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Industrial Biotransformation : Principles of Biotransformations Advantages of Biocatalysis
Industrial Biotransformation : Principles of Biotransformations Advantages of Biocatalysis
Industrial Biotransformation : Principles of Biotransformations Advantages of Biocatalysis
Biocatalysis or biotransformation encompasses the use To perform (large-scale) synthesis of drugs, materials,
of biological systems to catalyze the conversion of one or chemicals, one can principally follow three different
compound to another. The catalyst part of the biological approaches with various degrees of complexity:
system can thereby consist of whole cells, cellular extracts,
1. Use a purely chemical strategy.
or isolated enzyme(s). Up to the second decade of the twen-
tieth century, processes based on biotransformations were 2. Use a chemoenzymatic route combining chemical
classified under various denominations such as ‘‘zymotech- and biocatalytic steps. In this case, the biocatalyst
nology’’ or ‘‘technical biology’’. The term biotechnology was is preferentially used to perform the key reaction(s)
first used in the year 1917 (1). Already then, in the early requiring high selectivity or specificity or to replace
times of industrial biotechnology, the leading representa- environmentally intolerable reaction steps.
tives of the new science promoted the idea of using biolog- 3. Use a biological total synthesis by fermentation or
ical systems to create more efficient, more selective, and multistep biotransformation.
environmentally friendlier processes for the conversion of
Enzymes and whole-cell biocatalysts have several
raw materials into industrial products, thereby substi-
attractive properties, which make them privileged
tuting problematic chemical transformations. The concept
catalysts for organic synthesis; these are as follows:
of a more sustainable use of the limited resources was
thus one of the driving forces for the rapid development • They have high chemo-, regio-, and stereo-selecti-
of industrial biotechnology long before the now worldwide vities.
accepted political vision of sustainable development was • They require mild reaction conditions. Therefore, bio-
promoted by national and international conferences and catalysis offers great chances and advantages for
organizations. successful applications (also in cases where either
Thanks to genetic engineering techniques introduced in the substrates or the products of the reaction are
1973, enzymes from all kinds of biological sources can now chemically labile).
be recloned and overexpressed in easily mass cultivatable
• Biocatalysis is normally performed in an aqueous
microorganism or cell cultures. With this technology, even
environment but can, in many cases, also be con-
enzymes that have been rare up to the present can be
ducted in solvent mixtures, liquid–liquid two-phase
produced in large quantities and at affordable costs. In
systems, and even in pure organic solvents. A rel-
addition, whole-cell biocatalysts or production organisms
evant practical example is the use of esterases and
can now be genetically improved by directed engineering
lipases to catalyze esterifications in organic solvents
of metabolic pathways.
such as vinyl acetate.
An OECD-report of 1998 (2) analyzes the state of
the art and the future development needs for industrial • There is no, or only limited use of protecting groups,
biotechnology. Some important conclusions in this report for example, for the chemoenzymatic synthesis of
summarizing the state of the art are the following: complex carbohydrates and glycoconjugates.
Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology, edited by Michael C. Flickinger
Copyright © 2010 John Wiley & Sons, Inc.
1
2 INDUSTRIAL BIOTRANSFORMATION
The majority of the described enzymes belong to the EC as hydrolases (lipases, esterases, proteases) are still
classes 1, 2, and 3. the preferred biocatalysts for the preparation of opti-
Very detailed information on some 4000 EC-classified cally active compounds.
enzymes (organized in fact sheets) can be found in 2. Searching and screening for suitable novel microbial
the excellent Springer Handbook of Enzymes (4). The biocatalysts from natural sources, for example, by
same information is also available in electronic form via selective enrichment techniques from environmental
BRENDA (Braunschweig Enzyme Databank): http://www. samples, starting from soil or sewage samples, or
brenda-enzymes.de/. alternatively and in rare cases, by testing extracts
Useful information on commercially available enzymes of plant or animal tissues. This laborious and more
can be searched electronically in a variety of ways via demanding approach is essential when no suitable
the Enzyme Explorer (http://www.sigma-aldrich.com/ enzyme or microbe is commercially accessible for the
enzymeexplorer/. conversion of a defined substrate to a defined product
Practical examples for preparative biotransformations using a particular reaction type. Both approaches 1
(with checked procedures) can be found, for example, in and 2 are complementary.
