"INDUSTRIAL BIOTRANSFORMATION". In Encyclopedia of - Lonza

INDUSTRIAL BIOTRANSFORMATION*

ORESTE GHISALBA1 ,HANS-PETER MEYER2 , andROLAND WOHLGEMUTH3

1Ghisalba Life Sciences,Reinach, Switzerland

2Lonza AG, Visp, Switzerland3Research Specialties,

Sigma-Aldrich GmbH,Buchs, Switzerland

INTRODUCTION

Biocatalysis or biotransformation encompasses the useof biological systems to catalyze the conversion of onecompound to another. The catalyst part of the biologicalsystem can thereby consist of whole cells, cellular extracts,or isolated enzyme(s). Up to the second decade of the twen-tieth century, processes based on biotransformations wereclassified under various denominations such as ‘‘zymotech-nology’’ or ‘‘technical biology’’. The term biotechnology wasfirst used in the year 1917 (1). Already then, in the earlytimes of industrial biotechnology, the leading representa-tives of the new science promoted the idea of using biolog-ical systems to create more efficient, more selective, andenvironmentally friendlier processes for the conversion ofraw materials into industrial products, thereby substi-tuting problematic chemical transformations. The conceptof a more sustainable use of the limited resources wasthus one of the driving forces for the rapid developmentof industrial biotechnology long before the now worldwideaccepted political vision of sustainable development waspromoted by national and international conferences andorganizations.

Thanks to genetic engineering techniques introduced in1973, enzymes from all kinds of biological sources can nowbe recloned and overexpressed in easily mass cultivatablemicroorganism or cell cultures. With this technology, evenenzymes that have been rare up to the present can beproduced in large quantities and at affordable costs. Inaddition, whole-cell biocatalysts or production organismscan now be genetically improved by directed engineeringof metabolic pathways.

An OECD-report of 1998 (2) analyzes the state ofthe art and the future development needs for industrialbiotechnology. Some important conclusions in this reportsummarizing the state of the art are the following:

• Economic competitiveness has been established fora variety of biotechnological applications to achievecleanliness.

∗Dedicated to Klaus Kieslich, pioneer of industrial biotransfor-mations.

Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology, edited by Michael C. FlickingerCopyright © 2010 John Wiley & Sons, Inc.

• Industrial penetration of biotechnology is increasingas a consequence of advances in recombinant DNAtechnologies.

• Biotechnological operations have led to cleaner pro-cesses with lowered production of wastes and, insome cases, lower energy consumption.

• The fine chemicals industry is one of the industrialsegments where the impact of biotechnology is feltmost strongly.

PRINCIPLES OF BIOTRANSFORMATIONS

Advantages of Biocatalysis

To perform (large-scale) synthesis of drugs, materials,or chemicals, one can principally follow three differentapproaches with various degrees of complexity:

1. Use a purely chemical strategy.2. Use a chemoenzymatic route combining chemical

and biocatalytic steps. In this case, the biocatalystis preferentially used to perform the key reaction(s)requiring high selectivity or specificity or to replaceenvironmentally intolerable reaction steps.

3. Use a biological total synthesis by fermentation ormultistep biotransformation.

Enzymes and whole-cell biocatalysts have severalattractive properties, which make them privilegedcatalysts for organic synthesis; these are as follows:

• They have high chemo-, regio-, and stereo-selecti-vities.

• They require mild reaction conditions. Therefore, bio-catalysis offers great chances and advantages forsuccessful applications (also in cases where eitherthe substrates or the products of the reaction arechemically labile).

• Biocatalysis is normally performed in an aqueousenvironment but can, in many cases, also be con-ducted in solvent mixtures, liquid–liquid two-phasesystems, and even in pure organic solvents. A rel-evant practical example is the use of esterases andlipases to catalyze esterifications in organic solventssuch as vinyl acetate.

• There is no, or only limited use of protecting groups,for example, for the chemoenzymatic synthesis ofcomplex carbohydrates and glycoconjugates.

Application Range of Biocatalysis

According to the enzyme classification (EC) system ofthe nomenclature committee of the International Unionof Biochemistry and Molecular Biology (IUBMB) (3),enzymes are classified into six main classes (Table 1).

1

2 INDUSTRIAL BIOTRANSFORMATION

The majority of the described enzymes belong to the ECclasses 1, 2, and 3.

Very detailed information on some 4000 EC-classifiedenzymes (organized in fact sheets) can be found inthe excellent Springer Handbook of Enzymes (4). Thesame information is also available in electronic form viaBRENDA (Braunschweig Enzyme Databank): http://www.brenda-enzymes.de/.

Useful information on commercially available enzymescan be searched electronically in a variety of ways viathe Enzyme Explorer (http://www.sigma-aldrich.com/enzymeexplorer/.

Practical examples for preparative biotransformations(with checked procedures) can be found, for example, inthe highly acclaimed handbook edited by Stanley Robertset al. (5), and also in (6). For German speaking scientists,there is an excellent ‘‘Bioorganikum’’ available (7).

An additional and highly recommendable electronicinformation source for the synthetic chemist is the Catal-ysisNet (www.catalysisnet.com), which is based on theAbstracts of the Warwick Biotransformation Club andon current biotransformation abstracts maintained underthe supervision of Nicholas Turner (University of Manch-ester).

A new biotransformation search feature has beenadded recently to the SciFinder software by the ChemicalAbstract Service, which allows to search functional groupbiotransformation reactions in a substructure search. Thebiotransformation database assembled by Klaus Kieslichand the Warwick Biotransformation Club has providedthe nucleus of this data mining service.

The actual scope of bioreactions is summarized inTable 2.

Screening for Biocatalysts

Major driving forces for present and future R&D in indus-trial biocatalysis are the following:

• The continued demand for chiral building blocks andenantiomerically pure compounds, in general.

• A growing interest in areas of synthesis where bio-catalysts offer clear advantages over purely chemicalstrategies. The most prominent examples are, atpresent, peptide synthesis, protein modification, syn-thesis of complex carbohydrates, oligosaccharides,and glycoconjugates. The latter area presents manychemical, ecological, and economical problems to theclassical organic chemistry. The multifunctionality ofthe products, calling for sophisticated and laboriousprotection group strategies, has a great potential forselective and direct biocatalytic synthetic methodolo-gies.

• International efforts to promote sustainable develop-ment and environmentally friendly technologies.

Three main screening strategies have been followedso far:

1. Testing and evaluating inexpensive commerciallyavailable (bulk) biocatalysts or suitable microorgan-isms. Enzymes that do not require coenzymes, such

as hydrolases (lipases, esterases, proteases) are stillthe preferred biocatalysts for the preparation of opti-cally active compounds.

2. Searching and screening for suitable novel microbialbiocatalysts from natural sources, for example, byselective enrichment techniques from environmentalsamples, starting from soil or sewage samples, oralternatively and in rare cases, by testing extractsof plant or animal tissues. This laborious and moredemanding approach is essential when no suitableenzyme or microbe is commercially accessible for theconversion of a defined substrate to a defined productusing a particular reaction type. Both approaches 1and 2 are complementary.

