45Industrial Applications of Enzymes in Emerging AreasAnne van den Wittenboer, Lutz Hilterhaus, and Andreas Liese

45.1Industrial Processes Using Catalytically Promiscuous Enzyme Activities

Most industrial enzymatic processes can be classified as employing enzymaticsubstrate and/or condition promiscuity (definition according to Reference [1]) sincethe substrates and the reaction conditions applied do not equate to the naturalsubstrates and/or environmental conditions of these enzymes. Therefore, thiscriterion is also valid for the industrial processes already described in other chaptersand thus this chapterwill only focus on industrial processes that take advantage of thecatalytic promiscuity of the applied enzymes. According to Hult and Berglund [1] theterm �catalytic enzyme promiscuity� refers to enzymes that catalyze differentreactions by different catalytic mechanisms within the same active site. This appliesto enzymes from different enzyme classes and some applications are described inprevious chapters. One example is the use of pyruvate decarboxylase from Saccha-romyces cerevisiae for acyloin condensation of benzaldehyde and acetaldehyde to yield(R)-phenylacetylcarbinol, an intermediate for ephedrine synthesis, which isdescribed in detail in Chapter 24.

In addition, lipase-catalyzed amidation is an example of catalytic promiscuity thatis applied by BASFAG in the kinetic resolution of racemic amines [2, 3]. Enantiopure(R)-phenylethylamine, which is an intermediate for pharmaceuticals and pesticidesand can also be used as chiral synthon in asymmetric synthesis, is produced byenantioselective amidation of ethylmethoxyacetate on applying the lipase fromBurkholderia plantarii immobilized on polyacrylate. Loss of activity caused by theuse of organic solvent (MTBE–ethylmethoxyacetate) (MTBE¼methyl tert-butylether) could be decreased about 1000-fold by freeze-drying a solution of the lipasetogether with fatty acids, for example, oleic acid. The E-value of this reaction isover 500 and the (R)-phenylethylmethoxyamide can be easily hydrolyzed to obtainthe (R)-phenylethylamine. The non-converted (S)-enantiomer can be racemizedusing a palladium catalyst [4]. The enzymatic reaction is carried out in a plug-flowreactor connected to a falling film evaporator with subsequent distillation(Scheme 45.1).

Enzyme Catalysis in Organic Synthesis, Third Edition. Edited by Karlheinz Drauz, Harald Gr€oger,and Oliver May.� 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

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An example of artificially generated catalytic promiscuity is the use of halohydrindehalogenase (EC 3.8.1.x) in the cyanation of halohydrins. The hydrolase, whichnaturally catalyzes the epoxidation of halohydrins, was evolved to catalyze a C�Cbond formation using NaCN. Codexis Inc. is carrying out a process with this enzymefor the production of 4-cyano-3-hydroxybutyric acid esters, building blocks for thesynthesis of statins, which is described in detail in Chapter 38.

45.2Chemoenzymatic Industrial Processes

Multistep one-pot processes offer the opportunity to decrease the number of work-upand purification steps, thereby improving the overall process efficiency and economyand contributing tomore sustainable processes. The challenge in combining enzymecatalysis with chemical reaction steps in one-pot processes is, often, the limitedcompatibility of the enzymatic and chemical reaction steps in terms of reactionconditions that is, reaction medium, temperature, and so on [6]. This limitation canbe overcome either by use of enzymes that are stable under typical chemical synthesisconditions (e.g., organic solvents, elevated temperatures) or by application ofchemical reaction steps/chemocatalysts in aqueous solutions; examples of bothcases are discussed in the following.

45.2.1Chemoenzymatic Dynamic Kinetic Resolution of Secondary Alcohols

A process for the chemoenzymatic dynamic kinetic resolution (DKR) of secondaryalcohols applying lipases for enantioselective acylation and ruthenium catalysts forracemization of the remaining enantiomerwas patentedbyMerck&Co., Inc. [7].Oneexample of this process is the production of (R)-[3,5-bis(trifluoromethyl)phenyl]ethan-1-ol (6)and respective esters (Scheme 45.2). The product is an intermediate forthe synthesis of substance P (neurokinin-1) receptor antagonists that are used in thetreatment of inflammatory diseases, psychiatric disorders, and emesis [8].

Scheme 45.1 Enantioselective amidation of 1-phenylethylamine carried out at BASF AG [5].