the highly acclaimed handbook edited by Stanley Roberts
3. A third approach, related to the second, is the
et al. (5), and also in (6). For German speaking scientists,
search for completely novel enzymes catalyzing
there is an excellent ‘‘Bioorganikum’’ available (7).
difficult reaction types, where no indications or prior
An additional and highly recommendable electronic
knowledge on their existence has been available.
information source for the synthetic chemist is the Catal-
Typical examples for this approach from the last
ysisNet (www.catalysisnet.com), which is based on the
decades are the discovery and investigation of
Abstracts of the Warwick Biotransformation Club and
enzymes catalyzing Baeyer–Villiger oxidations (8)
on current biotransformation abstracts maintained under
or Diels–Alder-type reactions (9).
the supervision of Nicholas Turner (University of Manch-
ester).
Enzyme screening and bioprocess development were
A new biotransformation search feature has been
important skills and key factors in the success of the
added recently to the SciFinder software by the Chemical
Japanese industrial biotechnology. On the basis of a work-
Abstract Service, which allows to search functional group
ing group analysis, Cheetham came to a very harsh
biotransformation reactions in a substructure search. The
statement on this issue: ‘‘in Europe screening is under-
biotransformation database assembled by Klaus Kieslich
developed and underresourced both in absolute terms and
and the Warwick Biotransformation Club has provided
by comparison with current activities in Japan, and espe-
the nucleus of this data mining service.
cially when compared with the resources devoted to genetic
The actual scope of bioreactions is summarized in
manipulation’’ (10). Has there been a change 20 years
Table 2.
later? In hindsight, future biologists may consider screen-
Screening for Biocatalysts ing to have been a rate-limiting step in the development of
biotechnology in Europe in the late twentieth century (10),
Major driving forces for present and future R&D in indus- particularly with respect to screening efforts directed to
trial biocatalysis are the following: the discovery of completely new, so far unknown subclasses
of enzymes. Unfortunately, in our view, this situation has
• The continued demand for chiral building blocks and
not really improved during the last 20 years.
enantiomerically pure compounds, in general.
With modern polymerase chain reaction (PCR) tech-
• A growing interest in areas of synthesis where bio- niques, it has become possible to enrich and investigate
catalysts offer clear advantages over purely chemical the genetic information (DNA) even from the VBNCs
strategies. The most prominent examples are, at (viable but not culturable microorganisms) present in soil
present, peptide synthesis, protein modification, syn- samples. A general approach begins with the isolation of
thesis of complex carbohydrates, oligosaccharides, nucleic acids directly from soil and environmental sam-
and glycoconjugates. The latter area presents many ples, from primary enrichment cultures, and from purified
chemical, ecological, and economical problems to the microorganisms across the whole spectrum of biodiver-
classical organic chemistry. The multifunctionality of sity. In the next stages of the process, gene libraries
the products, calling for sophisticated and laborious (genes cloned in suitable expression hosts) are constructed
protection group strategies, has a great potential for from the purified DNA and subjected to high-throughput
selective and direct biocatalytic synthetic methodolo- automated (robotics) activity screening systems.
gies.
• International efforts to promote sustainable develop- Technical Aspects of Biocatalysis
ment and environmentally friendly technologies.
To set up a practical bioconversion process screening and
Three main screening strategies have been followed optimization, work is required at the following different
so far: levels:
1. Testing and evaluating inexpensive commercially 1. Screening for suitable microorganisms or plant cells,
available (bulk) biocatalysts or suitable microorgan- and so on, and/or enzymes with the required catalytic
isms. Enzymes that do not require coenzymes, such properties and selectivities.
INDUSTRIAL BIOTRANSFORMATION 3
Table 2. Types of Substrate Conversions (Selection) that have been Successfully Performed by Biocatalysisa
Type of Reaction
Reductions of
Aromatic carbonyl compounds, α-substituted carbonyl compounds, α-hydroxy ketones, β- and γ -substituted carbonyl compounds,
ketones and α- and β-diketones, β-keto acids and esters, γ -keto esters, masked carbonyl compounds, activated double bonds
(hydrogenation), acyclic and cyclic ketones, reductive amination of keto acids.