3. A third approach, related to the second, is thesearch for completely novel enzymes catalyzingdifficult reaction types, where no indications or priorknowledge on their existence has been available.Typical examples for this approach from the lastdecades are the discovery and investigation ofenzymes catalyzing Baeyer–Villiger oxidations (8)or Diels–Alder-type reactions (9).

Enzyme screening and bioprocess development wereimportant skills and key factors in the success of theJapanese industrial biotechnology. On the basis of a work-ing group analysis, Cheetham came to a very harshstatement on this issue: ‘‘in Europe screening is under-developed and underresourced both in absolute terms andby comparison with current activities in Japan, and espe-cially when compared with the resources devoted to geneticmanipulation’’ (10). Has there been a change 20 yearslater? In hindsight, future biologists may consider screen-ing to have been a rate-limiting step in the development ofbiotechnology in Europe in the late twentieth century (10),particularly with respect to screening efforts directed tothe discovery of completely new, so far unknown subclassesof enzymes. Unfortunately, in our view, this situation hasnot really improved during the last 20 years.

With modern polymerase chain reaction (PCR) tech-niques, it has become possible to enrich and investigatethe genetic information (DNA) even from the VBNCs(viable but not culturable microorganisms) present in soilsamples. A general approach begins with the isolation ofnucleic acids directly from soil and environmental sam-ples, from primary enrichment cultures, and from purifiedmicroorganisms across the whole spectrum of biodiver-sity. In the next stages of the process, gene libraries(genes cloned in suitable expression hosts) are constructedfrom the purified DNA and subjected to high-throughputautomated (robotics) activity screening systems.

Technical Aspects of Biocatalysis

To set up a practical bioconversion process screening andoptimization, work is required at the following differentlevels:

1. Screening for suitable microorganisms or plant cells,and so on, and/or enzymes with the required catalyticproperties and selectivities.

INDUSTRIAL BIOTRANSFORMATION 3

Table 1. The Six Enzyme Classes

IUBMB Enzyme Class Reactions Catalyzed/Special Requirements

EC 1 Oxidoreductases Reductions/oxidations at —CH—OH, —C=O, —C=C—, and so on Two-substrate reactionsCosubstrate required

EC 2 Transferases Transfer of functional groups such as C1, aldehyde, keto, acyl, glycosyl, and so on.Two-substrate reactions, one substrate with activated functional group →A–(B) + C = A − C + (B).

EC 3 Hydrolases Hydrolyses/condensations of esters, glycosides, ethers, peptides, amides, and so on. →A–B + H2O = AH + BOH. Hydrations → A–B + H2O = HA − BOH. Transesterifications.Two-substrate reactions, one substrate being H2O. No cofactors required.

EC 4 Lyases Additions/eliminations. Cleavage of C–C–, C–O–, C–N–bonds. One-substrate reactions→,two-substrate reactions ←. No cofactors required.

EC 5 Isomerases Racemization, cis–trans isomerization, epimerization. One-substrate reactions. No cofactorsrequired.

EC 6 Ligases Formation of C–O–, C–S–, C–N–, C–C–bonds. Two-substrate reactions. Cosubstrate ATPrequired.

Enzymes can generally catalyze their reactions in both directions. This is of special interest for the preparative use of hydrolytic enzymes such aslipases/esterases, where the reverse reaction can be performed in organic solvents.

Table 2. Types of Substrate Conversions (Selection) that have been Successfully Performed by Biocatalysisa

Type of ReactionReductions ofAromatic 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 ofUnactivated saturated carbons, (polyunsaturated) fatty acids, epoxidation and dihydroxylation of alkenes, aromatic compounds

(→ unsaturated diols), hydroxylated compounds and aldehydes, diols (and lactonization), enzyme-catalyzed Baeyer–Villigeroxidations, organic sulfides (sulfoxidation).

Hydrolysis ofEsters 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 ofNitriles, amides, C = C bonds.

Transesterifications (reversion of hydrolytic reactions) of(Racemic) alcohols with nonactivated esters, (racemic) alcohols with activated esters, irreversible transesterification with

vinyl-carboxylates.

Enzymatic ring closure and openingLactone formation from hydroxy esters, ring opening of anhydrides.

Asymmetric glycosylationAcylation of amines

Biocatalytic C–C-formationAdditions and eliminations

Isomerization ofSugars, sugar phosphates.

Racemization ofAmino acids.

Biotransformations of organometallic compoundsReductions and oxidations, hydrolysis and esterification.

Multienzymatic approachesOf special interest for cofactor-dependent systems with recycling enzymes and multistep one-pot enzymatic synthesis.

aCompiled from literature.


2. Screening for the optimum conditions for culturegrowth and production of the desired enzyme(s).This can be very demanding work in cases whereenzyme expression is not constitutive. In this case,the best and most economical process parameters,such as the conditions for enzyme induction and theright harvesting time must be identified.

3. Screening for the optimum reaction conditions withwhole cells, crude, or purified enzymes includingengineering and technological aspects such as cofac-tor regeneration, immobilization, type of reactor, andso on.

4. Optimization of the biocatalyst by directed evolutionor other enzyme engineering technologies in order toobtain the ideal biocatalyst under the given processconditions.

In order to perform bioreactions, the biocatalyst can beoperated under various process conditions (also refer toFig. 2):

• batch or fed-batch application of whole cells in freeor immobilized form in aqueous environment;

• continuous application of whole cells in immobilizedform in aqueous environment;

• application of whole cells in two-liquid phase, multi-phase systems, or in micelles;

• use of acetone-dried or permeabilized cells;• batch or fed-batch application of free crude or purified

enzymes in aqueous or organic environment;• use of polyethyleneglycol (PEG)-modified enzymes in

organic solvents;• continuous application of immobilized enzymes;• use of enzyme membrane reactors; the method of

choice for systems with cofactor recycling or for reac-tions with expensive enzymes.

For economic reasons, if ever possible, whole-cell bio-catalysis is used to perform biotransformations. This ispossible when the following criteria are met: (i) no dif-fusion limitations for substrate(s) and/or product(s) and(ii) no side or follow-up reactions due to the presence ofother cellular enzymes. If these conditions are not ful-filled, the use of isolated enzymes—or in special cases ofpermeabilized cells—is indicated.

Immobilization can be achieved by adsorption or cova-lent fixation of the biocatalyst to a solid support (e.g.surface-modified polymer or glass beads), by entrapmentor by encapsulation in gel beads (e.g. agarose, polyacry-lamide, alginate, etc.). Only a limited set of methods hasfound real technical applications. The first large-scaleapplications of immobilized enzymes were established forthe enantioseparation of D- and L-amino acids by Chi-bata, Tosa and coworkers at Tanabe Seiyaku Company.The Japanese achievements in the large-scale applicationof immobilized systems are very well documented in anexcellent multiauthor publication edited by Tanaka, Tosa,and Kobayashi (11). Some enzyme suppliers sell importantindustrial enzymes not only in the free form (solution orpowder) but also in immobilized form on solid supports.

