Table Of Content

Content 1 Introduction 1 2 HistoryofIndustrialBiotransformations–DreamsandRealities 3 2.1 Fromthe“flowerofvinegar”totherecombinantE.coli– Thehistoryofmicrobialbiotransformations 3 2.2 FromgastricjuicetoSweetzymeT– Thehistoryofenzymaticbiotransformations 11 2.3 Advantagesofbiotransformationsoverclassicalchemistry 25 References 27 3 EnzymeClassification 31 3.1 TheEnzymeNomenclature 31 3.2 TheEnzymeClasses 33 EC1 Oxidoreductases 34 EC2 Transferases 39 EC3 Hydrolases 43 EC4 Lyases 48 EC5 Isomerases 51 EC6 Ligases 54 References 56 4 BasicsofBioreactionEngineering 57 4.1 Definitions 58 4.2 Biosynthesisandimmobilizationofbiocatalysts 64 4.3 Characteristicsofthedifferentenzymeclasses 79 4.4 Kinetics 83 4.5 Basicreactortypesandtheirmodeofoperation 86 References 90 5 Processes 93 Index Indexofenzymename 397 Indexofstrain 400 Indexofcompany 403 Indexofstartingmaterial 406 Indexofproduct 414 1 Introduction The main incentive in writing this book wastogatherinformationonone-step biotransformations that are of industrial importance. With this collection, we wanttoillustratethatmoreenzyme-catalyzedprocesseshavegainedpracticalsig- nificancethantheirpotentialusersareconsciousof.Thereisstillaprejudicethat biotransformations are only needed in cases where classical chemical synthesis fails.Eventheconvictionthattherespectivebiocatalystsarenotavailableand,if so, then too expensive, unstable and only functional in water, still seems to be widespread. We hope that this collection of industrial biotransformations will in future influence decision-making of synthesis development in such a way that it might lead to considering the possible incorporation of a biotransformation step inaschemeofsynthesis. We therefore took great pains in explicitly describing the substrates, the cata- lyst, the product and as much of the reaction conditions as possible of the pro- cessesmentioned.Whereverflowschemeswereavailableforpublicationorcould begeneratedfromthereactiondetails,thiswasdone.Detailsofsomeprocesspa- rameters are still incomplete, since such information is only sparingly available. We are nevertheless convinced that the details are sufficient to convey a feeling fortheprocessparameters.Finally,theuseoftheproductsisdescribedandafew process-relevantreferencesaremade. We would go beyond the scope of this foreword, should we attempt to thank all those who were kind enough to supply us with examples. Of course, we only published openly available results (including the patent literature) or used per- sonallyconveyedresultswiththeconsentoftherespectiveauthors.Weareaware of the fact that far more processes exist and that by the time the book is published, many process details will be outdated. Nonetheless, we believe that this compilationwith its overviewcharacter willserve theabove-mentioned purpose. This awareness could be augmented if the reader, using his or her experience, wouldtakethetroubleoffillingouttheprintedworksheetattheendofthisbook with suggestions that could lead to an improvement of a given process or the incorporationofafurtherindustrialprocessintothecollection. Requesting our industrial partners to make process schemes and parameters moreaccessibledidnotpleasethemverymuch.Evenso,weareaskingourpart- nersonce again todisclose moreinformationthanthey have doneinthepast.In manyinstances,farmoreknowledgeofindustrialprocesseshasbeengainedthan ispubliclyavailable.Ourobjectiveistobeabletomakeuseofthese“wellknown secrets”aswell.Wewouldliketoexpressourgratitudetoallthosewhosupplied us with information in a progress-conducive manner. Thanks also go to those whodidnotrejectourrequestscompletelyandatleastsupplieduswithaphoto- graphincompensationfortheactuallyrequestedinformation. The book begins with a short historical overview of industrial biotransformations.Sincetheprocessorderofthecompilationisinaccordancewiththeenzyme nomenclature system, the latter is described in more detail. We also include a chapter on reaction engineering to enable an easier evaluation of the processes. 