Oxidations/hydroxylations of
Unactivated saturated carbons, (polyunsaturated) fatty acids, epoxidation and dihydroxylation of alkenes, aromatic compounds
(→ unsaturated diols), hydroxylated compounds and aldehydes, diols (and lactonization), enzyme-catalyzed Baeyer–Villiger
oxidations, organic sulfides (sulfoxidation).
Hydrolysis of
Esters of acyclic alcohols, hydroxy and enol esters, esters of cyclic alcohols, esters of cyclic diols and triols, esters of acyclic diols,
cyanohydrin acetates, acyclic monoesters, acyclic diesters, cyclic mono and diesters, amides and lactams.
Hydrations of
Nitriles, amides, C = C bonds.
Biocatalytic C–C-formation
Additions and eliminations
Isomerization of
Sugars, sugar phosphates.
Racemization of
Amino acids.
Multienzymatic approaches
Of special interest for cofactor-dependent systems with recycling enzymes and multistep one-pot enzymatic synthesis.
a Compiled from literature.
4 INDUSTRIAL BIOTRANSFORMATION
2. Screening for the optimum conditions for culture The cross-linked enzyme crystals (CLEC) technology
growth and production of the desired enzyme(s). was developed and launched by Altus Biologics Inc. (a
This can be very demanding work in cases where daughter company of Vertex Pharmaceuticals Inc.). This
enzyme expression is not constitutive. In this case, technology takes well-known soluble enzymes, which
the best and most economical process parameters, denature in harsh environments and stacks them in a
such as the conditions for enzyme induction and the solid crystal form—adding the cross-linking of chemical
right harvesting time must be identified. bonds for further strength (12,13). For a short review with
3. Screening for the optimum reaction conditions with focus on applications refer to (14). A related approach is
whole cells, crude, or purified enzymes including the cross-linked enzyme aggregates (CLEA). Since this
engineering and technological aspects such as cofac- more general method is also applicable to enzymes that
tor regeneration, immobilization, type of reactor, and cannot be crystallized, it is easier to handle and much
so on. cheaper (15).
4. Optimization of the biocatalyst by directed evolution An important technical issue is the large-scale appli-
or other enzyme engineering technologies in order to cability of cofactor-dependent enzymatic systems. It is
obtain the ideal biocatalyst under the given process generally accepted that, for example, NADH-requiring oxi-
conditions. doreductases can easily be used in whole-cell biocatalysis
such as baker’s yeast-mediated reductions. The cofac-
In order to perform bioreactions, the biocatalyst can be tor recycling step is thereby simultaneously performed
operated under various process conditions (also refer to within the intact cell, driven by the reduction equivalents
Fig. 2): introduced via the external carbon and energy source (glu-
cose). For preparative-scale synthesis, repetitive batch
• batch or fed-batch application of whole cells in free has proven to be an easy-to-handle technique. The mul-
or immobilized form in aqueous environment; tiple reuse of the enzyme(s) is possible after recovering
• continuous application of whole cells in immobilized them from the reaction mixture by means of concentra-
form in aqueous environment; tion with ultrafiltration equipment. Continuous processes
• application of whole cells in two-liquid phase, multi- often show higher space-time yields compared with batch
phase systems, or in micelles; processes. For the application of enzymes in continuous
processes, and especially for cofactor-dependent systems,
• use of acetone-dried or permeabilized cells;
the enzyme membrane reactor (EMR) concept has been
• batch or fed-batch application of free crude or purified developed and successfully applied by Kula and Wandrey
enzymes in aqueous or organic environment; (16,17), and others. It has also been successfully applied in
• use of polyethyleneglycol (PEG)-modified enzymes in industry, for example, by Degussa (now Evonik), Tanabe
organic solvents; Seiyaku, Sepracor, Sigma-Aldrich and others (18–20).