The cross-linked enzyme crystals (CLEC) technologywas developed and launched by Altus Biologics Inc. (adaughter company of Vertex Pharmaceuticals Inc.). Thistechnology takes well-known soluble enzymes, whichdenature in harsh environments and stacks them in asolid crystal form—adding the cross-linking of chemicalbonds for further strength (12,13). For a short review withfocus on applications refer to (14). A related approach isthe cross-linked enzyme aggregates (CLEA). Since thismore general method is also applicable to enzymes thatcannot be crystallized, it is easier to handle and muchcheaper (15).

An important technical issue is the large-scale appli-cability of cofactor-dependent enzymatic systems. It isgenerally accepted that, for example, NADH-requiring oxi-doreductases can easily be used in whole-cell biocatalysissuch as baker’s yeast-mediated reductions. The cofac-tor recycling step is thereby simultaneously performedwithin the intact cell, driven by the reduction equivalentsintroduced via the external carbon and energy source (glu-cose). For preparative-scale synthesis, repetitive batchhas proven to be an easy-to-handle technique. The mul-tiple reuse of the enzyme(s) is possible after recoveringthem from the reaction mixture by means of concentra-tion with ultrafiltration equipment. Continuous processesoften show higher space-time yields compared with batchprocesses. For the application of enzymes in continuousprocesses, and especially for cofactor-dependent systems,the enzyme membrane reactor (EMR) concept has beendeveloped and successfully applied by Kula and Wandrey(16,17), and others. It has also been successfully applied inindustry, for example, by Degussa (now Evonik), TanabeSeiyaku, Sepracor, Sigma-Aldrich and others (18–20).

At the time when the EMR-concept was introduced,most of the cofactors were still very expensive compoundsand therefore contributed significantly to the overall pro-cess costs. Therefore, for application in continuous pro-cesses, the molecular weight enlargement by covalentcoupling to PEG (in order to allow very high recyclingnumbers → upto 600,000 cycles have been reported) wasa conditio sine qua non for the economic viability of suchprocesses at this time. In the meantime, however, thanksto the rapid progress in fermentation technologies, at leastNAD(H) and ATP have become affordable compounds, atleast when purchased in bulk (i.e. multikilogram) quanti-ties. Under these conditions, these cofactor cost drop to 1US$ per gram or even less.

As an example, a more recent EMR-process for theenantioselective reduction of 2-oxo-4-phenylbutyric acid(OPBA) to (R)-2-hydroxy-4-phenylbutyric acid (HPBA,ee >99.9%) with NADH-dependent D-lactate dehydroge-nase (D-LDH) from Staphylococcus epidermidis and FDH(formate dehydrogenase)/formate for cofactor recyclinguses free NADH instead of PEG-NADH (21,22) (Fig. 1).Within the selected residence time of 4.6 h, a cycle numberfor the cofactor of 1000 can easily be achieved. Insteadof NADH, the less-expensive NAD+ is used. A secondreason for the application of the native coenzyme is thefact that the activity of D-LDH is reduced to one-tenthwhen PEG-enlarged coenzyme is used instead of thenative coenzyme. HPBA produced by this method with


O

COOH

OH

COOHD-LDH

NADH NAD+

FDHCO2 HCOOH

OPBA (R)-2-HPBA

Figure 1. Enantioselective enzymatic reduction of 2-oxo-4-phenylbutyric acid to (R)-2-hydroxy-4-phenylbutyric acid (usingEMR technology).

a space-time yield of 165 g/L/d is of considerably higherenantiomeric purity. After a second round of process opti-mization, the space-time yield of the D-LDH/FDH-basedprocess is around 400 g/L/d (unpublished data). Theprocess shows competitive economy in comparison withchemical ‘‘enantioselective’’ hydrogenation approaches(23) (either heterogeneous hydrogenation with a modifiedplatinum catalyst (yielding ee 82–91%) or homogeneoushydrogenation with a soluble chiral rhodium disphosphinecomplex (yielding ee 96%) (22).

On the other hand, performance, product quality andeconomics of this EMR-process are comparable with analternative biocatalytic approach using whole cells ofreductase-containing Proteus vulgaris or Proteus mirabilis(22). In this case, carbamoyl methylviologen is used as anelectron mediator and the oxidized mediator is reduced(recycled) by formate with formate dehydrogenase alsopresent in Proteus strains. HPBA is a versatile build-ing block, for example, for the angiotension-convertingenzyme (ACE) inhibitor benazepril/cibacen and relatedpharmaceuticals.

An attractive variation of the EMR-concept is thecharged ultrafiltration membrane enzyme reactor pro-moted by the groups of Kulbe and Chmiel (24,25). This con-cept applies negatively charged ultrafiltration membranesto efficiently retain the native cofactor in the reactor whenNAD(P)(H)-dependent enzymes coupled to cofactor recy-cling systems are used. The successful application of thisapproach was described, for example, for the simultane-ous conversion of glucose/fructose mixtures into gluconicacid and mannitol via the enzymes glucose dehydrogenaseand mannitol dehydrogenase [both NAD(H)-dependentenzymes; cofactor turnover number 150,000]. An addi-tional process improvement for the cofactor retention isthe use of nanofiltration instead of ultrafiltration mem-branes (26).

The classical EMR-concept can be extended for thebioconversion of poorly soluble substrates to an emulsionmembrane reactor. This reactor consists of a separatechamber for emulsification (with a hydrophilic ultrafiltra-tion membrane), an EMR-loop with a normal ultrafiltra-tion module, and a circulation pump. This approach hasbeen successfully demonstrated for the enzymatic reduc-tion of poorly soluble ketones (27). Using this device, forexample, for the enantioselective reduction of 2-octanoneto (S)-2-octanol (ee >99.5%) with a carbonyl reductasefrom Candida parapsilosis under NADH-regenerationwith FDH/formate, the total turnover number wasincreased by factor 9 as compared with the classical EMR.

INDUSTRIAL APPLICATIONS OF BIOTRANSFORMATIONS

History and Companies

Biocatalysis has been commercially used for several thou-sand years by mankind and one can claim that industrialbiotechnology is much older than biotechnology used forthe pharmaceutical industry, for example. Our ancestorsin Mesopotamia, Egypt, Mexico, and the Sudan have beenusing Acetobacter and yeast concomitantly for the conver-sion of sugars to alcohol and acetic acid, small moleculebiotechnology nota bene? Table 3 gives some landmarksand selected milestones of industrially relevant biocat-alytic processes.

Large-scale biocatalysis is mainly but not exclusivelyused for the production of fine chemicals. Table 4 showsexamples of companies using biocatalysis among othertechnologies.

The reason for using biocatalysis is cost optimizationthrough the high selectivity of enzymes. The precision ofenzymes allows process simplification and waste streamsreduction, especially with the increasing complexity andfunctionalization of the target products. Costs can be fur-ther reduced through a variety of process improvementsto reach high space-time yields using biological and engi-neering principles. In simple words, one has to reachthe highest possible product concentration in the shortestpossible time (value creation) during a biocatalytic process.All following steps in the downstream processing (DSP)must be designed to minimize losses during isolation andpurification (value conservation). Process intensificationincluding process integration between value creation andvalue conservation is the key to cost control.