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Immobilized lipase B fromCandida antarctica (Novozym 435�) selectively acylates(R)-[3,5-bis(trifluoromethyl)phenyl]ethan-1-ol (6) to yield (R)-[3,5-bis(trifluoro-methyl)phenylethyl] acetate (8), while the remaining (S)-6 is racemized by aruthenium catalyst. The acyl donor residue, which is acetone in the case ofisopropenyl acetate (7), is removed by continuous distillation under reduced pres-sure. Racemic 6 is supplied via reduction of the corresponding prochiral ketone 4,which is performed in situ by chemical hydrogenationusing transitionmetal catalysts(e.g., the racemization catalyst) in the presence of an alcohol (e.g., isopropanol) ashydrogen donor that is not converted by the lipase. The whole reaction is carried outwithout an additional solvent. The product work up consists of distillation of theremaining acyl donor (7), removal of catalysts by filtration, and subsequent crystal-lization of the product (R)-[3,5-bis(trifluoromethyl)phenylethyl] acetate (8), which isobtained with an isolated yield of 77% and an e.e. >99.5%.

45.2.2Chemoenzymatic Deracemization of Amines and Amino Acids

Chemoenzymatic platform processes for deracemization of several chiral amines aswell as amino acids were developed by Ingenza Ltd. Deracemization is accomplishedby stereoinversion of the undesired enantiomer, combining an enantioselectiveoxidase with a non-selective reducing chemical catalyst [9]. Chiral amines areimportant intermediates for various pharmaceuticals and agrochemicals andare also widely applied as chiral auxiliaries and resolving agents [10]. One exampleis the production of (R)-2-phenylpyrrolidine (10) (Scheme 45.3). Starting from the

Scheme 45.2 Chemoenzymatic dynamic kinetic resolution of (R,S)-[3,5-bis(trifluoromethyl)phenyl]ethan-1-ol (6) developed by Merck & Co., Inc.

45.2 Chemoenzymatic Industrial Processes j1839

racemic mixture a monoamine oxidase selectively oxidizes the (S)-enantiomer,forming an intermediate pyrrole (11) that is again reduced to the racemic pyrrolidine(10), thus affording accumulation of the (R)-enantiomer. To obtain an oxidase thataccepts also this secondary amine, a monoamine oxidase from Aspergillus niger(MAO-N) was evolved by an approach that combined random and saturationmutagenesis. In the final process MAO-N mutant is immobilized on Eupergit andthe reaction is performed in aqueous solution in thepresence of 250mMNH3:BH3 asreductant. Thework up consists of a pH shift to pH10 and subsequent extraction intoMTBE. Isolated yield and e.e. (R) are 80% and 98%, respectively [10]. In addition,tertiary amines such as the alkaloid (�)-crispine A were successfully deracemizedusing thisMAO-Nmutant (yield 95%, e.e. (R) 97%) [11]. The resulting (R)-crispine Ahas cytotoxic activity against certain cancer cell lines and is also a useful synthon forother pharmaceuticals, for example, anti-depressants [12, 13].

An analogous approach is applied to produce unnatural L- and D-amino acids usingD-amino acid oxidases (D-AAO) and L-amino acid oxidases (L-AAO), respectively.These enzymes are naturally highly enantioselective and display a broad substraterange with respect to the side chain. In accordance to the reaction in Scheme 45.3 theAAO selectively oxidizes one of the enantiomers to yield the respective imineintermediate, which is again chemically reduced to the racemic amino acid. Unlikethe aforementioned process, chemical reduction is achieved by catalytic transferhydrogenation using Pd–C in the presence of ammonium formate [9].

45.2.3Chemoenzymatic Synthesis of Xolvone

The synthesis of the solvent Xolvone (1,5-dimethyl-2-piperidone) is an example of achemoenzymatic process that does not consist of a kinetic resolution or deracemiza-tion [14, 15]. Xolvone is a non-flammable, completely water-miscible, and biode-

Scheme 45.3 Chemoenzymatic deracemization of 2-phenylpyrrolidine developed at Ingenza Ltd.

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gradable solvent that is used as precision cleaning solvent for electronics cleaning,industrial degreasing, andmetal cleaning. Furthermore, it is used in the formulationof inks and industrial adhesives and as reaction solvent in the production of polymersand chemicals.

