Chapter V ||
LARGE-SCALE
O R G A NI C S Y N T H E S I S
U S I N GC E L L F R E E E N Z Y M E S
G e o r g eM . W h i t e s i d e s
1 l n tr oduc t ion 901
,tt)
: x a mp l e sg O2
e n z y m e sE
2 Synt hes es
Ut iliz ingC e l l -F re E
g e w Pro c e s s eUsti l i z i ngC el l -Free
in De v e l o p i nN
3 Co n s ider at ions
En zy m icCat aly s is9 0 5
Enzymelsolation 905
En z y m eS t abiliz a ti o n9 0 6
En z y m icA c t iv it y 9 1 I
Co fac t orRequir em e n ts9 l 6
R e a c t orDes ign 91 8
Alternativesand Prospectives920
INTRODUCTION
I
,l
a;
,t
with which cell-freeenzymescan be appliedas catalystsin
The effectiveness
p ra cti calor ganics y nt h e ti cp ro c e d u redse p e n d sb o th o n scal eand reacti ontype.
havebeen widely employedin a variety of types
Cell-freeenzyrnepreparations
transformadegradative
of small-scale
syntheses.and usedin severallarge-scale
ti o n s n ot inv olv ingc ofa c to rsT. h e p a rti a lh y d ro l y s i sof mi l k protei nsby renni n
and the chi l l proofi ngof
a n d o f s oil pr ot einsby p ro te a s e -c o n ta i n idnegte rg e nts.
. n zymesarenot commonl y
b e e rb y papar n.pr ov id ee x a mp l e os f th e l a tte rc l a s sE
reactionsthat requirecofactorsor that carryout reactions
utilized in large-scale
hy
rro re c onr plext han d ro l y s i so r i s o me ri z a ti o nT.h e b orderbetw eenthe regi ons
ly
and thosei n
o f synt hes is
in whic h e n z y mi cc a ta l y s i h
s a sb e e ns u c cessful used
of
p
ri
rn
a
ri
l
y
e
c
o
n
o
rni
cs
l
n
syntheses
b
y
w l i i ch i t hasnot hasbe e nd e fi n e d
[1].
small quantities of rnaterialsfbr researchor laboratory use. leasibility and
co n ve niencar
e eus ual l ymo rei mp o rta n tth a n e n z y n rec ost:for exampl e.the fact
pos
s ible
but di ffl cul t
th a t i t is
t o joi n d e o x y ri b o n u c l e o ti dc eh a i n se nzymati cal l y.
W o r k s u p p o r t e db y ' N S I : ( R A N N ) u n d e r G r a n t N o . G I 3 4 2 8 4 .
901
9O2
L A R G FS C A L EE N Z Y M I C
SYNTHESIS
or impossibleto join them by conventional synthetic chenrical techniques,renders
the economics of the production and utilization of the appropriate ligasentoot
, y contrast,econonics
[ 2 ] . I n t h e s y n t h e s i so f l a r g e q u a n t i t i e s o f a s u b s t a n c e b
becomes an important consideration. and the factors defining the cost of a
reaction sequencerequiring cell-free enzymic catalysis production and isolation
of the enzyme, enzyme operating lifetime and specific activity, cofactor replacem e n t , r e a g e n t s ,s u p p o r t s ,a n d e q u i p m e n t p e c u l i a r t o e n z y m i c r e a c t o r s m a y b e
such that alternative synthetic procedures utilizing chen'ricaland/or rnicrobiological transformations may appearmore attractive than a cell-freeenzyrnic route.
The purpose of this chapter is to illustrate briefly examples or representative
reactions for the large-scalesynthesisor processingof organic materialsin which
cell-free enzymic synthesis presently plays a role, to discuss considerations
important to the successful design of new processes(particularly processes
i n v o l v i n g t r a n s f o r m a t i o n sm o r e c o m p l e x t h a n t h o s e n o w e m p l o y e d ) . a n d t o o u t line criteria useful in identifying situations in which cell-free enzynric catalysis
m i g h t o f f e r a d v a n t a g e so v e r o t h e r s y n t h e t i c s t r a t e g i e s C
. ertain of the considerat i o n s i m p o r t a n t i n u s i n g e n z y n t i c c a t a l y s i si n l a r g e - s c a l a
epplications particularl y t h o s e i n v o l v i n g t h e i s o l a t i o n a n d s t a b i l i z a t i o no f e n z y m e s a r e c e n t r a l r o t h r ' r r
use in synthesis at any scale,although the requirements for successfullarge-scalc
u t i l i z a t i o n a r e a l m o s t a l w a y s m o r e s t r i n g e n tt h a n t h o s e f o r s m a l l - s c a l er e a c r i r - r r r s :
others-cofactor regeneration,reactor design are unique to large-scaleproc..s'.':.
W e a r e c o n c e r n e dh e r e w i t h t h e q u e s t i o n o f t h e a p p h c a t i o n o f e n z r , ' n r erso r l r r .
synthesis of relatively small quantities of materials. this issue is discuss.'drrr
d e t a i l e l s e w h e r e i n t h i s b o o k ( P a r t I . C h a p t e r s I V a n d V I ) . R a r h c . r .r r c ' r i r l l
c o n s i d e r t h e d i s t i n c t p r o b l e m s t h a t m u s t b e s o l v e d i n s y n t l - r e s i z i nlga r . g ct l u l r l t i e s ( a r b i t r a r i l y d e f i n e d a s r a n g i n g f r o m o n e o r s e v e r a lm o l c s t t r u . n r r r r e r e i . i i
s c a l e ) o f a s u b s t a n c eu s i n g e n z y m i c c a t a l y s i s .I n c o r p o r a t i o n o t ' t h . . t c c h r l r r l r r i , :
r e q u i r e d t o s o l v e t h e s e p r o b l e m s i n t o t h e b o d y o f s y n t h e t i c o r s a n r cc h e n l r t r r
r e q u i r e s a b l e n d i n g o f c h e m i c a l p r o c e d u r e sw i t h t h o s e t ' r o r r .cr n z r n r o l o g r r n J
microbiology. These biological techniques are unfanriliar to nr()st s\n1lrr'rr.
organic chemists. Nonetheless,despite the fact that synthesis requiring cell-t-r....
e n z y m i c c a t a l y s i sm a y b e t e c h n i c a l l y m o r e c o m p l e x t h a n n r o r e f a n t i l i a rl 1 p t . s . l
c h e m i c a l o r f e r m e n t a t i o n s y n t h e s e st,h e s e l e c t i v i t yo t f e r e d b y e n z y m r c c a t a l l , s i s .
particularly for the many biologically, medicinally. and nutritionally inrporrlnr
c l a s s e so f m a t e r i a l sc o n t a i n i n g c a r b o h y d r a t e s p
, e p t i d e s .a n d n u c l e o t i d e s .c a l l n ( ) r .
in many cases,be equaled by conventional organic or 1-ermentationsynthesis.
2 SYNTHESES
U T I L I Z I N GC E L L - F R E EE N Z Y M E S :E X A M P L E S
Existingprocesses
thatutilizeenzymes
for the production
of discrete
chernical
compounds on a large scale have in common severalfeatures that sintplify thcir
operation: little or no enzyme purification is required; immobilization is
2 EXAMPLES 903
achieved simply using inexpensive supports; the reactions catalyzed are uncomp l i c a t e d [ 3 ] . R e a c t i o n s u t i l i z i n g L - a m i n o a c i d a c y l a s e ,p e n i c i l l i n a m i d a s e ,a n d
aspartase illustrate types of enzymic catalysis that are actually used in larges c a l ew o r k a n d t h a t p r o v i d e e x a m p l e so f t y p e s o f e n z y r n i ct r a n s f o r m a t i o n st h a t
c a n b e a p p l i e d r e l a t i v e l ye a s i l yi n c h e m i c a ls y n t h e s i s .