• continuous application of immobilized enzymes; At the time when the EMR-concept was introduced,
• use of enzyme membrane reactors; the method of most of the cofactors were still very expensive compounds
choice for systems with cofactor recycling or for reac- and therefore contributed significantly to the overall pro-
tions with expensive enzymes. cess costs. Therefore, for application in continuous pro-
cesses, the molecular weight enlargement by covalent
For economic reasons, if ever possible, whole-cell bio- coupling to PEG (in order to allow very high recycling
catalysis is used to perform biotransformations. This is numbers → upto 600,000 cycles have been reported) was
possible when the following criteria are met: (i) no dif- a conditio sine qua non for the economic viability of such
fusion limitations for substrate(s) and/or product(s) and processes at this time. In the meantime, however, thanks
(ii) no side or follow-up reactions due to the presence of to the rapid progress in fermentation technologies, at least
other cellular enzymes. If these conditions are not ful- NAD(H) and ATP have become affordable compounds, at
filled, the use of isolated enzymes—or in special cases of least when purchased in bulk (i.e. multikilogram) quanti-
permeabilized cells—is indicated. ties. Under these conditions, these cofactor cost drop to 1
Immobilization can be achieved by adsorption or cova- US$ per gram or even less.
lent fixation of the biocatalyst to a solid support (e.g. As an example, a more recent EMR-process for the
surface-modified polymer or glass beads), by entrapment enantioselective reduction of 2-oxo-4-phenylbutyric acid
or by encapsulation in gel beads (e.g. agarose, polyacry- (OPBA) to (R)-2-hydroxy-4-phenylbutyric acid (HPBA,
lamide, alginate, etc.). Only a limited set of methods has ee >99.9%) with NADH-dependent D-lactate dehydroge-
found real technical applications. The first large-scale nase (D-LDH) from Staphylococcus epidermidis and FDH
applications of immobilized enzymes were established for (formate dehydrogenase)/formate for cofactor recycling
the enantioseparation of D- and L-amino acids by Chi- uses free NADH instead of PEG-NADH (21,22) (Fig. 1).
bata, Tosa and coworkers at Tanabe Seiyaku Company. Within the selected residence time of 4.6 h, a cycle number
The Japanese achievements in the large-scale application for the cofactor of 1000 can easily be achieved. Instead
of immobilized systems are very well documented in an of NADH, the less-expensive NAD+ is used. A second
excellent multiauthor publication edited by Tanaka, Tosa, reason for the application of the native coenzyme is the
and Kobayashi (11). Some enzyme suppliers sell important fact that the activity of D-LDH is reduced to one-tenth
industrial enzymes not only in the free form (solution or when PEG-enlarged coenzyme is used instead of the
powder) but also in immobilized form on solid supports. native coenzyme. HPBA produced by this method with
INDUSTRIAL BIOTRANSFORMATION 5
chemicals differ from commodities in price and annual se complex and expensive. As a matter of fact, many
manufacturing volumes of up to 10 tons. biocatalytic processes are run in standard chemical
The global white (or industrial) biotechnology market reactors without any adaptations. This is almost always
of ∼50 billion US$ is smaller than the red biotechnology the case if one uses, for example, commercially available
(pharmaceutical) market (>70 billion US$), but industrial hydrolytic enzymes such as lipases or proteases.
white biotechnology represents a larger long-term busi- Some more complex biocatalytic procedures are per-
ness potential than red biotechnology. It is estimated that formed with whole cells because cofactors need to be
at least 20% of the global chemicals (2292 billion US$ pre- regenerated and may require fermentation equipment.
sently) could be produced by biotechnological means in As mentioned earlier, biocatalysis is often limited due to
2020. substrate or product inhibition. The former may be over-
come by suitable substrate-feeding regimes in fed-batch
Manufacturing Facilities processes. The latter, however, is technically more diffi-
cult to avoid. Ideally, it would be necessary to continuously
As discussed earlier, the advantage of biocatalysis lies in remove the product at a rate similar to the formation
the unmatched precision in the production and assembly rate using an efficient, nontoxic, regenerable, and cheap
of complex and chiral molecules due to the enantio-, regio-, process, which has a high specificity for the targeted
and substrate selectivity. This precision of biotransforma- substance. This is the concept of ISPR (in situ product
tion is based on the unique properties of catalytic proteins recovery) in combination with a biocatalytic process (also
(enzymes). in Fig. 2). The use of liquid–solid or liquid–liquid bipha-
The process has to be adapted to the biological premises, sic systems is thereby of much interest for simple and
unless the opposite is possible, which is the preferred straightforward industrial processes, if the biocatalyst is
way. Large-scale processes must be carried out in a well- compatible with these phases (32).