In a process carried out by Du Pont de Nemours & Co, nitrilase from Acidovoraxfacilis catalyzes the hydrolysis of 2-methylglutaronitrile (12), yielding 4-cyanopenta-nonic acid ammonium salt (13) that is subsequently converted into 1,5-dimethyl-2-piperidone (14) by chemical hydrogenation (Scheme 45.4). Compared to the enzy-matic hydrolysis, which produces 4-cyanopentanonic acid ammonium salt with aregioselectivity greater than 98% (conversion 100%), direct chemical hydrogenationof 2-methylglutaronitrile yields a mixture of 1,3- and 1,5-dimethyl-2-piperidone thathas a lower boiling point than the single isomer.

The process is carried out in consecutive stirred-batch reactions on a scale of 500 l,whereas nitrilase expressed in Escherichia coli is used in the form of whole cellsimmobilized in alginate beads. After recovery of the immobilized catalyst 4-cyano-pentanonic acid ammonium salt is directly catalytically hydrogenated within theaqueous solution in presence of added methylamine. Space–time yield and enzymeproductivity are 1896 g l�1 and 3500 gproduct/gbiocatalyst, respectively.

45.3Industrial Application of Enzymes in Material Science

In material science, enzymes can be used for polymerization reactions as well as formodification/functionalization of materials. Biocatalytic polymerization is per-formed either in vivo within the framework of a biosynthetic, metabolic pathwayor in vitro by isolated enzymes that catalyze the polymerization of artificial substratemonomers, which is defined as �enzymatic polymerization� [16]. The former hasbeen applied industrially for over 20 years for the fermentative production of poly(hydroxyalkanoate)s and is not within the focus of this book (for some recent reviewssee References [17–20]. Concerning the latter, many reports on enzymatic polymer-ization, that is, polycondensation and ring-opening polymerization (ROP) in thesynthesis of homopolymers, block polymers, and graft polymers, have been

Scheme 45.4 Chemoenzymatic synthesis of Xolvone carried out at Du Pont de Nemours & Co. [5]

45.3 Industrial Application of Enzymes in Material Science j1841

published but, although the enzymatic polymerization offers great opportunities interms of regio- and stereoselectivity, polymerization is still mainly performed bytraditional chemical processes. Nevertheless, some enzymatic polymerizations havebeen carried out on a large scale (Section 45.3.1) or companies hold patents onrespective processes (Section 45.3.2). Enzymatic treatment of textiles (Section 45.3.3)is an example of enzymatic material processing on an industrial scale.

45.3.1Enzymatic Large-Scale Production of Poly(hexane-1,6-diyl adipate)

Baxenden Chemicals Ltd. carried out a large-scale process for the enzymaticsynthesis of poly(hexane-1,6-diyl adipate) (17) [21]. Immobilized Candida antarcticalipase B (Novozym 435�) was used as catalyst in the polycondensation of adipic acidand hexane-1,6-diol (Scheme 45.5). The reaction was carried out in a scale up to twotons in a 500-l stirred-tank reactor (60 �C), with constant removal of water by vacuum(60mbar). After enzymatic polymerization the carrier-bound lipase was removed byfiltration and recycled. The obtained polyester was compared with a conventionallyderived product (Dynacol�) and was found to have more attractive physical prop-erties (e.g., crystallization behavior).

45.3.2Enzymatic Synthesis of Aqueous Polyamide Dispersions

BASF AG has patented different methods for the production of aqueous polyamidedispersions applying lipase-catalyzed polycondensation [22] and ring-opening poly-merization [23]. Aqueous polyamide dispersions are widely applied as componentsin, for example, hot-melt adhesives, coating formulations, cosmetic and paintformulations, or printing inks. In contrast to conventional chemical multistageprocesses the enzymatic synthesis is carried out directly in aqueous solution, thusavoiding the need for additional dispersion and distillation steps that are technicallycomplex and energy demanding. Scheme 45.6 depicts two examples of the patentedreactions.

The reactions are carried out in miniemulsions using dispersants (e.g., non-ionicemulsifiers, anionic emulsifiers), where the starting materials are present as a

Scheme 45.5 Polycondensation of hexane-1,6-diol and adipic acid catalyzed by Novozym 435�.