L-Amino acid acylase is used in a process for resolving racemic amino acids
[4] A racemic mixture of n- and L-rV-acylarninoacid (obtained by chemical
synthesis) is passed through a column containing enzyme inrmobilized by
a d s o r p t i o n o n D E A E c e l l u l o s e .T h e L - e n a n t i o r n e ri s h y d r o l y z e d t o f i e e a m i n o
acid. Separation of the D-acylarnino acid from the free L-ar.ninoacid is easily
a c c o r n p l i s h e da. n d t l i e D - a c y l a r n i n oa c i d i s s u b s e q u e n t l ye p i m e r i z e da n d r e t u r n ed to the column.
N H,^
Ac
I
L - RC
CHC
lO 2H
+
NHAc
I
D -R C H C O2 II
II
I
II
*N
Lamino acid
acylase
I
I
epimerization
I
I
Y
F{:
,
I
I Cto.
,
L- R
R.CH
NHAc
o-nlHco,H
Pe n i cillinam idas eis used to remove the side chain of penicillin G in certain
p ro ce dur esf or t he p r o d u c t i o n o f 6 - a m i n o p e n i c i l l a n i ca c i d ( 6 - A P A ) [ 5 ] . T h i s
o
ll
PIICH2CHN
-sr
\
r
c o 2H
penicillin
-_-------------amidase
I{ ,N
\r--r-S
r
{,,
"J-'r
6-APA
materialis in turn usedas a startingmaterialfor a varietyof semisynthetic penicillins. Aspartasecatalyzesthe addition of ammoniato fumarate ion, yielding
o p ti ca llyac t iv eL- as pa rtiac c i d [6 ,7 ].
904
L A R G E . S C A L EE N Z Y M I C S Y N T H E S I S
C0 2 - N H 4 *
H;N
-O2C
.'s'artasc
>
fo;
n
N[{o.
- o r cr4,
/ \nnr.
The most successfultechniqueused to immobilizeaspartase
is one that, although not formally "cell-free,"offersgreatpromisefor many typesof enzymecatalyzedsyntheses.
The enzymeis not isolated.Instead,an intact microorganism containingthe enzymeis incubatedwith acrylamideand appropriatecrosslinking agent and radical initiator, and the entire suspension
polymerized[6] .
The resultingpolyacrylamidegel containingimmobilized,polymer-filledcells,
is broken up, and transferredto a column. The resultingpreparationshows
g ood s t abilit yan d a c ti v i ty .(A i 0 X 1 0 0 -c mc o l urnn,contai ni ngpackedcel l sof
Escherichiacoli in polyacrylamide,
cangenerateabout 700 g of asparticacid per
hour) [7] .
An obviousadvantage
of this techniqueis its simplicity:no enzymeisolation
i s r equir ed.Les s o b v i o u sa d v a n ta g easl s o a p p arentl yattend i t: the enzyrre
i m m obiliz edby p o l y me ri z a ti o o
n f th e i n ta c t o rgani smi s very stabl e.It i s possi ble, alt houghno t e s ta b l i s h e dth, a t th i s s ta b i l i t yrefl ectspermeati onof the cel l
i nt er ior by ac r y l a m i d ew
. i th s u b s e q u e npt o l y m eri zati onto a pol yacry' l anri de
gel t hr oughoutt h e c e l l m a tri x . T h i s g e l rn u s th a veporessuffi ci entl ysnral lthat
a ny pr ot eas es
pr e s e nat t tl i e ti m e o f p o l y rn e ri z ati on,
or rel eased
by the pol ypteri z at ion,ar e ut r ab l eto a tta c kth e a s p a rta s e .land d i ti on,the envi ronnrent
provi ded
for the enzyme its naturalmatrix with a low concentrationof polyacrylarrricle
m ay dis c our age
co n fo n n a ti o n aclh a n g eth
s a t l o w eri ts acti vi ty.
T his pr oc edur ei.n i ts p re s e n fo
t rm , i s n o t a p p l i cabl to
e anumberof' cl asses
of
enzyme catalyzedreactions.Any processthat requiresdiffusionof enzyrlesor
o ther t nac r om o l e c u l e(e
s .g .,ri b o s o rrrapl ro te i n synthesi s)
w i l l be hi ndereclsr
p r ohibit edby t he p o l y m e rm a tri x . T h e c e l l w a l l of the rni croorgani snr
nri ghti n
p rinc ipleals or es tri c td i ffu s i c tno f c e rta i ns u b s t rates
or productstg or l l .r' rl re
i n t er ior of t he c e l l . T h e c o n d i ti o no f th e c e l l wal l i n thesepreparati ons
l s .()t
kn own. T he c at a l y ti ca c ti v i tyo f th e a s p a rta sper eparati on.
i s how ever.i ncreasc-cl
b y an ac t iv at iotpi ro c e d u rci n v o l v i n gi.n te r a l i a .h eati ng:thi s acti vati onnrayserve
to disrupt cell merrrbranes
sufficientlyto pernrit free passage
of substrates
alcl
p roduc t s A
. c r y la m i d ere a c tsra p i d l yw i th s e v e rafuncti
l
onalgroupsof enzyrl cs.
p ar t ic ular lyt he -SH rn o i e ty o f c y s te i n ea n d o ther nrercaptan-contai niprsl
ng ccu les ,and is it s e l f c a p a b l eo f mo d i ty i n go r d e stroyi ngthe acti vi ty of certai p
e l l z y n l e s o l l v c rsyh o r tc t l t l t a c t[ 8 ] . P o l y a c r , v . ' l a ngtei dl scI i a v cr.' c l a t i v e p
l yg o r 1 1 . * ,
a n d r t lec hat lic aclh a ra c te ri s ti cas n
, d a re n o t i d eal for processes
requi ri ngt he
p roc es s ing
of v er y l a rg ev o l u m e so f s o l u ti o n .F i n al l y.the acceptabi l i ty
ro rnan)'
o f t he wor ld' s f o o d a n d d ru g a d rn i n i s tra ti obno di esof materi al s.
i ntendecltgr
h u m anus e.pr ep a re db y a s te p i n v o l v i n gi n i rn o bi l i zed
w hol ecel l si s not enti rel l ,
3
NEW PROCESSES
905
that involverelativelysimplecatalytictransforfor processes
clear.Nonetheless,
the immobilizationof intact organsubstrates,
mation of low-molecular-weight
of utilizingenzymiccatalysis.
method
practical
and
attractive
ismsoffersa very
sufficientlydevelopedto be usedin comThesethree examplesare processes
alsoexist [9],
syntheses
synthesis.Many examplesof smaller-scale
rnercial-scale
ancl representativeexamples may be found in Metfutds in L'nzymc.tlogy,
BittchemicalSltntheses,and similarsources,aswell asin other partsof this volume.