controlled manner. For this purpose, different process
operation modes have been developed (Fig. 2, see also
Reactor for Biocatalytic Reactions
the section titled ‘‘Technical Aspects of Biocatalysis’’).
Substrate and product inhibition, the stability of No special equipment is needed for biocatalysis in many
enzymes, growth or cofactor requirements, by-product cases and ‘‘ordinary’’ stirred tanks, used in large-scale
formation must be well mastered during enzymatic chemical synthesis with temperature and pH control, are
processes, although the sensitivity tends to vary from sufficient. Thus, in this article, we concentrate on a few
reaction to reaction. However, this does not mean that selected process units that are used in biocatalysis. Fer-
large-scale biocatalytic transformation processes are per mentors (Fig. 3) are used for the production of the enzymes,
5000 B.C. Preservation of food and alcoholic drinks on Egyptian pictures, vinegar production
800 B.C. Casein hydrolysis with chymosin for cheese production. Homer’s Iliad mentions stomach enzymes for cheese making
1670 ‘‘Orleans’’ process for the industrial biooxidation of ethanol to acetic acid
1680 Antoni van Leeuwenhoek, first to see microorganisms with his microscope
1833 Payen and Persoz investigate germinating barley and formulate basic principles of enzymes
1874 Christian Hansen started an enzyme laboratory and company
1878 Kühne coined the term enzymes
1890 Takamine isolates bacterial amylases (what later became Miles Laboratories)
1894 Emil Fischer elaborated the essentials of enzyme catalysis
1897 Buchner discovers yeast enzymes converting sugar into alcohol
1897 Eduard Buchner published fermentation with cell-free extracts from yeasts
1899 Berzelius acknowledges the catalytic reaction of starch hydrolysis by diastase
1907 Röhm founded a company producing ‘‘Oropon’’ for tanning (mixture pancreatic extract and ammonium salts)
1926–1935 Sumner, Northrop, and Kunitz prove that enzymes are proteins
1930 Regioselective biooxidation of sorbite to sorbose for the Reichstein Vitamin C synthesis
1940 Sucrose inversion using an invertase
1950 Bioconversion of steroids
1969 First industrial use of an immobilized enzyme (aminoacylase)
1970 Hydrolysis of penicillin to 6-aminopenicillanic acid
1985 Enzymatic process for the production of acrylamide
1990 Hydrolysis by protease (trypsin) of porcine insulin to human insulin
1995 3000 tons per year plant for the biotransformation of to nicotinamide
Asahi Chemical Industries, Astra Zencca, BASF, Bayer, Bristol Myers Squibb, Ciba (now BASF), Dow Pharma, Dr Reddys, DSM,
Glaxo Smith Kline, Evonik, Givaudan, Kaneka, Lonza, PCAS & Proteus, Marukin Shoyu, Mercian, Mitsubishi Chemical Company,
Monsanto, Nicholas Piramal, Pfizer, Sankyo, Sanofi Aventis, Schering, Sigma-Aldrich, Takasago, Tanabe, Takeda.
INDUSTRIAL BIOTRANSFORMATION 7
if they are not commercially available. They are stirred In addition to liquid-liquid extraction and crystalliza-
vessels that allow a sterile (monoseptic) operation. This tion as shown in figure 5, a variety of other unit operations
means that only the organism with the desired enzyme like chromatographic separations (Fig. 6), membrane
used for the biocatalysis is allowed in the bioreactor. How- separations (Fig. 7) and drying operations (Fig. 8) are
ever, in many cases, normal stirred-tank reactors, as used standard processes for product recovery and purification.
in chemistry, can be applied.