1 Introduction The mainpart of the book, as you would expect,is the compilation of the industrial biotransformations. The comprehensive index will allow a facile search for substrates,enzymesandproducts. We sincerely hope that this book will be of assistance in the academic as well astheindustrialfield,whenonewantstogetaninsightintoindustrialbiotransfor- mations. We would be very thankful to receive any correction suggestions or further comments and contributions. At least we hope to experience a trigger effectthatwouldmakeitworthwhileforthereadership,theauthorsandtheedi- torstohaveasecondeditionsucceedingthefirst. We are indebted to several coworkers for screening literature and compiling data, especially to J(cid:252)rgen Haberland, Doris Hahn, Marianne Hess, Wolfgang Lanters,MonikaLauer,ChristianLitterscheid,NagarajRao,DurdaVasic-Racki, MurilloVillelaFilho,PhilomenaVolkmannandAndreaWeckbecker. We thank especially Uta Seelbach for drawing most of the figures during long nights, as well as Nagaraj Rao and the “enzyme group” (Nils Brinkmann, Lasse Greiner, J(cid:252)rgen Haberland, Christoph Hoh, David Kihumbu, Stephan Laue, ThomasStillgerandMurilloVillelaFilho). And last but not least we thank our families for their support and tolerance duringthetimethatweinvestedinoursocalled(cid:28)bookproject’. 2 2 History of Industrial Biotransformations – Dreams and Realities DURDAVASIC-RACKI FacultyofChemicalEngineeringandTechnology UniversityofZagreb HR-10000Zagreb,Croatia Throughoutthe history ofmankind,microorganismshavebeen oftremendous social and economic importance. Without even being aware of their existence, manusedthemintheproductionoffoodandbeveragesalreadyveryearlyinhis- tory. Sumerians and Babylonians practised beer brewing before 6000 B.C., references to wine making can be found in the Book of Genesis, and Egyptians used yeast for baking bread. However, the knowledge of the production of chemicals such as alcohols and organic acids by fermentation is relatively recent and the firstreportsintheliteratureappearedonlyinthesecondhalfofthe19thcentury. Lactic acid was probably the first optically active compound to be produced industriallybyfermentation.ItwasaccomplishedintheUSAin1880[1].In1921, Chapman reviewed a number of early industrial fermentation processes for organicchemicals[2]. Inthecourse oftime,itwasdiscoveredthat microorganismscould modifycer- tain compounds by simple, chemically well-defined reactions which were further catalyzed by enzymes. Nowadays, these processes are called “biotransformations”. The essential difference between fermentation and biotransformation is that there are several catalytic steps between substrate and product in fermentation while there are only one or two in biotransformation. The distinction is also inthefactthatthechemicalstructuresofthesubstrateandtheproductresemble oneanotherinabiotransformation,butnotnecessarilyinafermentation. 2.1 From the “flower of vinegar” to the recombinant E. coli – The history of microbial biotransformations 2.1Fromthe“flowerTofvinegar”tohtherecombienantE.coli story of microbial biotransformations is closely connected with vinegar productionwhichdatesbacktosome2000yearsB.C. Vinegarproductionisperhapstheoldestandbestknownexampleofmicrobial oxidation which may illustrate some of the important developments in the field ofbiotransformationsbylivingcells(figure1). 