3
CONSIDERATIONS IN
DEVELOPING
NEW
PROCESSES
CATALYSIS
EN Z Y M I C
U T IL I Z IN G C E L L - F R EE
in designingand developinga practiFive principalfactorsmust be considered
cal synthesisbasedon enzymic catalysis:(1) the availability,easeof isolation,
and purificationof the enzyme(s)required;(2) the lifetime of the enzyme(s)
under operatingconditionsand the efficacityof stabilizationprotocols:(3) the
intrinsic activity of the enzyrne(s),and the efficiency with which enzymic
activities can be coupled in reactionsrequiring multiple enzylxe-catalyzed
steps;(a) the cofactorsrequiredfor enzymic activity; and (5). the designof
We considereachof
reactorscompatiblewith the transformationto be achieved.
thesesubjectsin turn.
Enzyme lsolation
For synthetic
Enzymesare, in principle,availablefrorn all living orgarlislns.
wo rk requir inglar geq u a n ti ti e so f e n z y me sc, e rta i nty pesof sourcesarebetter
Y easts,bacteri a,
th a n o ther s .T he m os t c o n v e n i e nst o u rc e sa re m i c ro o rgani sms.
technif-ermentation
well-established
using
quantity
grown
in
be
and fungi can
yeast
and
Baker's
lines-particularly
used
comrnonly
certain
ques;alternatively,
ot'
trunlbers
quantities
large
of
Starter
available.
E. coli-are commercially
Collection.
Culture
Type
Arlerican
the
from
standardcultures are available
In addi ti on.
(Se ea l s oA ppendix1, P a rt I fo r a l i s t o f o th e rc u l tu r ecol l ecti ons.)
that
is partisources
plant
or
animal
over
offer an advantage
microorganisms
that
narnely
required;
are
enzyrnes
quantities
of
cularly valuablewhen large
strai
ns
to
devel
op
be
used
o
fte
n
g
e
n
e
ti
c
c
a
n
ma n i p u l a ti o n
se l e ct iv m
e ut at ionand
enzyme
and
i
nterest,
of
e
n
z
y
m
es
o
f
th
e
th a t p roduc er elat iv e l yl a rg eq u a n ti ti e s
than with higherorganinduction is easierto accomplishwith microorganisms
as
of rnicroorganisms enzymesourcesis that
isms.A final, significantadvantage
the conveniencewith whicii they can be handledlias.in the past.made them
enzyrnologyand biology.Thus,largequantities
favoritesubjectsfor mechanistic
of manyi nterestn n d p roperti es
e n th e p re p a ra ti o a
o f d a ta ar e alr eadyava i l a b l o
most commonl y
i n g e n z y m es .Unf or tu n a te l y ,s o m e o f th e mi c ro organi sms
particularly
Salmonella
exploited as enzyme sourcesfor mechanisticwork,
quantity
growth
in
typhimuriurn, are sufficiently pathogenicto make their
906
L A R G ES C A L EE N Z Y M I C
SYNTHESIS
i n c o n v e n i e n t o r h a z a r d o u s :a l t e r n a t i v es o u r c e sf o r t h e s e e n z v m e s c a n . h o w e v e r .
u s u a l l yb e f o u n d .
Plant and animal sources of enzymes are less convenient than rnicrobial
s o u r c e s ;t h e y m a y , h o w e v e r , o f f e r t h e o n l y s o u r c e o f e n z y m e so f i n t e r e s t .P l a n t
s o u r c e s ,i n p a r t i c u l a r , m a y b e c o m e i n c r e a s i n g l ya t t r a c t i v e a s t h e t e c h n i q u e sf o r
p l a n t t i s s u ec u l t u r e d e v e l o p [ 1 0 ]
I s o l a t i o n o f c n z y r n e sl o r l a r g e - s c a l se y n t h e t i c w o r k p r e s e n t se c o n o r l i c p r . o b i e n r s n o t e t r c o u n t e r e dr n s n i a l l - s c a l p
e r c p a r a t i o n .I n g c n e ' r a le. n z y n r e i s o l a t i o r r
p
u
r
i
f
i
c
a
t
i
o
n
and
i s a n e x p e n s i v ep r o c e s s .a n d t h e l e s sm a n i p u l a t i o n r e q u i r e d i n
r e a c h i n g a u s e a b l ep r e p a r a t i o n . t h e b e t t e r . T h e l e a s t c o m p l e x t e c h n i q u e c o n ceptually is the use of whole cells [6, I I 13]. Although this technique will
c e r t a i n l y b e w i d e l y c x p l o i t e d w h e n a p p l i c a b l e i. t i s n o t y e t c l e a rw h e t h e r i t c a r r
b e e m p l o y e d f o r o t h e r t h a n v e r y s i r t r p l et r a n s f o r n r a t i o n s .N e r t l c a s t c o n r p l e x
i s t o u s e t l t c c r u d e p r o t e i n m i x t u r e o b l a i n e d o n b r e a k i n gc e l l s l v i t h a n r r n i t t t u n r o f p u r i f i c a t i o n . T y p i c a l l y . a u t o l y s i s . l y s i s w i t h a n a d c l e d e n z \ , n r e. ( ) r
mechanicalor osmotic disruption of cellsyields a rnixture of cell contponents
c o n t a i n i n g , i n a d d i t i o n t o t h e e n z y m e ( s ) o f i n t e r e s t , v a r i o u s p r o t e a s c s .n u c l e i c
a c i d s .l i p i d s , c a r b o h y d r a t e s ,a n d p r o t e i n s t h a t r n a y b i n d o r d e g r a d et h e p r o d u c t s
o r s t a r t i n g r n a t e r i a l so f t h e r e a c t i o n o f i n t e r e s t , t h e e n z y l l e s r e q u i r e d t o e f 1 - e c t
t h e d e s i r e d t r a n s f o r m a t i o n s .o r e s s e n t i a cl o f a c t o r s ( S c h e m e I ) . O f t e n . h o w e v e r .
e i t h e r n o o r s u r p r i s i n g l yl i t t l e f u r t l i e r t r e a t n t e n t i s r e q u i r e d t o o b t a i n L r s e a b l e
p r e p a r a t i o n sT. h u s , i f t h e e n z y m e o f i n t e r e s ti s t h e r m a l l y s t a b l e .b r i e f h e a t i n g r n u y
d e n a t u r en t a n y o f t h e o t h e r p r o t e i n s p r e s e n t .E n z y r n e i r n r n o b i l i z a t i o nb y a d s o r p t i o n , e n t r a p m e n t , o r c o v a l e n t a t t a c h m e n t o f c r u d e p r e p a r a t i o n st o s u p p o r t sn r a v
d e c r e a s et h e r n o b i l i t y o f t l t e p r o t e a s e ss u f l - i c i e n t l yt h a t t h e 1 ,b e c o r n e u n i r r r p o r t a n t i n d e t e r t t i i n i n ge r t z y n r i cl i f c t i n r e s :a l t c r n a t i v e l y .a d d i t i o n o l ' p r o t c a s ci n l r i b i t o r s m a y d e c r e a s et h e i r a c t i v i t y ( s e e b e l o w ) . N u c l e i c a c i d s c a n b e r e r l o v e d b . v
precipitation (protamine sulfate, streptoniycin) or enzyntically degracled.