When using hydrolytic enzymes, the reactor configu- Examples of Biocatalysis Products
ration can be very simple as shown in the picture below Products resulting from biocatalytic processes range from
(Fig. 4). These reactor series are used for the annual commodity products to high-value pharmaceuticals. For
production of several thousand tons of nicotinamide. a review of the potential applications and industrial
The product recovery processes are key steps after the examples of the six enzyme classes, see also (19,20,33,34).
biocatalytic reactions and make use of conventional unit Table 5 shows a few examples of biocatalytic products.
operations and employ currently established and available Considering products that have market relevance, one
techniques (see figure 5 for an example of a plant layout). realizes that the majority depends on hydrolytic enzymes.
F2
F1
F1
F1
Figure 3. (a) The top of a 15 m3 bioreactor used for fermentation and/or whole-cell biocatalysis.
The two photographs (b and c) show the interior of a 75 m3 fermentor used for fermentation and
biocatalysis. The shaft and the turbines have been removed for a better view. The details show
that the baffles are constructed as heat exchangers to maintain optimal temperature ranges for
exothermal high cell density fermentation and biocatalysis. [Courtesy Lonza Biotec sro Kouřim,
CZ]
Biomass
Precursor separator
feed Solvent
evaporation
Liqiud–liqiud
extraction
Figure 5. The plant layout for the fed-batch process Biotransformation
developed by Givaudan for the biocatalysis of ferulic
acid to vanillin. The biomass is grown on glucose. At
a given moment during fermentation the substrate Vacuum
(ferulic acid) is started to be added according a feed
regime. The biocatalytic step (Fig. 14) is carried out Centrifug-
with an actinomycete strain (Streptomyces setoni), Vacuum ation
and high titers of >15 g/L were reached, despite the drying
cellular toxicity of vanillin. The DSP consists in a Crystallizer Carbon
two-step organic solvent extraction. treatment
COOH
+
COOH
Erwinia herbicola
HO
(Tyrosine phenol lyase) NH2
HO
HO
+ OH
+ H 2O
NH3 L-Dopa
O
Figure 8. Products can be dried by several methods, and spray
dryers are versatile units. The picture shows the bottom of large N
spray dryer with a performance of 130 kg of water per hour. Nitrile hydratase NH2
[Courtesy Lonza Biotec sro Kouřim, CZ]
N N
of the process, and low enzyme and coenzyme consumption Nictotinonitrile Nictotinamide
per unit mass of product.
Figure 10. Conversion of nicotinonitrile to nicotinamide in a
The following are also of high importance:
three-step continuous biocatalytic reaction developed by Lonza
with Rhodococcus rhodochrous J1 cells immobilized in polyacry-
• The number of reaction steps involved, environmen- lamide.
tal aspects—‘‘greenness’’.
• Biocatalysis versus chemical conversion. When the
number of reaction steps can be drastically reduced, 2. Potential alternatives: plug-flow reactor, packed-
chances are in favor of biocatalytic strategies. bed reactor, fluidized-bed reactor, two-liquid-
Enantio- and stereoselectivity can be strong points phase reactor, membrane reactor (especially for
in favor of biocatalysis. cofactor-requiring free enzymes), and so on.
• Reactor configuration: standard versus tailored. Deci- • Mode of operation: (fed)-batch versus continuous.
sion is based on the kinetics of the biocatalytic Parameters for decision: kinetics, stability, and form
process; availabilty and experience; desired mixing; of the biocatalyst; desired substrate conversion, prod-
and mass transfer properties. uct concentration (solubility); need of process con-
1. Standard case: stirred-tank reactor for batch ope- trol, inhibitory/toxic effect of substrate or product.
ration—in industry great reluctance to change to Examples for fed-batch bioprocesses: acrylamide pro-
other configurations. cess (Nitto Chemicals), L-carnitine process (Lonza).
12 INDUSTRIAL BIOTRANSFORMATION
OAc
OAc 16 g OH
Lipase OF
+
6°C, 1.07 equiv.