Because of a well-filled toolbox chemistry is, in general,still faster in new process development and biocatalyticprocesses typically represent the second generation (28).Counterexamples are special reaction platforms like theenzymatic reduction of ketones, which have advancedrapidly and can compete with the best chemical meth-ods available (29). The biocatalytic process (if there isone) usually turns out to be cheaper and more efficient.However, the development and implementation is oftenslower, as commercial enzymes are not yet readily avail-able. Another obstacle is that products and processes candiffer very much, unlike the production of, for example,monoclonal antibodies with mammalian cell culture oreven microbial protein production.

Chemical Market

According to a study published by CHEManager 2008(30), the global chemistry market is estimated at 2292billion US$ and is expected to grow to 3235 US$ by 2015and 4000 billion US$ by 2020. Of the 2290 billion US$,the global industrial biotechnology market is believed torepresent only about 50 billion US$ (not including bio-fuels). Thus, the chemical market is the main target forbiocatalysis.

Of these 50 billion US$ biotechnology products, about25% are fine chemicals and the growth of the share ofbiotechnologically produced fine chemicals is expected togrow from 8% to 60% between 2001 and 2010 (31). Fine


chemicals differ from commodities in price and annualmanufacturing volumes of up to 10 tons.

The global white (or industrial) biotechnology marketof ∼50 billion US$ is smaller than the red biotechnology(pharmaceutical) market (>70 billion US$), but industrialwhite biotechnology represents a larger long-term busi-ness potential than red biotechnology. It is estimated thatat least 20% of the global chemicals (2292 billion US$ pre-sently) could be produced by biotechnological means in2020.

Manufacturing Facilities

As discussed earlier, the advantage of biocatalysis lies inthe unmatched precision in the production and assemblyof complex and chiral molecules due to the enantio-, regio-,and substrate selectivity. This precision of biotransforma-tion is based on the unique properties of catalytic proteins(enzymes).

The process has to be adapted to the biological premises,unless the opposite is possible, which is the preferredway. Large-scale processes must be carried out in a well-controlled manner. For this purpose, different processoperation modes have been developed (Fig. 2, see alsothe section titled ‘‘Technical Aspects of Biocatalysis’’).

Substrate and product inhibition, the stability ofenzymes, growth or cofactor requirements, by-productformation must be well mastered during enzymaticprocesses, although the sensitivity tends to vary fromreaction to reaction. However, this does not mean thatlarge-scale biocatalytic transformation processes are per

se complex and expensive. As a matter of fact, manybiocatalytic processes are run in standard chemicalreactors without any adaptations. This is almost alwaysthe case if one uses, for example, commercially availablehydrolytic enzymes such as lipases or proteases.

Some more complex biocatalytic procedures are per-formed with whole cells because cofactors need to beregenerated and may require fermentation equipment.

As mentioned earlier, biocatalysis is often limited due tosubstrate or product inhibition. The former may be over-come by suitable substrate-feeding regimes in fed-batchprocesses. The latter, however, is technically more diffi-cult to avoid. Ideally, it would be necessary to continuouslyremove the product at a rate similar to the formationrate using an efficient, nontoxic, regenerable, and cheapprocess, which has a high specificity for the targetedsubstance. This is the concept of ISPR (in situ productrecovery) in combination with a biocatalytic process (alsoin Fig. 2). The use of liquid–solid or liquid–liquid bipha-sic systems is thereby of much interest for simple andstraightforward industrial processes, if the biocatalyst iscompatible with these phases (32).

Reactor for Biocatalytic Reactions

No special equipment is needed for biocatalysis in manycases and ‘‘ordinary’’ stirred tanks, used in large-scalechemical synthesis with temperature and pH control, aresufficient. Thus, in this article, we concentrate on a fewselected process units that are used in biocatalysis. Fer-mentors (Fig. 3) are used for the production of the enzymes,

Table 3. The Long Development to Modern Biotransformation

5000 B.C. Preservation of food and alcoholic drinks on Egyptian pictures, vinegar production800 B.C. Casein hydrolysis with chymosin for cheese production. Homer’s Iliad mentions stomach enzymes for cheese making1670 ‘‘Orleans’’ process for the industrial biooxidation of ethanol to acetic acid1680 Antoni van Leeuwenhoek, first to see microorganisms with his microscope1833 Payen and Persoz investigate germinating barley and formulate basic principles of enzymes1874 Christian Hansen started an enzyme laboratory and company1878 Kuhne coined the term enzymes1890 Takamine isolates bacterial amylases (what later became Miles Laboratories)1894 Emil Fischer elaborated the essentials of enzyme catalysis1897 Buchner discovers yeast enzymes converting sugar into alcohol1897 Eduard Buchner published fermentation with cell-free extracts from yeasts1899 Berzelius acknowledges the catalytic reaction of starch hydrolysis by diastase1907 Rohm founded a company producing ‘‘Oropon’’ for tanning (mixture pancreatic extract and ammonium salts)1926–1935 Sumner, Northrop, and Kunitz prove that enzymes are proteins1930 Regioselective biooxidation of sorbite to sorbose for the Reichstein Vitamin C synthesis1940 Sucrose inversion using an invertase1950 Bioconversion of steroids1969 First industrial use of an immobilized enzyme (aminoacylase)1970 Hydrolysis of penicillin to 6-aminopenicillanic acid1985 Enzymatic process for the production of acrylamide1990 Hydrolysis by protease (trypsin) of porcine insulin to human insulin1995 3000 tons per year plant for the biotransformation of to nicotinamide

Table 4. Examples of Companies Developing Biotransformation Processes on a Large Scale

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.


if they are not commercially available. They are stirredvessels that allow a sterile (monoseptic) operation. Thismeans that only the organism with the desired enzymeused for the biocatalysis is allowed in the bioreactor. How-ever, in many cases, normal stirred-tank reactors, as usedin chemistry, can be applied.

When using hydrolytic enzymes, the reactor configu-ration can be very simple as shown in the picture below(Fig. 4). These reactor series are used for the annualproduction of several thousand tons of nicotinamide.

The product recovery processes are key steps after thebiocatalytic reactions and make use of conventional unitoperations and employ currently established and availabletechniques (see figure 5 for an example of a plant layout).

In addition to liquid-liquid extraction and crystalliza-tion as shown in figure 5, a variety of other unit operationslike chromatographic separations (Fig. 6), membraneseparations (Fig. 7) and drying operations (Fig. 8) arestandard processes for product recovery and purification.

Examples of Biocatalysis Products

Products resulting from biocatalytic processes range fromcommodity products to high-value pharmaceuticals. Fora review of the potential applications and industrialexamples of the six enzyme classes, see also (19,20,33,34).

Table 5 shows a few examples of biocatalytic products.Considering products that have market relevance, onerealizes that the majority depends on hydrolytic enzymes.

F1

Product

F1

F2

F2

Fed-batch process. One-timefed- or or repeated batch process.

ISPR(in situ product recovery). Aninhibitor is continuously removed.

Immobilized enzymes in fluidizedor fixed bed reactors. Most productiveif operated in continuous mode.

Recycling of enzyme or cells. Actuallya method to immobilize enzyme inwhole-cell biocatalytic processes

F2

F1

Multistage continous culture. Externalor internal multistage bioreactor.

