is promising, be-
Nitrilases have attracted considerable synthetic cause their atom and step economy can be im-
interest for the direct, mild and selective conversion proved. Aldolase-catalyzed synthesis of lactols and
of nitrile functional groups to carboxylic acids,56 N-polyhydroxylated compounds and their subse-
because nitrilase-catalyzed reactions avoid the quent conversion to the correspondding products
usual harsh chemical reaction conditions of strong has been performed in a highly diastereoselective
acids and bases at high temperature and therefore manner.67–70
preserve labile functional groups. Nitrilases have Molecules of increasing complexity can be
also been used for the synthesis of chiral carboxylic built by multi-step enzyme conversions in one-pot
acids and nitriles by hydrolytic kinetic resolution of or by multiple enzymes expressed in one cell. This
racemic nitriles57 or by enantioselective desymmetri- is of practical industrial interest in the synthesis of
zation of prochiral dinitriles.58 compounds, where the chemical total synthesis is
The increasing structural and functional char- not competitive.71 Robust processes to obtain the
acterization of transferases has also raised the inter- desired complex compound require however the de-
130 R. WOHLGEMUTH, Molecular and Engineering Perspectives of the Biocatalysis …, Chem. Biochem. Eng. Q. 25 (1) 125–134 (2011)

velopment of advanced analytical in-process con- Engineering perspectives

trols.72
Shifting the molecular perspective from the re- The value creation process is not complete
action type to the product types, biocatalysts have with the discovery of a new bioconversion from a
not only been applied for the synthesis of small mo- starting material to the product. The development
lecular weight compounds, but also as catalysts for of a suitable industrial process involves upstream
polycondensations, ring-opening polymerizations, technologies, the actual bioconversion and down-
oxidative polymerizations and polymer modifica- stream processing like product recovery and purifi-
tions.73 Polyphenols, polyanilines and vinyl poly- cation, all of which can have a significant influence
mers have been prepared by oxidoreductase-cata- on the final design of the overall process. Process
lyzed oxidative polymerization, while polysaccha- analysis, process design and improvement of equip-
rides, cyclic oligosaccharides and polyesters have ment, processes and their operation aiming at pro-
been obtained by transferase-catalyzed polymer cess intensification play a key role for manufactur-
synthesis. Hydrolase-catalyzed polycondensations ing products at industrial scale. Sustainable process
or ring-opening polymerizations have been used for engineering for the production of larger amounts of
the preparation of polyesters, polycarbonates, poly- product can be achieved in different ways, as
amides, polyphosphates and polythioesters. shown in Fig. 4, either by engineering larger reac-

F i g . 4 – Sustainable Process Design from starting material to product

R. WOHLGEMUTH, Molecular and Engineering Perspectives of the Biocatalysis …, Chem. Biochem. Eng. Q. 25 (1) 125–134 (2011) 131

tors and equipment or by parallelization of the same gen and substrate supply.83 The choice of the
reactor and equipment size. Reduced material re- reaction medium depends on the solvent compati-
quirements and increased speed of process develop- bility of the biocatalyst and the influence on the
ment for classical reaction scale-up through reactors downstream and purification process steps. The
and equipment of increasing size can be achieved substrate and product inhibition has been overcome
by microscale techniques using microwell-plates, by different engineering designs. The direct liq-
microbioreactors or microreactors.74–76 The appli- uid-liquid extraction of the product from a
cation of microreactor technology to biocatalytic whole-cell catalysed Baeyer-Villiger oxidation with
processes extends to synthesis77 and is of much in- controlled substrate addition can lead to significant
terest for cases where aqueous and organic two-phase differences in phase separation times depending on
systems require efficient mixing and toxic reagents the utilized reaction medium. In the SFPR process
as e.g. in the hydroxynitrile lyase-catalyzed synthe- design, the lactone product is obtained after
sis of cyanohydrins in microreactors from the corre- adsorber separation from the reaction mixture and a
sponding aldehydes and in-situ generated HCN.78
washing step.83 The downstream processing and the
A prerequisite is the scale-up of the production purification of the two chiral regioisomeric lac-
processes for the starting material as well as for the tones, obtained as products in a 1:1 mixture in
biocatalyst. While the starting material is not con- nearly enantiopure form (ee > 98 %) and good
sidered here, the scale-up of the biocatalyst produc- yield, has been a major bottleneck. Simulated Mov-
tion already requires a key decision concerning the
ing Bed Chromatography has been established as
form of the biocatalyst to be used (isolated enzyme,
robust large-scale purification technology achieving
immobilized enzyme, whole cell). Other key deci-
sions include the reactor type, reaction medium, re- a tremendous reduction in solvent consumption.84
action conditions, the sequence and timing of pro- The decision whether to use whole cell bio-
cess steps, technology and equipment for down- catalysts or isolated functionally pure enzymes de-
stream processing and purification. The identifica- pends on various parameters like the location of the
tion of reaction and downstream processing bottle- enzyme in the cell, the substrate-product transport
necks to be overcome has been essential for the de- abilities of the cell, possible interfering side
velopment of large-scale processes. reactions by other enzymes of the whole cells and
The scale-up of the biocatalytic Baeyer-Villiger the type of biocatalyst or biocatalytic reaction. Pro-
oxidation of racemic bicycloheptenone to the two cess simplification is therefore another major pro-
regioisomeric lactones (-)-(1R,5S)-3-oxa-bicyclo cess design goal in order to allow the use of exist-
[3.3.0]oct-6-en-2-one and (-)-(1S,5R)-2-oxabi- ing industrial large-scale equipment and process
cyclo-[3.3.0]-oct-6-en-3-one, the corresponding control.85,86 The simple reactor configurations, used
down-stream processing and product purification il- in large-scale chemical synthesis, have been a
lustrate the importance of the engineering aspects major factor for the success of hydrolase-catalyzed
and their consideration in the early phases of the processes at large scale.14–16,87 In case of the iso-
process design.79–84 lated enzymes operational stability, mass transport,
While whole cells were chosen as the form of separation and cost issues are important for reaction
the biocatalyst, microbial cell growth, enzyme in- engineering. The retention of enzymes within the
duction and biotransformation have been devised as reaction space by membranes has been well estab-
three separate parts, thereby allowing independent lished in the operation of fed-batch or continuous
parameter optimization. The bottleneck of substrate biocatalytic processes at a production scale of
and product inhibition for the Baeyer-Villiger Mono- several hundred tons per year.88 Production pro-
oxygenase has been overcome by the process de- cesses using immobilized enzymes89 are also well
sign of a resin-based substrate feed and product re- established and the most adequate immobiliza-
covery process (SFPR)79,81,82 or by direct substrate tion method can be selected from a variety of op-
feeding below the inhibitory concentration and sol- tions.90
vent extraction of the product.80 The choice of the
reactor type and specification is another key deci- An ideal product recovery scheme would be
sion for which experimental data on the process and based on an ideal bioprocess with complete conver-
its parameters like the required amounts and con- sion to a single product, so that no product-starting
centrations of biocatalyst and oxygen and the de- material or product-side product separation would
gree of mixing are useful.81 Technical improve- be needed. Real product recoveries require however
ments for oxygen mass transfer have been devel- a versatile toolbox of downstream technologies
oped using sinter-metal spargers, which have been to meet the demand for high-yield processes with
experimentally tested. The decision for the stirred minimum amount of work, energy and number of
tank reactor has been guided by the required oxy- steps.
132 R. WOHLGEMUTH, Molecular and Engineering Perspectives of the Biocatalysis …, Chem. Biochem. Eng. Q. 25 (1) 125–134 (2011)