AcO OAc 22 h
AcO OAc AcO OAc
46.3 g (88%)
>99% ee
96% GC*
* 2.6% cis-diacetate
Antipsoriatic retiferol
0.6% trans-meso-diacetate
HO OH
Figure 11. Enzymatic conversion of triacetoxy cyclohexane to the homochiral diacetate was car-
ried out with a lipase isolated from Candida rugosa, developed in the laboratories of Hoffmann–La
Roche. The product was produced on a multikilogram scale.
HO OH
OH
O
OH O
O OH
O
UDP O
O OH
O OH
O
O
OH UDPGA-transferase
O Acylglucuronide (desired product)
(foal liver homogenate)
Mycophenolic acid +
HO OH
+
O OH
UDP-glucuronic acid
O
OH
O O
O
O
O OH
O-Glucuronide
Figure 12. Enzymatic process for the production of mycophenolic-derivative studied for human
transplantation.
INDUSTRIAL BIOTRANSFORMATION 13
Examples for continuous operation: production of protect the environment by producing (bio)degradable
L-tert leucine and other amino acids (Degussa). products, to avoid or reduce pollution and repair damages
• Level of process integration. Parameters for deci- to the environment. The quality of life is increased and
sion: number of process steps (subsequent reactions); preserved by the introduction of new pharmaceuticals
nature of the reactions. and foods. This can be achieved by the utilization of
renewable resources, by the wise and economic use
Many of current large-scale applications use immobi- of nonrenewable resources, and by the protection of
lized enzymes. In particular, the Japanese industry has, ecosystems with increased use of biological processes.
for a long time, pioneered this sector of biotechnology. In The necessary changes in technologies can only be
the multiauthor publication edited by Tanaka, Tosa, and achieved by linking with economical, ecological, and social
Kobayashi (11) and in a more recent review, (48) a num- goals.
ber of well-established continuous production processes Two conclusions in the earlier-mentioned 1998
with immobilized biocatalysts are described in detail. OECD-report (2) read as follows:
Complementary informations on some of these Japanese
bioprocesses as well as additional case studies for other • ‘‘The key area of opportunity is improved and novel
bioprocesses have been compiled by Cheetham (49). biocatalysts. The search is on to examine the remain-
The most recent and most complete compilation of ing unexplored biological diversity which presumably
existing industrial biotransformations (with process data holds a great wealth of biocatalytic potential and
and scale of operation of the listed processes) was recently of new bioactive compounds and biomaterials. The
published by Liese, Seelbach, and Wandrey (20). This pub- introduction of biotechnology into many industrial
lication clearly demonstrates that already the application processes will be increasingly dependent on the devel-
opment of recombinant biocatalysts’’.
spectrum of biocatalysis for industrial processes is much
broader than generally perceived. Thus, the hope seems • ‘‘Bioprocess engineering and integrated bioprocess-
justified that the contribution of biotechnology in medium ing also continue to be critical factors for the com-
to large-scale production processes can be significantly mercialization of biotechnology’’.
increased in future, provided that the necessary efforts
In a later OECD-report of 2001 on the application of
to enlarge the biocatalytic toolbox are made by academia
biotechnology to industrial sustainability (50), the main
and industry.