Figure 2. The principal process modes arebatch (not shown), fed-batch or continuousoperation, which can still be further combinedwith each other. ISPR, enzyme or cell immo-bilization, or bioreactions in two phases arepromising examples of developments. As men-tioned above, a biocatalytic process consists inone or several biocatalytic steps, which can beintegrated with downstream processing stepsor repeated if several steps are involved.


(a) (b) (c)

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 andbiocatalysis. The shaft and the turbines have been removed for a better view. The details showthat the baffles are constructed as heat exchangers to maintain optimal temperature ranges forexothermal high cell density fermentation and biocatalysis. [Courtesy Lonza Biotec sro Kourim,CZ]

Figure 4. The picture shows a series of 16 m3 chemical reac-tors used for the continuous three-step biocatalysis (hydration)of a nitrile to the corresponding nicotinamide using an immobi-lized biomass. The installation results in very high volumetricproductivities due to very stable and active biocatalyst.

A broad range of large-scale biocatalytic methodologieshave been developed for the production of L-amino acidsby Evonik or DSM, for example. However, processes toproteinogenic amino acids are in competition with fermen-tative processes. Especially, whole pathway engineeringat present allows grafting whole sets of genes to designan organism capable of producing a desired amino acid orother product.

Hydrolytic enzymes are applied by numerous com-panies (BASF, Ciba, Dowpharma, Lonza, Sigma-Aldrichetc.). Several pharmaceutical intermediates are producedin this way at a large scale using hydrolases to hydrolyzethe corresponding esters. (S)- or (R)-nitrilases to makechiral carboxylic acids via either conventional or dynamicresolutions. A dynamic resolution is possible if one startswith cyanohydrins, because these can be racemized rapidlyenough in situ. Enantioselective hydrolysis by the nitri-lase produces one α-hydroxycarboxylic acid enantiomerand the unreacted cyanohydrin can be racemized. The

Table 5. Some Examples of Biotransformation ProductsUsed in the Ton Scale

Acrylamide ∼250 000 tonsAspartame 10 000 tonsNicotinamide 15 000 tonsL-Carnitine Several hundred tonsL-DOPA >150 tonsLysine 700 000 tons7-ACA 4000 tons6-APA 10 000 tons(S) Naproxen >1000 tonsLysine >1000 000 tonsGlucose–Fructose syrup 12 000 000 tonsVitamin C >100 000 tonsCitric acid 1000 000 tons

Note that there are many more commodity products produced byfermentation.

desired acid can then be accumulated in high yield andwith high enantiomeric excess. BASF uses this designto make (R)-mandelic acid and derivatives on a multi-ton scale (35). R-selective hydroxynitrile-lyases have beenapplied for the large-scale synthesis of cyanohydrins (36).

It was mentioned earlier that the main reason to choosebiocatalysis is the cost advantage over chemical synthe-sis. A good example proving that biocatalysis can havea manifold better productivity compared with chemicalsynthesis and even result in a reduced ecological foot-print is L-Dopa (33,37,38). L-Dopa is a metabolic precursorof dopamine, a drug for the treatment of parkinsonism,and is also of interest in other therapeutic indications.The annual production is over 250 tons and we estimatethat over two-third of this is produced using biocatalyticprocedures (Fig. 9).

An example of a very large, large-scale biocatalysisprocess is the conversion of nicotinonitrile to nicoti-namide (39,40), an essential nutrient in animal and humannutrition. There are several chemical processes for theproduction of nicotinamide but Fig. 10 shows the biocat-alytic step in a cascade of four highly selective, continuous,catalytical reactions and used in the production of severalthousand tons of nicotinamide per year.


Table 6. Glossary and Some Definitions of Terms Used in this Article

API: Active pharmaceutical ingredientBiocatalyst: Biological agents such as microorganisms or enzymes that activate or speed-up a chemical reactionBioconversion: Chemical conversion of a substance using biological methods (enzymes or whole cells, biocatalysis or

biotransformation)Biocatalysis: Chemical conversion of a substance (educt, defined starting material) into a desired product with the aid of a free or

immobilized enzyme. The difference between biocatalysis and biotransformation is that biocatalysis uses an isolated enzymeBiotransformation: Chemical conversion of a substance (educt, defined starting material) into a desired product with the aid of a

(usually) living whole cell, containing the necessary enzyme(s). Unless necessary for the context, we use only the termbiotransformation

Biosynthesis: De novo production of an entire molecule by a living organism. Unlike biotransformation, which acts on a startingsubstance or educt, the biosynthesis is not dependent on educts or starting substances, but only on nutrients

Building blocks: Compounds that can combine with other compounds to form a new moleculeClone: A population of genetically identical microorganisms derived from a single parent cellCloning (of a gene): Terminus technicus to describe the transfer of genes from one (micro) organism to another. Inserting a population

of DNA molecules, known to contain the DNA of interest, into a population of vector DNA molecules in such a way that each vectormolecule contains only a single DNA molecule from the original population

DNA: Acronym for deoxyribonucleic acid, usually 2′-deoxy-5′-ribonucleic acid. DNA contains the genetic information of an organismand the code in the genes contain the blueprints to form the different proteins

Educt: Chemical starting compound for a chemical or biological conversion to a productEnzyme: Proteins that act as biological catalysts (biocatalysts), initiating all biochemical reactions. Enzymes are classified into six

enzyme classes according to their mechanism (also refer to Table 1)Fermentation: Process for the cultivation of microorganisms in special vessels (fermentors) that allow the ‘‘monoseptique’’

propagation of a desired microorganism, only the desired microorganism is allowed to grow using sterile fermentation technology.Thus the result of a fermentation is microbial biomass, which contains the desired enzyme used in the following biotransformation.The biotransformation can take place during fermentation or after the fermentation in a separate vessel, which often does not needto be operated in a sterile manner

Fine chemicals: Value-added intermediates and active substances used, for example, in pharmaceuticalsWhite biotechnology: A term for industrial biotechnology, which includes a vast area of products, processes, and industries.

Biocatalyis/biotransformation is one of many process technologies used in ‘‘white biotechnology’’ Reference 53Gene: Basic unit of the hereditary material DNA, an ordered sequence of nucleotide bases that encodes a product. The product of a

gene finally is a protein. Genes are the blueprints for the enzymes used in biotransformationGenome: The entire complement of genetic material in a (micro) organismGreen chemistry: This term for sustainable (chemical) industrial manufacturing processes striving for, for example, minimal waste

production and energy consumption. Biosynthesis and biotransformation are assumed to play a key role in green chemistry in thefuture

Host: Microorganism that is used for the expression of foreign DNA, consequently the production of a foreign protein (enzyme), whichis encoded in a gene. The expression of a foreign gene for the production of a foreign protein is also called heterologous expression,because the gene does not belong naturally to the producing organism (host).