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dispersed phase with an average droplet size <1mm within the aqueous phase. Inbothmentioned caseswater-immiscible organic solvents (e.g., toluene) are optionallyadded to obtain organic phase droplets that contain the substrates. The appliedenzyme catalyst was Candida antarctica lipase B (CALB), which was used as freeenzyme, but other lipases and esterases were also found to be suitable catalysts. Allreactions were carried out at 60 �C at a pH between 3 and 9. The resulting polyamidedispersions of polymer 20 and polycaprolactam (22) had a solid content of 9 and11wt%, respectively. The corresponding polyamide powderswere obtained by dryingof the aqueous suspensions after removal of enzyme catalyst and emulsifiers. Theproduced polyamides had a weight-average molecular weight (Mw) of 5200 gmol�1

(polymer 20) and 212 000 gmol�1 (polycaprolactam, 22).

45.3.3Enzymes Applied in the Textile Industry

The processing of textiles and garmentsmade fromnatural fibers, synthetic fibers, orblends of natural and synthetic fibers is increasingly applying enzymes, mainlyhydrolases and some oxidoreductases. Compared to traditional chemical treatmentsthe enzyme-based processes are more environmentally benign, because they enablecleaner processes by reducing energy consumption and production of wastewater.Moreover, they help to reduce the waste of raw material since they are less abrasive,that is, more fabric is obtained from the same amount of fiber material [24, 25]. Fourmajor areas of enzymatic textile processing are described in the following andTable 45.1 gives an overview of respective commercial enzyme-based products:

1) De-sizing is the removal of the so-called �size� (mainly starch, starch derivatives,waxes) that is used for coating of fibers before weaving in order to prevent theirbreaking and which needs to be removed prior to further processing (dyeing,

Scheme 45.6 Enzymatic polyamide synthesis in aqueous dispersions developed at BASF AG.

45.3 Industrial Application of Enzymes in Material Science j1843

bleaching, etc.). Chemical de-sizing uses strong acids, bases, or oxidizing agents,which also affect the fabric. Enzymatic alternatives for de-sizing are mainlyamylases and lipases, which enable complete removal of size without affectingthe fabric [26].

2) Scouring of cotton and other natural fibers is the removal of hydrophobic non-cellulosic impurities such as, for example, pectins, fats, waxes, and so on toachieve a better and more even wettability of the textile. The enzymatic processapplying pectinases and lipases uses lower temperatures and less water than thetraditional chemical scouring process by alkaline or detergent treatment, which isone of themost energy- and water-consuming steps in cotton processing [24–26].

3) Bleach clean-up, that is, the removal of hydrogen peroxide after the bleachingprocess, has been performed enzymatically for over 20 years. The use of catalasesandperoxidases, instead of traditional chemical clean-up using reducing reagentsand hot baths, saves huge amounts of wastewater and energy [27]. More recently,biobleaching, for example, the removal of color chromophores and pigments bylaccases,was introduced,which is reducing theneed for environmentallyharmfulchemicals such as hydrogen peroxide as well as wastewater and energy [26].Another enzymatic bleaching approach is the in situ formation of hydrogenperoxide using glucose oxidase [28]. A special case of biobleaching is theapplication of enzymes for denim finishing, the so-called �stonewashingeffect� [27]: cellulases are used for abrasion and loosening of the indigo dye,esterases and laccases are used to adjust the indigo shade by bleaching.

4) Biopolishing of cotton and other cellulosic fibers yields smoother and glossiertextiles and reduces the pilling tendency of the fabric. Cellulases are used to

Table 45.1 Examples of commercial, enzyme-based products used for textile processing.

Application Enzyme Trade name Company

De-sizing Amylase Aquazym� NovozymesOptisize� GenencorTexamyl Inotex

Scouring Pectinase Scourzyme� L NovozymesPrimaGreen� EcoScour GenencorTexazym SCW Inotex

Bleach clean-up Catalase Terminox� Ultra NovozymesOxy-Gone� T400 Genencor

Oxidase Texazym Dox InotexBiobleaching Laccase DeniLite� Novozymes

PrimaGreen� EcoFade LT00 GenencorEsterase PrimaGreen� EcoLight 1 Genencor

Denim finishing (�stone-washing�) Cellulase DeniMax� NovozymesIndiAge� Genencor

Biopolishing Cellulase Cellusoft� NovozymesPrimaFast� GenencorTexazym AP Inotex

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hydrolyze and thereby remove protruding microfibrils (hairs or fuzz) from thefiber surface that form the �pill� (balls of fuzz) which presents a serious qualityproblem. Compared to traditional singeing with a gas flame, the enzymatictreatment saves energy and only attacks the protruding fibers [27].

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