2 HistoryofIndustrialBiotransformations–DreamsandRealities E O OH + O2 + H2O OH ethanol oxygen acetic acid water Fig.1 Vinegarproduction(E=biocatalyst). A prototype bioreactor with immobilized bacteria has been known in France since the 17th century. The oldest bioreactor using immobilized living microorganisms, a so-called generator, was developed in 1823 [3,4]. Even today, acetic acidisstillknownas“vinegar”ifitisobtainedbyoxidativefermentationofetha- nol-containingsolutionsbyaceticacidbacteria[5]. In 1858, Pasteur [6] was the first to demonstrate the microbial resolution of tartaric acid. He performed fermentation of the ammonium salt of racemic tartaric acid, mediated by the mold Penicillium glaucum. The fermentation yielded (–)-tartaricacid(figure2). COOH HO H H OH COOH (–)-tartaric acid (S,S)-tartaric acid Fig.2 Pasteur’sproductofthefirstresolutionreaction. This was also the first time that a method in which microorganisms degrade oneenantiomeroftheracematewhileleavingtheotheruntouchedwasused. In 1862, Pasteur [7] investigated the conversion of alcohol to vinegar and con- cluded that the pellicle, which he called “the flower of vinegar”, “serves as a transportofairoxygentoamultitudeoforganicsubstances”. In1886,BrownconfirmedPasteur’sfindingsandnamedthecausativeagentin vinegarproductionasBacteriumxylinum.Healsofoundthatitcouldoxidizepro- panoltopropionicacidandmannitoltofructose(figure3)[8]. Bacterium O xylinum OH OH propan-1-ol propionic acid CH2OH CH2OH H OH Bacterium H OH H OH xylinum H OH HO H HO H HO H O H CH2OH CH2OH mannitol fructose Fig.3 ReactionscatalyzedbyBacteriumxylinum,thevinegarbiocatalyst. 4 2.1 Fromthe“flowerofvinegar”totherecombinantE.coli In1897,Buchner[9]reportedthatcell-freeextractspreparedbygrindingyeast cellswithsandcouldcarryoutalcoholicfermentationreactionsintheabsenceof livingcells.Thisinitiatedtheusageofrestingcellsforbiotransformations. NeubergandHirsch[10]discoveredin1921thatthecondensationofbenzalde- hydewithacetaldehydeinthepresenceofyeastformsopticallyactive1-hydroxy- 1-phenyl-2-propanone(figure4). O O OH OH pyruvate decarboxylase OH Saccharomyces cerevisiae chemical + O O HN 1 2 CO2 3 4 1 = benzaldehyde 2 = 2-oxo-propionic acid 3 = 1-hydroxy-1-phenylpropan-2-one 4 = 2-methylamino-1-phenylpropan-1-ol Fig.4 L-Ephedrineproduction. The obtained compound was further chemically converted into -(–)-ephe- D drinebyKnollAG,Ludwigshafen,Germanyin1930(figure5)[11]. Fig.5 Knoll’spatentof1930. 5 2 HistoryofIndustrialBiotransformations–DreamsandRealities The bacterium Acetobacter suboxydans was isolated in 1923 [12]. Its ability to carryoutlimitedoxidationwasusedinahighlyefficientpreparationof -sorbose L from -sorbitol(figure6). D CH2OH CH2OH CH2OH CH2OH O H -KMnO4 or HO HO Acetobacter O O NcaatOalCysl/tn oicrkel HO H2/cat HO suboxydans HO acetone, H+ O air oxidation OH OH OH O HO HO HO O CHO CH2OH CH2OH H2C O D-glucose D-sorbitol L-sorbose diacetone-L-sorbose O COOH COOH COOMe O O O O HO O H3O+ HO MeOH, H+ HO MeO- HO H O OH OH O HO HO HO H2C O CH2OH CH2OH CH2OH diacetone-2-keto L-gulonic acid 2-keto-L-gulonic acid methyl-2-keto-L-gulonate L-ascorbic acid H3O+, D Fig.6 Reichstein-Gr(cid:252)ssnersynthesisofvitaminC(L-ascorbicacid). -Sorbosebecameimportantinthemid-1930’sasanintermediateintheReich- L stein-Gr(cid:252)ssnersynthesisof -ascorbicacid[13]. L In 1953, Peterson at al. [14] reported that Rhizopus arrhius converted proges- teroneto11(cid:97)-hydroxyprogesterone(figure7),whichwasusedasanintermediate inthesynthesisofcortisone. O O Rhizopus HO arrhius O O O progesterone 11a -hydroxyprogesterone O O OH HO OH O OH O O cortisol cortisone Fig.7 Microbial11(cid:97)-hydroxylationofprogesterone. 