Immobilized enzymes: The half-life and stability of enzymes can be prolonged by ‘‘associating’’ enzymes using cross-linking,covalently binding them to, for example, polymer resins, by encapsulating them

Metagenomics: Environmental genomics is the study of genomes recovered from environmental samples as opposed to clonal cultures.This relatively new field of genetic research allows the genomic study of organisms that are not easily cultured in a laboratory

ISPR (in situ product recovery): Removal of the inhibiting biotransformation product from the solution during the reaction. Byremoving the product, which inhibits cellular growth and/or an enzyme activity used for biotransformation, the productivity can bemaximized

NCE: New chemical entityProduct inhibition: Finely tuned enzyme activities are often inhibited at higher concentrations of the resulting product of their

catalytic activity. Unlike educt inhibition, which can be overcome using well-regulated feeding techniques, product inhibition ismore difficult to deal with in biotransformation

Secondary metabolite: A product of the secondary metabolism. Secondary metabolites are typically produced in tiny amounts, verycomplex (sometimes with ‘‘dozens’’ of chiral centers) and biologically very active. Secondary metabolites (e.g. vancomycin) are notessential for normal growth, development, or reproduction of a microorganism. On the other hand, the products of the primarymetabolism (e.g. ethanol sugar fermentation) are essential for the survival of the microorganism

Sequence: The order of amino acids in a protein or the order of nucleotides of a gene in DNA. Knowing the sequence allows thecloning, heterologous expression, and production of a protein in different hosts or microorganisms

Strain: A genetically homogeneous population of (micro)organisms of common origin. A strain can be biochemically ormorphologically differentiated from other strains. Microbial strains are able to produce enzymes with distinctive chemo-, regio-,and enantioselectivity. Another characteristic of microbial strains is that they divide and grow very quickly, and are well suited forrapid production of large amounts of enzymes. The growth of microorganisms (see, e.g. in photograph) can be exponential

Strain collection: Microorganisms can be stored for several years in small ampoules in frozen form (–80◦C) or lyophilized. Individualstrains can be revived easily for tests. Thus the larger a collection of different strains, the higher the success rate to for a newbiotransformation candidate

System biology (in silico biology): Computational models of biological systems, where varying streams of biochemical information areintegrated and modeled in silico. System biology is applied from drug discovery to metabolic pathway simulation


Figure 5. The plant layout for the fed-batch processdeveloped by Givaudan for the biocatalysis of ferulicacid to vanillin. The biomass is grown on glucose. Ata given moment during fermentation the substrate(ferulic acid) is started to be added according a feedregime. The biocatalytic step (Fig. 14) is carried outwith an actinomycete strain (Streptomyces setoni),and high titers of >15 g/L were reached, despite thecellular toxicity of vanillin. The DSP consists in atwo-step organic solvent extraction.

Biotransformation

Carbontreatment

Solventevaporation

Precursorfeed

Vacuum

Crystallizer

Biomassseparator

Vacuumdrying

Centrifug-ation

Liqiud–liqiudextraction

Figure 6. Chromatography steps are increasingly used for thepurification of small molecules issued from biocatalysis processes.The column in the photograph is a new large chromatographycolumn of 2 m in diameter ready to be installed. [Courtesy LonzaBiotec sro Kourim, CZ]

Calcitriol is a pharmacologically active metabolite ofVitamin D3, exhibiting far broader activity beyond regulat-ing calcium and phosphorus metabolism. For the synthesisof a modified compound of Vitamin D3, an antipsoriaticretiferol, Hoffmann–La Roche developed a biocatalyticdesymmetrization using a lipase shown in Fig. 11 (41).

Kittelmann et al. (42) from Novartis developed a processfor the production of the acylglucuronide of mycopheno-lic (MPA) acid, which is biologically active and requested

for pharmacological studies in transplantation research(Fig. 12). In human metabolism, the ratio of the acylglu-curonide to the 7-O-glucuronide is reported to be 1:80in favor of the 7-O-glucuronide. The preparative-scalesynthesis of the minor acylglucuronide was therefore agreat challenge.

The chemoenzymatic route to 2′-deoxynucleotidesjointly developed by Novartis, Yuki Gosei, Lonza, and theKyoto University (Fig. 13), or the chemoenzymatic route to2′-O-(2-methoxyethyl) ribonucleotides/-nucleosides (bothfor antisense applications) are another example (43,44).The starting materials for the 2′-deoxynucleotides routewere cheap bulk mononucleotides (IMP, GMP, AMP).

Givaudan developed a large-scale biotransformationprocess for natural vanillin by using Streptomycessetonii (45). The microbiological biocatalysis processconverts ferulic acid to vanillin (Fig. 14). Ferulic acid isan abundant natural product, but the acid often occursin the form of a glycoside. Typical products are vanilllicalcohol, vanillic acid, guaiacol, and so on. Guaiacol is aphenolic, smoky-type molecule, which contributes to thecharacteristic flavor of the vanilla extracts.

Ciba developed a series of chemoenzymatic processeswith lipases (Fig. 15 and 16) for the production of pho-toinitiators [Reinhold Oehrlein, Ciba, personal communi-cation].

Wandrey (46) and Tramper (47) have defined criteriafor the optimization and the viability of biochemicalconversions in comparison with chemical processes.Process design includes the choice of an appropriatesubstrate, of a catalyst, and of methods of downstreamprocessing in order to obtain the desired product ina defined purity. The aims of process selection andoptimization (especially in the pharmaceutical sector) areto find process conditions defined by high conversion ofthe substrate, high selectivity of the reaction, high opticalpurity of the product, high space-time yield (productivity)


Figure 7. A 100 m2 nanofiltration spiral-wound micro-filtration unit used for cell and enzyme concentration.[Courtesy Lonza Biotec sro Kourim, CZ]

Figure 8. Products can be dried by several methods, and spraydryers are versatile units. The picture shows the bottom of largespray dryer with a performance of 130 kg of water per hour.[Courtesy Lonza Biotec sro Kourim, CZ]

of the process, and low enzyme and coenzyme consumptionper unit mass of product.

The following are also of high importance:

• The number of reaction steps involved, environmen-tal aspects—‘‘greenness’’.

• Biocatalysis versus chemical conversion. When thenumber of reaction steps can be drastically reduced,chances are in favor of biocatalytic strategies.Enantio- and stereoselectivity can be strong pointsin favor of biocatalysis.

• Reactor configuration: standard versus tailored. Deci-sion is based on the kinetics of the biocatalyticprocess; availabilty and experience; desired mixing;and mass transfer properties.1. Standard case: stirred-tank reactor for batch ope-

ration—in industry great reluctance to change toother configurations.

HO

OH

NH2

COOH

COOH

O

L-Dopa

Erwinia herbicola

(Tyrosine phenol lyase)

H2O

HO

HO

NH3

+

++

Figure 9. This scheme describes the L-DOPA synthesis usingwhole cells of Erwinia herbicola developed in a joint effort betweenJapanese academic groups and Ajinomoto (37,38).

N

N

N

NH2

O

Nitrile hydratase

Nictotinonitrile Nictotinamide

Figure 10. Conversion of nicotinonitrile to nicotinamide in athree-step continuous biocatalytic reaction developed by Lonzawith Rhodococcus rhodochrous J1 cells immobilized in polyacry-lamide.

2. Potential alternatives: plug-flow reactor, packed-bed reactor, fluidized-bed reactor, two-liquid-phase reactor, membrane reactor (especially forcofactor-requiring free enzymes), and so on.

