Table Of ContentContent
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 pub-
lished, 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 biotransforma-
tions.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 indus-
trial 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., refer-
ences 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 “biotransforma-
tions”. The essential difference between fermentation and biotransformation is
that there are several catalytic steps between substrate and product in fermenta-
tion 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 microor-
ganisms, 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 tar-
taric 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