A m m o n i u r n s u l f a t e o r o r g a n i cs o l v e n t f r a c t i o n a t i o n s e r v e st o r e n r o v es o n t eo f - t h e
u n w a n t e d p r o t e i n s f r o m t h e c r u d e p r e p a r a t i o n .T h e s e a n d r e l a t e d e l e n r e n t a r y '
p u r i f i c a t i o n t e c h n i q u e sa r e d e s c r i b e di n a v a r i e t y o f s o u r c e s [ 1 4 . 1 5 ] . A i o n e .
t h e y m a y p r o v e i n s u f f i c i e n t o r u n s a t i s f a c t o r yi n m a n y c a s e s ; t h ei m p o r t a n t p i - r i n 1
I o r e a h z e ,h o w e v e r . i s t h a t i t i s n o t n e c e s s a r i l yr e q u i r e d ( o r e v e n d e s i r a b l e ) t o
p u r i f y a n e n z y m e t o h o m o g e n e i t y f o r i t t o b e u s e l ' u li n a s y n t h e t i c p r o c e d u r c .
a n d i t i s u s u a l l y w e l l w o r l h t h e e f f o r t t o e x a r n i n et h e a c t i v i t y a n d s t a b i h t y o f '
crude preparations.
E n z y m eS t a b i l i z a t i o n
T h e s t a b i l i z a t i o n o f e n z y r n e p r e p a r a t i o n si s a t o p i c o f c e n t r a l i n r p o r t a n c ei n
enzymic synthesis: even relatively inaccessibleand expensive enzylnes can. in
principle, be used in a practical synthesis provided the lifetime of the enzyrxe
u n d e r o p e r a t i n g c o n d i t i o n s i s l o n g . U n f o r t u n a t e l y , i n m o s t i n s t a n c e s ,l e s s i s
3
NEWPROCESSES
907
Cells
II
or osmoti c
m echani cal
n u to l y s i s ,l y s o z y me,
I
disruPtion
i
BrokenCells
Celldisruption
l*n
l_^z
Protease
inhibition
I
I
I
Precipitationor
hydrolysisof
nucleicacids
I
I
P h c l ' l r S o 2 FP: h N H C \
NH'
D N A seand
e to ta mi n es; tre p to rnyci n;
RNAse
V
CrudeProteinMixture I
lnitial separation
I
I
I
I
I
Catciumphosphategel precipitation;
a n d /o ra cetonefi acti onal
tN H 4 )2 SO4
p te c i p i ta ti o n s .
C ru d ePro te i nM i x tu reIl
I
and/or
ion exclrange,
Gel penrteation,
Y
i
urilnlty chrornatography
Further
purification
I
Pure E,nzyme
EnzynrcIsolationkorrdure
Scheme1. Representative
s f t h e p r o c e s s e sl e a d i n g t o l o s s e si n e n z y m i c a c t i v i t y
k n o w n a b o u t r n e c h a n i s r no
than about the mechanism of the enzymic activity itself. Consequently, enzyme
stabilization is presently as much art as science. It is possible to point to a
number of experimental protocols that are known to increasethe stability of
enzymes, and in some casesto rationalize these procedureson plausible mechani s t i c g r o u n d s . I n a n y n e w c a s e ,h o w e v e r , t h e p r o c e s so f d e v i s i n ga s e t o f e x p e r i mental conditions resulting in good operating lifespans for an enzyme remains
l a r g e l ye m p i r i c a l [ 1 6 ]
Proteases
A major contributor to the loss of enzymic activity in crude enzyme-containing solutions (and in many "pure" preparations) is destruction of the enzyme
by proteases.Proteasesoccur in a wide range of molecular weights. and operate
o v e r a r a n g e o f v a l u e so f p H [ 1 7 - 1 9 ] . T h e a b i l i t y o f p r o t e a s e st o p e r s i s tt h r o u g h
9OB
L A R G E - S C A L EE N Z Y M I C S Y N T H E S I S
purification procedures120-221has led to an
even careful chrornatographic
only partiallyjokhg feelingamongenzymologists
that for everyproteinthereis
an almostidenticallyconstitutedprotease.
Therearethreeeffectivestrategies
for
dealingwith proteasecontaminationof an enzytne.First, the proteasecan be
i nhibit edby add i ti o no f s u b s ta n c eth
s a t i rre v e rsi blbl
y ock or compl etel yi nhi bi t
the proteaseactivesites.Two broadly effectivecornpoundsfor this purposeare
PhCH2S O 2F[ 2.3 J(c a l l e dp h e n y l n re th y l s u l l onyl f' l uori dei n bi ochenri cal
ci r(DIFP). Both inhibit serineproteases
cles) and diisopropylphosphofluoridate
(e. g. ,t r y ps in.c h y mo try p s i n );th e fo rrn e ri s l e s stoxi c than the l atter.w hi ch i s
also a very powerful acetyl cholinesterase
inhibitor. Benzamidineand related
s l atedto thrornbi n[2a]. P rotei n
m at er ials arals
e ou s e fu li n i n h i b i ti n gp ro te a s ere
p r ot eas einhibito rs [2 5 ] a re to o s p e c i fi cto b e usefulfor broad-spectrurn
protease inhibition. Second,immobilizationof the enzyme preparationusualll'
render spr ot eas ea c ti v i ty u n i mp o rta n t,s i n c e t he sol uti on protei n-protei enn
c ount er st hat pe rm i t a tta c kb y th e p ro te a s easreuni mportantw i th i mntobi l i zed
e nz y m es[ 26] . Th i rd . c a re fu lc l tro rn a to g ra p h parti
y- cul arl y'affi ni ty chronratography m ay r edu c ep ro te a s a
e c ti v i tyto i n s i g n i flcant
l evcl s.
A u t o x i d a t r o n :O x i d a t i o n R e d u c t i o nB u ff e r i n g
M any enz y m e sre q u i re-S H o r -S -S-rn o i e ti e sa sessenti al
structuralor catah' ti c
elem ent st 8] . B o th c a n b e o x i d i z e dra p i d l y u nder condi ti onscommonl venc ount er edin eu z y m i c re a c to rs Al
. th o u g h th e detai l edrnechani snts
of tl tese
ox idat ionsar e n o t k n o w n , s e v e raol f th e i r q u a l i tati vecharacteri sti cs
havebeen
i d ent if ied.T ir us ,th e i n i ti a l o x i d a ti o no f c y s te i neto cysti neoccursnrorerapi tl l r
I(-SH
R-S
R_S{I
R-S
I
[.-,el
L.-AI+,
RSO.r
R S OI
a I h i g h t h a n a t l o w p H . i s s t r o n g l y c a t a l y z e db y t r a c e s o f t r a n s i t i o n r n e t a l s
I p a r t i c u l a r l y F e ( l l l ) a n d C u ( l l ) ] . a n d c a n b c r e v e r s e dw i t h s u i t a b l e r e d u c i n e
a g e n t s . T h e s u b s e q u e n to x i d a t i o n o f c y s t i n e d i s u l f i d e l i n k a g e s( u l t i m a t e l l ' t t r
c y s t e i n e s u l f o n i c a c i d ) i s u s u a l l y s l o w e r ,b u t i s n o t e a s i l y r e v e r s e d A
. utoxidatirrn
r n a y a l s od e s t r o y p r o t e i n sb y r o u t e sn o t i n v o l v i n gS H g r o u p s [ 2 ] 1 .
T h e s i g n i f i c a n c eo f t h e s e o b s e r v a t i o n sf b r e n z y n r e st h a t a r e a c t i v ei n t h e f u l l y
reduced fomt (i.e., with functional cysteinesbut without intporlant cystine
g r o u p s ) i s c l e a r : t h e e n z y m e s h o u l d b e s t o r e d a n d u s e d u n d e r a n a e r o b i cc o n c l i tions (a nitrogen or argon atrnosphere); the reagents used shoulcl be f'rec of
transition-metalcontaminants: a suitable reducing agent should be present in
t h e s o l u t i o n t o r e v e r s ea n y a d v e n t i t i o u so x i d a t i o n . T h e f i r s t c o n d i t i o n i s d i t ' t l c u l t
3 N E WP R O C E S S E S9 0 9
to satisfy adequately:I ltl of air may contain enoughoxygento oxidize the
cysteinemoietiesof I mg of protein. The secondis also difficult to meet.