• Mode of operation: (fed)-batch versus continuous.Parameters for decision: kinetics, stability, and formof the biocatalyst; desired substrate conversion, prod-uct concentration (solubility); need of process con-trol, inhibitory/toxic effect of substrate or product.Examples for fed-batch bioprocesses: acrylamide pro-cess (Nitto Chemicals), L-carnitine process (Lonza).


OAc

OAc

AcO

OAc

AcO OAc

OH

AcO OAc

HO OH

CF3

OH

CF3

16 gLipase OF

+6°C, 1.07 equiv.22 h

Antipsoriatic retiferol

81.7 g (77% trans)1.35 L buffer pH 7.0200 ml cyclohexane

cis

Chromatographicfiltration

700 g silica gel

46.3 g (88%)>99% ee96% GC*

* 2.6% cis-diacetate 0.6% trans-meso-diacetate

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–LaRoche. The product was produced on a multikilogram scale.

UDP-glucuronic acid

O

O OH

O

O

OH

O

O OH

O

O

OH

OH

OH

OHO

O

OH

OH

OH

OHO

O

HO

O

O

O

O

O

OH

Mycophenolic acid+

UDPGA-transferase

(foal liver homogenate)Acylglucuronide (desired product)

O-Glucuronide

UDP

+

Figure 12. Enzymatic process for the production of mycophenolic-derivative studied for humantransplantation.


Examples for continuous operation: production ofL-tert leucine and other amino acids (Degussa).

• Level of process integration. Parameters for deci-sion: number of process steps (subsequent reactions);nature of the reactions.

Many of current large-scale applications use immobi-lized enzymes. In particular, the Japanese industry has,for a long time, pioneered this sector of biotechnology. Inthe multiauthor publication edited by Tanaka, Tosa, andKobayashi (11) and in a more recent review, (48) a num-ber of well-established continuous production processeswith immobilized biocatalysts are described in detail.Complementary informations on some of these Japanesebioprocesses as well as additional case studies for otherbioprocesses have been compiled by Cheetham (49).

The most recent and most complete compilation ofexisting industrial biotransformations (with process dataand scale of operation of the listed processes) was recentlypublished by Liese, Seelbach, and Wandrey (20). This pub-lication clearly demonstrates that already the applicationspectrum of biocatalysis for industrial processes is muchbroader than generally perceived. Thus, the hope seemsjustified that the contribution of biotechnology in mediumto large-scale production processes can be significantlyincreased in future, provided that the necessary effortsto enlarge the biocatalytic toolbox are made by academiaand industry.

OUTLOOK

On the way to sustainable production strategies, thechemical industry must play a key role in innovationand structural changes by introducing more biologicalprocesses and biobased thinking into its R&D effort.Biotechnology and genetic engineering greatly enlarge thearsenal of organic synthesis methods. This reengineeringhelps to preserve natural resources and ecosystems, to

protect the environment by producing (bio)degradableproducts, to avoid or reduce pollution and repair damagesto the environment. The quality of life is increased andpreserved by the introduction of new pharmaceuticalsand foods. This can be achieved by the utilization ofrenewable resources, by the wise and economic useof nonrenewable resources, and by the protection ofecosystems with increased use of biological processes.The necessary changes in technologies can only beachieved by linking with economical, ecological, and socialgoals.

Two conclusions in the earlier-mentioned 1998OECD-report (2) read as follows:

• ‘‘The key area of opportunity is improved and novelbiocatalysts. The search is on to examine the remain-ing unexplored biological diversity which presumablyholds a great wealth of biocatalytic potential andof new bioactive compounds and biomaterials. Theintroduction of biotechnology into many industrialprocesses will be increasingly dependent on the devel-opment of recombinant biocatalysts’’.

• ‘‘Bioprocess engineering and integrated bioprocess-ing also continue to be critical factors for the com-mercialization of biotechnology’’.

In a later OECD-report of 2001 on the application ofbiotechnology to industrial sustainability (50), the mainmessages are as follows:

• Global environmental concerns will drive increasedemphasis on clean industrial products and processes.

• Biotechnology is a powerful enabling technology forachieving clean industrial products and processesthat can provide a basis for industrial sustainability.

• Measuring the cleanliness of an industrial productor process is essential but complex; life cycle assess-ment (LCA) is the best current tool for making thisdetermination.

N

NN

N

NH2

OO

OH OH

P

O

O

-O

N

NN

N

NH2

OO

OH OH

P

O

O

OPOP

O

O

O

O

O

N

NN

N

NH2

OO

OH

P

O

O

OPOP

O

O

O

O

O

R-(SH)2

NMP

Phosphorylation

2 Pi

NTP

RTP-ReductaseLactobacillus leichmannii

RS2 + H2O

Phosphatase

Transferase

Base exchange

dNTP

Figure 13. Chemoenzymatic route for the synthesis of antisense nucleotides.


Figure 14. The biocatalysis of ferulic acid tovanillin, a process developed and realized at indus-trial scale by Givaudan (Switzerland). For a basicplant layout, refer Fig. 5. Besides reaching veryhigh productivities, also the right balance betweenvanillin and by-products, which is part of theknowhow of the man skilled in the art, were reached.

COOH

OH

OMe

OH

OH

COOH

OH

OMe

CHO

OH

OMe

CH2OH

OH

OMe

COOH

OH

OMe

Ferulic acid Vanillin Vanillic acid Protocatechuic acid

Vanillyl alcohol Guaiacol

O

O

OH

OH

O

OO

O

HO

2

O

O OO

OHOH

IRGANOX 245®

LIPASE70°C / 99%

200 mbar

+

Figure 15. Example of a lipase process developed by Ciba. Thebiocatalytic process has the advantage of low-reaction temper-ature, high conversion, high purity, simple workup, no metalcontaminants, and continuous operation.

• The main drivers for industrial biotechnological pro-cesses are economic (market) forces, governmentpolicy, and science and technology.

• Achieving greater penetration of biotechnology forclean environmental purposes will require joint R&Defforts by government and industry.

• For biotechnology to reach the full potential as a basisfor clean industrial products and processes, beyondits current applications, additional R&D efforts willbe needed.

• Because biotechnology, including recombinant DNAtechnology and its applications, has become increas-ingly important as a tool for creating value-addedproducts and for developing biocatalysts, there is astrong need for harmonized and responsive regula-tions and guidelines.

• Market forces can provide very powerful incentivesfor achieving environmental cleanliness objectives.

• Government policies to enhance cleanliness of indus-trial products and processes can be the single-mostdecisive factor in the development and industrial useof clean biotechnological processes.

• Communication and education will be necessary togain penetration of biotechnology for clean productsand processes into various industrial sectors.

The report contains 21 case studies from various indus-trial sectors and demonstrates the ecological and econom-ical viability of bioprocesses.

The further development of biotransformations hasobtained more weight recently by the simultaneousappearance of initiatives in atom and step economy,safety, and health as well in environment representedby the Green Chemistry initiatives. Safety, health,renewable resources, energy conservation, and wasteminimization will continue to be of both global and localconcern and the sequence of molecular transformationsfrom raw material to product is also characterized by thereplacement of hazardous reagents (51).