6 2.1 Fromthe“flowerofvinegar”totherecombinantE.coli This microbial hydroxylation simplified and considerably improved the effi- ciency of the multi-step chemical synthesis of corticosteroid hormones and their derivatives. Although the chemical synthesis [15] (figure 8) from deoxycholic acid that was developed at Merck, Germany, was workable, it was recognized thatitwascomplicatedanduneconomical:31stepswerenecessarytoobtain1kg ofcortisoneacetatefrom615kgofdeoxycholicacid.Themicrobial11(cid:97)-hydroxy- lationofprogesteronequicklyreducedthepriceofcortisonefrom$200to$6per gram. Furtherimprovementshaveledtoacurrentpriceoflessthan$1pergram [16]. In the 1950’s, the double helix structure and the chemical nature of RNA and DNA – the genetic code of heredity – were discovered. This discovery can be regarded as one of the milestones among this century’s main scientific achieve- ments. It led to the synthesis of recombinant DNA and gave a fillip to genetic engineering in the seventies’. Such developments quickly made the rDNA tech- nologyapartofindustrialmicrobialtransformations.Applicationofthistechnol- ogyfortheproductionofsmallmoleculesbeganin1983.Ensleyetal.[17]report- edontheconstructionofastrainofE.colithatexcretedindigo,oneoftheoldest known dyes. They found that the entire pathway for conversion of naphthalene to salicylic acid is encoded by genes of Pseudomonas putida. These genes can be expressed in E.coli. Their results led to the unexpected finding that a subset of these genes was also responsible for the microbial production of indigo. More- over, they showed that indigo formation was a property of the dioxygenase enzyme system that forms cis-dihydrodiols from aromatic hydrocarbons. Finally, theyproposedapathwayforindigobiosynthesisinarecombinantstrainofE.coli (figure9). GenencorInternationalisdevelopingacommerciallycompetitivebiosynthetic route toindigo using recombinant E.coli that can directlysynthesize indigofrom glucose[18].Andersonetal.in1985[19]reportedtheconstructionofametaboli- callyengineeredbacterialstrainthatwasabletosynthesize2-keto- -gulonicacid L (figure10),akeyintermediateintheproductionof -ascorbicacid(vitaminC). L BASF, Merck and Cerestar are building a 2-keto- -ketogulonic acid plant in L Krefeld, Germany. The start up of operation is scheduled for 1999. They devel- opedanewfermentationroutefromsorbitoldirectlytotheketogulonicacid[20]. Thismethodisprobablysimilartothemethoddescribedin1966[21]. The Cetus Corporation (Berkeley, California, USA) bioprocess for converting alkenes to alkene oxides emerged in 1980 [22]. This bioprocess appeared to be very interesting, thanks to the possibility of replacing energy-consuming petro- chemicalprocesses. TherewerehighhopesthatthedevelopmentofrecombinantDNAtechnology would speed up technological advances. Unfortunately, there is still a lot left to be done about the development and application of bioprocesses before the com- mercialproductionoflow-valuechemicalsbecomesfeasible[23].However,today even the traditional chemical companies like Dow Chemical, DuPont, Degussa- H(cid:252)ls AG etc., pressurized by investors and technological advances, are trying to usemicrobialorenzymatic transformationsinproduction.Theyaredoingthisto see whether natural feedstocks can bring more advantages than crude oil. One only needs to compare the cost of a barrel of oil with that of corn starch to see thatthelatterisquitecheaper[20]. 7 2 HistoryofIndustrialBiotransformations–DreamsandRealities HO COOCH3 PhCOO COOCH3 2) KOH 1) PhCOCl 3) D 4) Ac2O HO PhCOO COOCH3 Br COOCH3 HO 5) HOBr 6) CrO3 AcO AcO O Br COOCH378)) KZnOH O COOCH131) NaN3 O NH2 1 3 ) pHyNriOdi3ne 9) HOAc 12) HOAc 10) SOCl2 OH O O 14) tosyl chloride O 15) O3 O 16) KOH 17) C2H2 HO O HO O 19) Ac2O 18) H2 20) PBr3 HO O Br O COOH O O 21) KOAc 22) KOH 23) succinic anhydride AcO HO OH O OAc HO 24) CrO3, KOH 25) Ac2O O OAc 26) Br2 O OH HO OAc 27) Pyridine O 28) K2CO3 30) CrO3 O 29) Ac2O Br O O O HO HO O OAc O OH 31) hydrolysis O O cortisone acetate cortisone Fig.8 Chemicalsynthesisofcortisone. 8