Although buffers and reagentsmay be reasonablystrippedof transition-metal
ions by passingthem through a resin containingSH groupsbefore use, the
ions
enzymeitself usuallycannotbe. Sincemany proteinsbind transition-rnetal
strongly,it is often impracticalto try to keepsolutionscontainingenzymesfree
of transition metals.Thus, the major burden of protectrngenzymesagainst
deactivationby autoxidationusually falls on the reducingagentaddedto the
so l u ti o n.
The most popular and effective material for this purposeis dithiothreitol
(Clelland'sreagent,DTT), ordinarily added in concentrationsapproximately
1000 times greaterthan that of the enzyme [28] DTT is unfortunatelyan
is
but lesseffectiveZ-mercaptoethanol
expensivematerial,and the lessexpensive
often usedin its place.
en./sl
\s'
+ Hr^)a
HSI._
*rH
OFI
SH
/
...........--L.
Enr
,,n
ntY
OH
\s -s.--..r[..on
DTT
/t
//
SH
S'^\r,'
Enz
\
+
SH
orl
Jlnn
The stabilization of enzymes that require an intact cystine disulfide linkage
against oxidation is a problerr that has not been satisfactorily solved. The
p r e s e n c ei n s o l u t i o n o f a l a r g ee x c e s so f a n a d d e d m a t e r i a l c o n t a i n i n g a d i s u l f i d e
l isulfide in a protein. but the irreversibility
l i n k a g e s h o u l d s t a b i l i z ea n e s s e n t i ad
past
the
disulfide oxidation level presents obvious
of oxidation proceeding
problems. The coupling of two or rnore enzyme systems operating at different
o x i d a t i o n l e v e l sh a s n o t b e e n a t t e m o t e d .
E n v i r on r n e n t
are capableof influencingthe stabilityot'
A varietyof solutionvariables
enzylnes:pH. temperature;ionic strength,buffer composition.solventcharacter
(a n d i m m obiliz at ions u p p o rt,fo r i mmo b i l i z e de n z ymes).the natureof i nterof other sol uti on
fa ce sp r es entin s olut i o n ,a n d th e n a tu re a n d c o n c entrati on
p
re
s
u
m
e
d
to
i
nfl
uenci ngthe tera
re
act
by
M
os
t
of
th
e
s
e
v
a
ri
a
b
l
e
s
co mp o nent s .
concerningrnostof theseintertiary structureof the enzyme.No generalizations
910
L A R G ES C A L EE N Z Y M I C
SYNTHESIS
actions are presently possible: indeed, no generalizationsmay exist, since the
relative importance of various factors will differ for different proteins. It is,
however, worthwhile to question one assumption that is commonly tacit in
much experimental work, namely, that most enzymes exist in the cell in a
predominantly aqueous environment, and that, once removed f;om the cell,
e n z y m e s a r e l i k e l y t o b e r n o s t s t a b l ei n w a t e r . T h e r e i s l i t t l e q u e s t i o n t h a t f o r
enzymes that are clearly associatedwith membranes, the presenceof lipids in
s o l u t i o n m a y b e e s s e n t i a lf o r s t a b i l i t y a n d a c t i v i t y 1 2 9 1. A t a n o t h e r e x t r e m e ,
extracellular enzymes probably are most stable in water. For the interr-nediate
c l a s so f e n z y m e s t h a t e x i s t i n t h e i n t e r i o r o f t h e c e l l , i t i s b y n o m e a n s c l e a r
what solution composition would be expected most closelyto duplicate their
native environment. The fluid portions of cells may contain high concentrations
o f m a n y c o m p o n e n t so t h e r t h a n w a t e r , a n d d u p l i c a t i o n i n v i t r o o f t h e c e l l u l a r
microenvironmerrt of an enzyme, even in the uncommon event in which the
p h y s i c a l l o c a t i o n o f t h e e n z y m e w i t h i n t h e c e l l i s k n o w n . c a n n o t p r e s e n t l yb e
carried out by design. Thus. many of the standard empirical tricks used to
stabilize enzymes in solution may be effective by virtue of providing the type of'
m i x e d a q u e o u s - n o n a q u e o u se n v i r o n m e n t f o u n d i n t h e c e l l . I n f a c t . i t s e e n ) s
p o s s i b l et h a t i n v e s t i g a t i o no f t h e b a l a n c eo f h y d r o p h i l i c a n d l i p o p h i l i c c h a r a c t e r
that is most effective in stabilizing an enzylne rnight provide an rndirect
a p p r o a c ht o i n f - e r r i n gt h e c h a r a c t e ro f t h e c e l l i n t e r i o r .
B r i e f l i s t i n g a n d c o m m e n t o n s e v e r a lo f t h e s e t r i c k s i s w o r t h w h i l e . E n z y r n e s
may denature at interfaces [30] lnterfacial denaturation may often be controlled by allowing an inexpensive. inert protein (cornmonly bovine serun'l
a l b u m i n ) t o a d s o r b o n t h e s u r f a c eo f g l a s w a r e t o b e u s e d w i t h e n z y m e s o r b y
using polyethylene or Teflon rather than glass apparatus. Since interf-acial
d e n a t u r a t i o n o f s u s c e p t i b l ee n z y m e s m a y a l s o o c c u r a t t h e s o l u t i o n - a i r( o r i n e r t
gas) interface, gasesshould not be bubbled through an enzyme solution. nor
s h o u l d s t i r r i n g b e s o v i g o r o u sa s t o c a u s es t r o n g v o r t e x f o r m a t i o n . l n c o n t r o l h n g
t h e p H a n d i o n i c s t r e n g t ho f e n z y m e s o l u t i o n s ,i t i s i m p o r t a n t t o r e m e m b e r t h a t
b u f f e r c o m p o n e n t s [ 3 1 ] a n d s a l t s u s e d t o a d j u s t i o n i c s t r e n g t h i n f l u e n c em a n y
solution properties other than those of immediate concern. Thus, solutions
c o n t a i n i n g , f o r e x a m p l e , p h o s p h a t eb u f f e r o r t r i s b u f f e r o f t h e s a m e p H d o n o t
h a v e t h e s a m e v a l u e s o f s u r f a c e t e n s i o n s , i n t e r n a l p r e s s u r e s ,o r m a n y o t h e r
parameters that might influence the conformations of enzymes. The subject of'
hydrophobic interactions involving proteins is discussed in greater detail in
Chapter VIII of this volume and elsewhere[32-361. Addition of glycerol, sorbitol.
polyvinyl alcohol, or other neutral, hydrophilic polymers (often in high concent r a t i o n s ) . s t a b i l i z e sm a n y e n z y m e s [ 3 7 - 3 9 1 . I n a t l e a s t o n e i n s t a n c e , t h i ss t a b i l i zation has been correlated with a changein tertiary structure [39] . Polyelectrolytes may also strongly influence enzyme stability and activity [a0] . The
influence of detergents on enzyme stability has not been extensively investi-
3 N E WP R O C E S S E S 9 11
g a t e d . a n c lj u d i c i o u s c h o i c e o f s u r f ' a c ea c t i v e a g e n t sw i l l p r o b a b l y p r o v i d e s i g n i l - i c a n t s t a b i l i z a t i o n f c l r e n z y m e s r t o t c l a s s i f i e da s " m e i l t b r a l t e b o u t r c l " b u t s t i l l
a s s o c i a t e dw e a k l y w i t h r n e r n b r a n e s .l i p i d s , o r h y d r o p h o b i c p o r t i o n s o f o t h e r
p r o t e i n s i n v i v o . S t u d i e so f t h e s e n s i t i v i t y o f t h e a c t i v i t y o f p - a l a n i n e c a r b o x y p e p t i d a s et o d e t e r g e n t s t r u c t u r e s u g g e s t st h a t c o n s i d e r a b l ec a r e m a y b e r e q u i r e d
to achieve the best balance of hydrophilic and lipophilic interactions and
"structure-making" and "structure-breaking" properties fbr such stabilizatitln
l4t ^421.