messages are as follows:
NH2
N
N
N
N RS2 + H2O
2 Pi R-(SH)2
O
-O P O NH2
O NH2
O N
Phosphorylation N RTP-Reductase N
N Phosphatase
HO N Lactobacillus leichmannii
OH N N
NMP N Transferase
O O O
O O O
O P O P O P O O
O P O P O P O Base exchange
O O O O
O O O
HO OH
HO
NTP
dNTP
COOH
COOH
CHO COOH
OMe OH
OMe OMe
OH OH
OH OH
Ferulic acid Vanillin Vanillic acid Protocatechuic acid
CH2OH
Figure 14. The biocatalysis of ferulic acid to
vanillin, a process developed and realized at indus-
trial scale by Givaudan (Switzerland). For a basic OMe
plant layout, refer Fig. 5. Besides reaching very OMe
high productivities, also the right balance between OH
OH
vanillin and by-products, which is part of the
knowhow of the man skilled in the art, were reached. Vanillyl alcohol Guaiacol
Melt reactor
T = 100°C Figure 16. A transesterification pro-
l
no
p = 1–0.1 mbar cess was scaled up by Ciba for
ha
Enzyme
et
for example, engineering substrates, reaction media (52), at present for a bright future for industrial biotechnol-
process conditions, or from the expanding applications ogy (53). The research and development on biocatalytic
of known biocatalysts. In any case, progress in the reaction methodologies is thereby key to further advances
understanding of the molecular mechanisms of enzyme in biotransformations, where modular and scalable biocat-
action will be key for the further development of the alytic reaction steps are compatible with the development
science of synthesis with its challenges toward the more of chemical reactions (76,77). As catalytic asymmetric reac-
difficult and more complex target molecules (53). With the tions are a key research area in organic chemistry, it is
growing collection of biocatalytic reactions, the successful crucial that the best combination of preparative methods
retrosynthetic thinking can be applied to biocatalysis is selected in the design of a complete synthesis route.
as well. The introduction of biocatalytic reactions into As enzymes show a nearly perfect efficiency for asymmet-
synthetic organic reaction sequences is uniquely suited ric catalysis with high catalytic turnover numbers and
for cost reductions. In addition, biocatalysis offers high enantioselectivity under very mild reaction condi-
opportunities for quality improvements and for making tions, they are well suited to simplify methods and routes
pharmaceutical processes more sustainable (54) . in the science of synthesis.
Biocatalyst production technologies and integrated Multistep reaction sequences are of much fundamental
process engineering have been instrumental in the interest to overcome limitations in one reaction step and
establishment of biocatalytic reaction steps in chemical avoid intermediate product purification (78), by analogy
synthesis. The inherent properties of biocatalysts with cellular metabolic pathways.
make them the privileged catalysts for highly selective The production of bulk chemicals using biotransforma-
asymmetric molecular transformations like, for example, tions is entering into a new era of global sustainability and
hydrolysis reactions (55–58), oxidation reactions (59–63), is bound to make a significant contribution to solving two of
carbon–carbon bond formation reactions (64–67) as well the most urgent environmental problems, climate change,
as molecular unit transfer reactions (68,69). The universe and the depletion of fossil energy. This will require, how-
of six enzyme classes provides a tremendous goldmine ever, truly sustainable global commitment and effort of
for discovering improved versions of enzymes with known real innovation in science, industry, and society in order to
functions (70,71) as well as for finding completely novel make it happen. The resulting creation of value, also from
enzymes. Once the feasibility of a biocatalytic reaction the point of view of economy, should not be viewed only
has been proven, up- and downscaling experiments have from the short-term cost perspective, but more in terms
been useful for engineering the most adequate process of long-term knowledge building for a future bioeconomy.
design. In the case of the first large-scale biocatalytic The vision 2025, the strategic research agenda and the
Baeyer–Villiger oxidation, the debottlenecking of the implementation action plan of ESAB and the sustainable
substrate feed and product recovery, final purification, and chemistry technology platform (79) provide a synopsis on
overcoming thermodynamic limitations have been essen- ways to move in this direction. Investments into the area of
tial in establishing bioprocesses with high yields of enan- industrial biotransformations are not short-term oriented,
but will certainly belong to some of the finest long-term
tiopure products (72–74). These downscaling experiments
investments for the twenty-first century, which our global
in conjunction with new analytical techniques have proven
society can make.
useful also in the case of asymmetric synthesis of natural
The most recent OECD report (80) designs a policy
compounds (75). Spatial and temporal organization of
agenda for the Bioeconomy to 2030. To conclude this arti-
biocatalysts, reactants, or products is another interesting
cle, we cite some of the most important biotransformation-
engineering option for biocatalytic process design.
related technology and policy statements of this
The scientific and technological advances in the areas of
report:
biocatalyst development, bioprocess design and interfac-
ing with organic reactions, process analysis and product • As biotechnology evolves from a gene-based to
purification technology have created the proper mindset multidisciplinary science that takes into account
16 INDUSTRIAL BIOTRANSFORMATION
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