The interdisciplinary character of the dead endsand locks between chemistry, biology, and engineeringrequires a thorough understanding of the interfaces (32).Communication across scientific and technologicaldisciplines and with the value creation perspective isimportant for the development of a better synthesis of thefinal product in the bottle. If, in the last step of a longtotal synthesis, the protecting group cannot be cleavedoff without damage to the product, or in the case wherethe modification of a complex natural product from abiosynthetic approach is not known to occur by a biocat-alytic reaction, a whole synthetic route might become adead end. The search for novel reaction methodologiesis therefore highly relevant, but represents a majorchallenge and looks for further advances. Successfulproblem solution may come from various approaches as,


Melt

Enzyme

T = 75°Cp = 1 bar

T = 75°Cp = 1–0.1 mbar

T = 100°Cp = 1–0.1 mbar

Met

hano

lMelt reactor

Figure 16. A transesterification pro-cess was scaled up by Ciba forIrganox 245, a photoinitiator. Theprocess is carried out with an immo-bilized enzyme in a column. A fallingfilm evaporation (T = 75◦C, p =1–0.1 mbar) is useful here for thecontinuous separation of the prod-ucts and the substrates. The ethanoland the product were collected andremoved, while the substrate was fedback into the process. Ciba was able torun such an equipment continuouslyfor 6 months.

for example, engineering substrates, reaction media (52),process conditions, or from the expanding applicationsof known biocatalysts. In any case, progress in theunderstanding of the molecular mechanisms of enzymeaction will be key for the further development of thescience of synthesis with its challenges toward the moredifficult and more complex target molecules (53). With thegrowing collection of biocatalytic reactions, the successfulretrosynthetic thinking can be applied to biocatalysisas well. The introduction of biocatalytic reactions intosynthetic organic reaction sequences is uniquely suitedfor cost reductions. In addition, biocatalysis offersopportunities for quality improvements and for makingpharmaceutical processes more sustainable (54) .

Biocatalyst production technologies and integratedprocess engineering have been instrumental in theestablishment of biocatalytic reaction steps in chemicalsynthesis. The inherent properties of biocatalystsmake them the privileged catalysts for highly selectiveasymmetric molecular transformations like, for example,hydrolysis reactions (55–58), oxidation reactions (59–63),carbon–carbon bond formation reactions (64–67) as wellas molecular unit transfer reactions (68,69). The universeof six enzyme classes provides a tremendous goldminefor discovering improved versions of enzymes with knownfunctions (70,71) as well as for finding completely novelenzymes. Once the feasibility of a biocatalytic reactionhas been proven, up- and downscaling experiments havebeen useful for engineering the most adequate processdesign. In the case of the first large-scale biocatalyticBaeyer–Villiger oxidation, the debottlenecking of thesubstrate feed and product recovery, final purification, andovercoming thermodynamic limitations have been essen-tial in establishing bioprocesses with high yields of enan-tiopure products (72–74). These downscaling experimentsin conjunction with new analytical techniques have provenuseful also in the case of asymmetric synthesis of naturalcompounds (75). Spatial and temporal organization ofbiocatalysts, reactants, or products is another interestingengineering option for biocatalytic process design.

The scientific and technological advances in the areas ofbiocatalyst development, bioprocess design and interfac-ing with organic reactions, process analysis and productpurification technology have created the proper mindset

at present for a bright future for industrial biotechnol-ogy (53). The research and development on biocatalyticreaction methodologies is thereby key to further advancesin biotransformations, where modular and scalable biocat-alytic reaction steps are compatible with the developmentof chemical reactions (76,77). As catalytic asymmetric reac-tions are a key research area in organic chemistry, it iscrucial that the best combination of preparative methodsis selected in the design of a complete synthesis route.As enzymes show a nearly perfect efficiency for asymmet-ric catalysis with high catalytic turnover numbers andhigh enantioselectivity under very mild reaction condi-tions, they are well suited to simplify methods and routesin the science of synthesis.

Multistep reaction sequences are of much fundamentalinterest to overcome limitations in one reaction step andavoid intermediate product purification (78), by analogywith cellular metabolic pathways.

The production of bulk chemicals using biotransforma-tions is entering into a new era of global sustainability andis bound to make a significant contribution to solving two ofthe most urgent environmental problems, climate change,and the depletion of fossil energy. This will require, how-ever, truly sustainable global commitment and effort ofreal innovation in science, industry, and society in order tomake it happen. The resulting creation of value, also fromthe point of view of economy, should not be viewed onlyfrom the short-term cost perspective, but more in termsof long-term knowledge building for a future bioeconomy.The vision 2025, the strategic research agenda and theimplementation action plan of ESAB and the sustainablechemistry technology platform (79) provide a synopsis onways to move in this direction. Investments into the area ofindustrial biotransformations are not short-term oriented,but will certainly belong to some of the finest long-terminvestments for the twenty-first century, which our globalsociety can make.

The most recent OECD report (80) designs a policyagenda for the Bioeconomy to 2030. To conclude this arti-cle, we cite some of the most important biotransformation-related technology and policy statements of thisreport:

• As biotechnology evolves from a gene-based tomultidisciplinary science that takes into account


full cellular modules and their interactions withthe external environment, bioinformatics willplay an increasingly important role. This willinclude systems modeling and the production ofthree-dimensional models of a wide variety ofbiological components.

• Synthetic biology is emerging as a new field forimproving microorganisms, based on an engineeringapproach that enables the design and construction ofnew biological parts, devices, and systems, and theredesign of existing, natural biological systems foruseful purposes (http://www.synthetic-biology.info).The purpose of synthetic biology is to increase bio-logical efficiencies by designing a cell system for aspecific function, thereby eliminating the produc-tion of unwanted products that waste the cell’senergy.

• Industrial biotechnology (IB) is used in the produc-tion of chemicals and derived biomaterials, with addi-tional applications in mining and resource extraction.There are many industrial applications based onenzymes that are either produced by GM (geneticallymodified) microorganisms or selected using modernbiotechnology. Activity is also under way to combine anumber of biobased industrial processes into a singleproduction line known as biorefinery.

• Metabolic pathway engineering techniques will con-tinue to broaden the range of compounds that canbe produced through biotechnology. They are likelyto be extensively used before 2015 to economicallyproduce nonbiodegradable bioplastics, high-densitybiofuels, and pharmaceuticals. This is supported bythe significant amount of research currently underway and the entry of a number of large corporationsinto the field.

• The competitiveness of both biotechnological andalternative solutions will depend on several factors,including the amount of R&D in each option, the rel-ative cost of different technologies, and governmentsupport through subsidies, tax credits, or mandates.Major technological breakthroughs in a competingtechnology could divert private and public invest-ment away from some biotechnologies.

• The full potential of the bioeconomy in 2030 willnot develop automatically. Success will requireintelligent and flexible government policy andleadership to support research, markets, andcreate incentives for private firms to invest inbiotechnology.

• International collaboration will also be essential,both because the major markets for many industrialand primary production biotechnologies will bein developing countries and because collaborationwill be necessary to solve global problems suchas resource constraints and climate change.With appropriate policy and good leadership,the bioeconomy of 2030 should provide a higherquality of life and a more prosperous and environ-mentally sustainable future for all of the world’scitizens.

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