T h e s t o r a g es t a b i l i t y o f s o l u t i o n so f n r a n y e n z y n r e si s i n c r e a s e db y t l i t ' p r e s c n c c o t ' s u b s t r a t c s .c o f a c t o r s . o r p r o d u c t s . a n d b o t h s t a b i l i t y a n d a c t i v i t y o f
enzymes may require rnetal ions [43] The fornrer characteristic rnay reflect
either stabilization of tertiary structure or protectiorr of the activesite region
f r c t n i a t t a c k b y o x y g e n . r n e t a l i o n s , o r o t h e r s o l u t i o n c o l n p o n e n t s .T l i e f r e q u e n t
r e q u i r e r n e n t f b r s i g n i f i c a n t c o n c e n t r a t i o n so f f r e e M g ( l l ) o r o t h e r r n a i n - g r o u p
r n e t a l sf o r a c t i v i t l , p l a c e ss o m e l i r n i t a t i o n o n t h e e x t e n t t o w h i c h t h e a c t i v i t y o t t r a n s i t i o n r n e t a l i o n s c a n b e s u p p r e s s e db y a d d i n g c h e l a t i n g a g e n t s s u c h a s
E D T A . T h e a s s o c i a t i o nc o n s t a n t sb e t w e e n E D T A a n d C u ( l l ) a n d M g ( l l ) a r e
a b o u t 1 0 r e a n d l 0 e r r o l / l i t e r . r e s p e c t i v e l y[ 4 4 ] T h u s . i f t h e o r i g i n a lc o n c e n t r a t i o n o f C u ( l l ) i n a n e n z y m e s o l u t i o n i s 1 0 - a, 4 / . a d d i t i o n o f e n o u g h E D T A t o
r e d u c e t h e c o n c e n t r a t i o no f u n c o n r p l e x c dC L r ( l l )t o 1 0 - 1 2X I r . v i l hl a v ev er y l i t t l c
=
i n f l u e n c eo n t h e c o n c e n t r a t i o no f f r e e M g ( l l ) [ ( M g ( l l . l ) / ( M g ( l l ) ' E D T A ) l 0 r l .
A d d i t i o n o f e n o u g h m o r e E D T A t o r e d u c et l t e C i u ( l l ) c o n c e n t r a t i o nt o 1 0 - 1 4t U
w i l l . h o w e v e r . s i g n i f i c a n t l yp e r t u r b t h e c o n c e n t r a t i o n o f f r e e M g ( l l ) [ ( M g ( l l ) ) /
(Mg(lI).EDTA) = ll. Thus. there is a lower limit belowwliich it is not practical
t o r e d u c e t h e c o n c e n t r a t i o n o f t r a n s i t i o n - m e t a li o n s i n s o l u t i o n b y c h e l a t i o n .
Although this lirrrit is low. catalysisof autoxidation of enzyt-nesullhydryl groups
b y t h e s e n r e t a l s n r i g h t , i n p r i n c i p l e , s t i l l b e s i g n i t - i c a n t .F u r t h e r . t h e r t t e t a l EDTA cotnplexes lnay be autoxidation catalysts itt their own right. Thus.
a l t h o u g h a d d i t i o n o f c h e l a t i n ga g e n t ss h o u l d ( a n d d o e s ) g r e a t l y d e c r e a s et h e r a t e
o f r n e t a l - c a t a l y z e da u t o x i d a t i o n . i t m a y n o t c o r t r p l e t e l ye l i m i n a t ei t . l n f a c t . t h e
u s e o f E D T A i n s o l u t i o n sc o n t a i n i n gD T T m a y b e s u p e r f l u o u s .s i n c e t h e l a t t e r i s
p r o b a b l y a v e r y s t r o n g c h e l a t i n ga g e n ti n i t s o w n r i g h t .
E n z y m i cA c t i v i t y
T h e a c t i v i t y o f a n e n z y t n e t o b e u s e d i n a s y r t t h e t i c r e a c t i o n d e t e r m i n e st h e
amount of enzyme required to achievesynthesisof a given amount of product in
a set tirne. For calibration, I mg of an enzyme having specific activity of about
700 international units (1. U.) will catalyze the formation of I mole of product
per day, when operating at maximum velocity (a specific activity of I I. U. = I
trrnrolsubstrate transformed/(min) (mg) of protein at V^^*) [45] The specific
a c t i v i t y o f e n z y m e s v a r i e s w i d e l y . T a b l e 7 . 1 l i s t s s p e c i f i c a c t i v i t i e so f e n z y m e s
catalyzing reactions representative of those that might be useful in organic
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916
L A R G ES C A L EE N Z Y M I C
SYNTHESIS
synthesis. Many of these activities are for impure enzymes. and activities for
pure enzymes may vary widely depending on the source. Moreover, severalof
these transformations may involve more than one step. These data should thus
be considered only as lower limits. Nonetheless, the total range of specific
activities shown covers almost lAe. Enzymes are known that are appreciably
s l o w e r t h a n t h e s l o w e s t l i s t e d . l t i s d i f f i c u l t t o d r a w g e n e r a l i z a t i o n cs o n c e r n i n g
which types of transformations are likely to be fast and which slow. Proton
transfer, hydration and dehydration, and phosphate transfer are often fast;
individual steps in the biosynthesis of complex molecules may be very slow.
C l e a r l y , t h e p r a c t i c a l i t y o f a n y s c h e m ef o r s y n t h e s i z i n ga p p r e c i a b l eq u a n t i t i e so f
a substance using enzymatic catalysis rrust take into consideration the activity
of the enzyme'. enzyme-catalyzed reactions are. of course. faster than uncatalyzedreactions,but they may still be very slow.
Cofactor Requ irements
T h e r e q u i r e r n e n t o f r n a n v b i o s y n t h e t i c r e a c t i o n s f o r c o f a c t o r s p l a c e si r n p o r ' t a n t e c o n o r l i c r e s t r i c t i o n so n t h e u s e o f t h e s er e a c t i o n sf u r l a r g e - s c a l e
sl ntheses
(Chapter I of Part I of this book outlines the functionsof most of the comrnon
c o f a c t o r s ) . A l t h o u g h c e r t a i n c o f a c t o r s ( C o A . t h i a r n i n e p y r o p h o s p h a t e .b i o t i n .
tetrahydrofolic acid, 812) act as true catalysts-that is, they are regenerated
u n c h a n g e da t t h e c o n c l u s i o n o f t h e r e a c t i o n si n w h i c h t h e y a r e i n v o l v e d o t h e r s
(NAD*, NADP.. nucleoside triphosphates, flavins, pyridoxal phosphate) are involved as stoichiometric reagents. All of these cofactors are expensivr'. For
small-scale syntheses in whicli the costs of reagents is not a major col.)ccnt.
c o f a c t o r sc a n b e a d d e d i n q u a n t i t i e ss t o i c h i o m e t r i c a l l ye q u i v a l e n tt o t h o s e o l ' t h e
s t a r t i n g m a t e r i a l s , a n d c o n s i d e r e d t o b e c o n s u m a b l e r e a g e n t s .F o r l a r s e - s c a l e
s y n t h e s i s i. t w i l l b e n e c e s s a r yt o r e c y c l et h e c o f a c t o r st o a c h i e v ea n e c o l ' l o l t r i c a l l v
a c c e p t a b l ec o s t .