Value creation by integrated processes

The number of reaction steps, as well as prod-
uct recovery and purification steps in-between, to-
gether with the yield, raw material and labour costs,
is among the main factors determining the econom-
ics of an overall synthesis. It is therefore clear that
the replacement of multiple downstream processing
steps or multiple reaction steps by single-stage pro-
cesses can improve operational efficiency and cost
issues. Integration of process steps can also be done
between a reaction and a downstream processing
step, as Louis Pasteur has already demonstrated in
the biocatalytic tartaric acid resolution process.11,12
In a one-step reaction, the integration should not
add new problems but simplify the overall process
and make it better scalable and more robust so that
small deviations do not lead to a failed reaction. For
a two or multi-step reaction, a sequence of reactions
F i g . 5 – The interconnection of molecular and engineering
steps would ideally be integrated into a single step. aspects in the overall process
If this were not possible, one option would be to
make the optimum conditions for each of the reac-
tions compatible with each other so that the overall selective transformations and the reduction in the
reaction could be performed in one pot. In many extensive use of protective groups during the total
cases however, the biocatalytic reaction step comes synthesis of multifunctional molecules is clearly fa-
before or after a series of chemical reaction steps, vourable for biocatalytic reactions.
which have to be worked through one after another. The steady progress in building biocatalytic
Even then, a minimum of integration is required single-step reaction platforms, which are modular
and the choice of the right interfaces, as e.g. infor- and scalable,24 enables the evaluation of the com-
mation storage, reaction medium, protecting group patibility with the development of chemical reac-
chemistry and purification/separation processes, be- tion steps.91 Although thousands of new bio-
tween biocatalytic and classical organic synthetic catalysts have been discovered and characterized
reaction steps are important. In case of incompati- over the last 50 years, the continued search and de-
ble reaction conditions, new enzymes with the de- velopment of novel enzyme functions is key for
sired properties and activities may be engineered or new or improved synthetic methodologies,92 espe-
screened from nature. The goal of shifting thermo- cially where direct organic reactions are difficult or
dynamic equilibria to the product side can on the unknown. The tremendous progress in genomics
other hand justify the integration of an additional ir- and proteomics with the rapid sequencing technol-
reversible reaction step or an in-situ product re- ogy are providing access to a huge amount of struc-
moval/separation step. In any case, the value cre- tural information, while the functional assignments
ation in the overall process depends on various in- have not kept up the same pace. The further devel-
teractions between molecular and engineering as- opment of robust, reliable and meaningful func-
pects as shown in Fig. 5 and the further exploration tional enzyme assays and analytical technologies
of this exciting area looks promising. for biocatalytic reactions is therefore essential
for the discovery and optimization of novel bio-
catalysts or proteins with unknown functions.93 It
Outlook can be expected that the value of standardized func-
Biocatalysts, as nature’s privileged catalysts for tional information and EC numbers for novel en-
achieving high-performance transformations in liv- zymes, standardized reporting of biocatalytic reac-
ing biological systems with the resources and en- tions and rapid access will facilitate advanced
ergy available, are a good choice for developing se- studies building on these standardizations.94
lective chemical reactions with similar high molec- It is worthwhile for the synthesis of small mol-
ular economy and efficiency without negative ef- ecules to perform the comparison of different meth-
fects on biological systems. This is of industrial in- ods like total chemical synthesis, chemoenzymatic
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