P r o c e s s e sf o r c a t a l y t i c r e g e n e r a t i o n o f t w o c l a s s e so f c o l a c t o r s . n u c l e o s i d e
t r i p h o s p h a t e s a r t d N A D ( P ) H d e r i v a t i v e s ,h a v e b e e n d e v e l o p e dt o t h e S t u g eo t '
s u c c e s s f u l a b o r a t o r y d e m o n s t r a t i o n s .T h e m o s t i m p o r t a n t o f t h e f l r s t o t ' t h c s e
c l a s s e si s A T P . w h i c h i s i n s o m e s e n s et h e b i o c h e m i c a l e q u i v a l e n t o f D C C ' t r r
t o s y l c h l o r i d e , t h a t i s . p h o s p h a t e o r a d e n y l a t e t r a n s f - e rf i o m A T P t o o x l ' s e r r
c e n t e r s s e r v e st o a c t i v a t c o x y g e n a s a l e a v i n g g r o u p [ - 5 1 I F o r e x a n t p l e .c a t b o x y l g r o u p a c t i v a t i o nc a n p r o c e e d b y e i t h e r p a t h [ 5 1 . 5 2 ] . a n d a v e r y ,w
' idc'
r a n g e o f i m p o r t a n t s u b s t a n c e s - p r o t e i n s ,c a r b o h y d r a t e s .n u c l e o t i d e s . r r u c l e i c
a c i d s , t e r p e n e s ,a n d o t h e r s - r e q u i r e A T P a t o n e o r s e v e r a sl t a g e si n t h e i r b i o s v n thesis. NAD./NADH and NADP./NADPH are similarly widely used as hyciride
donors and hydride acceptors.
A u s e f u l s c h e m e f b r A T P r e g e n e r a t i o nm u s t c o m b i n e t w o f e a t u r e s : i t m u s t b e
a b l e t o a c c e p t e i t h e r A D P o r A M P a s t h e s t a r t i n g r n a t e r i a l ,s i n c e e i t h e r o r b o t h
m a y b e g e n e r a t e di n a p a r t i c u l a r b i o s y n t h e s i s a
. nd it must utilize an inexpensive
NEWPROCESSES 917
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tl
o-ofor ATP
agent.The most highly developedschemes
asphospliorylating
substance
regenerationare basedon the coupled activity of adenylatekinase [53] and
anotherkinaseutilizing the ultimatephosphatedonor assubstrate.In one regend reacti onof ketenew i th
e ra ti ons c hem e.X - P is a c e ty lp h o s p h a teg. e n e ra te by
AMP + arp {*q
I kI"*^
2ADP + 2X-P
IADP
2ATP + 2X
p h o s p h o r iac c i d [ 5 4 ] , a n d X k i n a s ei s a c e t a t ek i n a s e[ 5 5 ] ; i n t h e s e c o n dX - P i s
ca rb a m y lphos phat e .p ro d u c e db y re a c ti o nb e tw e enpotassi umcyanateand
p h o sp hat e.
her e.c ar ba m ypl h o s p h o k i n a si seu s e da sc atal yst[56] .The esti rnated
by a routc basedon acetyl
A T P (i n v e ry l a rg ec l u a n ti ti e s)
co st of r egener at ing
p h o sp hat e
is les st han S1 .0 0p e r p o u n d [5 7 ] ;d e ta i l e deconomi cesti matesbased
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o n c a r b a r n y l p h o s p h a t eh a v e n o t b e e n m a d e . S i m i l a r p r o c e s s e cs o u l d b e u s e d t t l
r e g e n e r a t en u c l e o t i d e t r i p h o s p h a t e s o t h e r t h a n A T P . o r A T P c o u l d b e u s e d .
t o g e t h e r w i t h a p p r o p r i a t ep h o s p h o t r a n s f e r a s etso. e f t e c t t h e s ep h o s p h o r y l a t i o n s .
918
L A R G E S C A L E E N Z Y M I CS Y N T H E S I S
XDP+ATP+XTP+ADP
Schemes
for regeneration
of cofactorsin the NADPH (NADH) seriesrely on an
alcoholdehydrogenase
to catalyzehydrogentransfer[58]
o
H
H2
-/\'\*/
J.CH3CH2OH
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M a n y p r o b l e m s ,n o t t h e l e a s to f w h i c h i s t h a t o f c o u p l i n g t h e c o f a c t o r r e g e n e r ation step to the enzymic synthesisstep(s), rernain to be solved before processes
for regenerating ATP and NAD(P)H derivatives can be used directly for largescale synthesis. Nonetheless, many of the fundarnental uncertainties in tl-rese
s c h e m e sh a v e b e e n r e s o l v e d , a n d i t s e e n l s c l e a r t h a t t h e s e c o f a c t o r s c a n b e
e c o n o m i c a l l y r e g e n e r a t e di n q u a n t i t y , i f r e q u i r e d f o r a l a r g e - s c a l es y n t h e s i s N
. o
a t t e n t i o n h a s b e e n d e v o t e d t o t h e p r o b l e m so f r e g e n e r a t i n go t h e r c o f a c t o r s .a n d
the practicality of these regenerationsremains to be demonstrated.
R e a c t o r D e s i g n[ 5 9 ]
Enzymesto be usedas catalystsin biosyntheticreactorswill be expensive.A
biosyntheticreactor using cell-freeenzymesas catalystsshouldbe designedto
conserveenzymrc activity: specifically,a continuous processshould retain
enzyrnicactivity in the synthetic reactor with high efficiency, and a batcfi
p roc es s houldpe rm i t e a s ys e p a ra ti oonf p ro d u c tsfrom catal ysts.
In ei thertype.
the r eac t ors hou l db e d e s i g n e to
d p e rm i t ma x i mumstabi l i zati on
and operati ng
lifetime of the enzymes.Two strategies
are availablefor designof biosynthetic
reactors:eitherthe enzymes
may be irnmoblllzedon an insolublesupport,or tlrel'
may be usedin solution,and reisolatedfrom products(by ultrafiltration,nricroe n c aps ulat ion,
or p o s s i b l yb y p re c i p i ta ti o n ).
O f t he tw o, i rnmobi l i zati on
seepts
the m or e ef f ec t iv ea p p ro a c hw h e na p p l i c a b l e :n ot
onl y areprobl emsof i sol ati on
minimizedby immobilization,but imrnobilizedenzyrnesautomaticallyenjoy r
high degreeof protection againsthydrolysisby proteasesand are subjectecl
neither to the potentialfor shearand surfacedegradationpresentduring ultrafiltration, nor to the rigors of precipitationor microcapsuleformation.The
subjectof enzymeimmobilizationis discussed
in ChapterIX of this volume;here
it is worthwhile to make only a few generalpoints about the characteristics
3 N E W P R O C E S S E S9 1 9
r e q u i r e d o f a s u p p o r t f o r i m m o b i l i z a t i o n s f o r l a r g e - s c a lsey n t h e t i c r e a c t o r s .T h e
support should present a balance of hydrophilic and lipophilic characterthat
perrnitsmaximum stabilizationof the active confbrmation of the enzyme: its
f u n c t i o n a l i z a t i o ns l i o u l d b e s t r a i g h t f o r w a r d .a n d t h e c o u p l i n g o f t h e e n z y m e t o
t h e s u p p o r t s h o u l d i d e a l l y p r e s e r v et h e c h a r g et y p e o f t h e a m i n o a c i d i n v o l v e d
( u s u a i l y t h e 7 - a r n i n og r o u p o f l y s i n e ) a n d p r o d u c e a p e r m a n e n tl i n k b e t w e e n
e n z y m e a n d s u p p o r t ; t h e s u p p o r t s h o u l d h a v e g o o d m e c h a n i c a ls t r e n g t h a n d
t l o w c h a r a c t e r i s t i c sf o r u s e i n l a r g e c o l u m n s a n d s t i r r e d o r f l u i d i z e d r e a c t o r s : i t
s h g u l d p e r r n i t r a p i d a c c e s so f s t a r t i n g m a t e r i a l s t o t h e e n z y l r e . a n d r a p i d d i f f u s i o n o f t h e p r o d u c t f r o n t t h e e n z y r n e : i t s h o u l d n o t b e s u b j e c tt o t n i c r o b i o l o g i c a l
d e g r a d a t i o n ,a n d , f o r c e r t a i n a p p l i c a t i o n s .s h o u l d t o l e r a t e s t e r i l i z a t i o n :i t s h o u l d
be inexpensive.
T h e s e c h a r a c t e r i s t i c sa r e . o f c o u r s e , a l s o d e s i r a b l ef o r s u p p o r t s t o b e u s e d f o r
i r n r n o b i l i z a t i o n so n a s n t a l l s c a l e . l t s e e m s r n o s t u n l i k e l y t h a t a s i n g l e s u p p o r t
w i l l e v e r s e r v ee v e r y n e e d a d e q u a t e l y ,a n d i t i s d i f f l c u l t t o j u d g e a p r i o r i t h e b e s t
s u p p o r t l b r a n e w a p p l i c a t i o n . N o n e t h e l e s s .c e r t a i n d i f t e r e n c e sb e t w e e n l a r g e a n d s m a l l - s c a l ep r o c e s s e sa r e c o m m o n . F o r o n e . t h e t r a t u r e o f l a r g e - s c a l ep r t l c e s s e sp l a c e sa g r e a t e rp r e n r i u m o n t h e m e c h a n i c a la n d f l o w c h a r a o t e r i s t i c os f t h e
s u p p o r t . T h e s e c o n s i d e r a t i o n sa l o n e a r e p r o b a b l y s u f f l c i e n t t o e x c l u d e t h e c r o s s e ork from wide
l i n k e d a g a r o s ea n d d e x t r a n g e l s c o m m o n l y u s e d i n s n t a l l - s c a l w
p r o c e s s e sS. e c o n d . s i n c e t h e e n z y m e p r e p a r a t i o n su s e d
applicationin large-scale
i n l a r g e - s c a l eb i o s y n t h e s i sw i l l p r o b a b l y b e l e s s p u r e t h a n t h o s e i n s r n a l l e rs y n t h e s e s .t h e c a p a c i t y o f t h e s u p p o r t i s o f g r e a t e ri n r p o r t a n c e i n t h e f o r m e r t h a n
t h e i a t t e r . S i n c e t h e a c t i v i t y p e r u n i t o f ' r e a c t o r v o l u t n e t l - r a tc a n b e a c h i e v e dw i t h
a n y e n z y t n e p r e p a r a t i o nw i l l d e p e n d u p o n t h e s u p p o r t c a p a c i t y ,a n d s i n c eb o t h
t h e e x p e n s eo 1 -t h e r e a c t o r a n d i t s o p e r a t i n gc h a r a c t e r i s t i c (sp a r t i c u l a r l y p r e s s u r e
d r o p a c r o s s l a r g e c o l u r r r n s )d e p e n d s u p o n t h e s i z e r e q u i r e d . t h e r e i s a s t r o n g
e c o n o r l i c i n c e n t i v e t o w o r k w i t h h i g h - c a p a c i t ys u p p o r t s . F i n a l l y . s i n c e t h e l i f e t i m e o f t h e e n z y m e u u d e r o p e r a t i n g c o n d i t i o n s i s c e n t r a l t o l a r g e - s c a l ep r o c e s s e ss. e l e c t i o no f a s u p p o r t a n d c o u p l i n g p r o c e d u r et h a t r n a x i n t i z e st h i s p a r a r n e t e ri s i r t t p o r t a n t .
T h e r e i s p r e s e n t l y n o c o l n m o n a g r e e m e n tc o n c e r n i n g t h e b e s t s u p p o r t s f b r
large-scaleinrrnobilizations. In the relatively rare applications in which pliysical
a d s o r p t i o n o n a n i n e x p e n s i v e .h i g h - s u r f a c ea r e a i n o r g a n i c ( d i a t o m a c e o u se a r t h )
o r o r g a n i c ( D E A E c e l l u l o s e )s u p p o r t i s s u c c e s s f u lt.h i s p r o c e d u r e w i l l p r o b a b l y
b e d i f f i c u l t t o b e a t . I n t h e m o r e f r e q u e n t a p p l i c a t i o n si n w h i c h t h e e n z y m e
must be rnanipulated more carefully. organic polyrner supports derived frorn
vinyl ntonomers seem likely to prove rnost useful. Supports derived frotn vinyl
p o l y m e r i z a t i o n h a v e t h e c o m b i n e d a d v a n t a g e so f r e l a t i v e l yl o w c o s t a n d g r e a t
llexibility in properties. Thus. polyacrylamide crosslinkedwith,A/,rv'-methylenebisacrylamide provides a hydrophilic polymer with fair but not outstanding
rlechanical properties. Inclusion of rnore hydrophobic rnonomers (2-hydroxy-
92O
L A B G E . S C A L EE N Z Y M I C S Y N T H E S I S
ethyl methacrylate,glycidyl methacrylate,methyl methacrylate),or cross
o1
O\
\H{H.,
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J
l i nk ing wit h nr o reh y d ro p h o b i cm a te ri a l s(h e x anrethyl ene
,V ,M-bi sacryl arni de)
properti es,
m ay pr ov ideir np ro v e ds tru c tu ra a
l n d n re c h a n i cal
al thoughpossi bl y
w it h s om edec r e a sien s u i ta b i l i tyo f th e re s u l ti ngpol yrri ermatri x as a support.
Coupling of the enzyme to the gel can be accornplished
either by inclLrding
appropriaternonomers(glycidyl methacrylate.rV-acryloxysuccinirnide)
in the
i n it ial poly m er i z a ti o no r b y fu n c ti o n a l i z a tionafter pol yrneri zati on.S i nce
acrylic acid, rnethacrylicacid. and acrylarnideare all readily availableand
su bjec t o f ac iled e ri v a ti z a ti oonf a w i d e ra n g eo f types,i t shoul dbe possi bl eto
tailor the resultingpolymersto have those propertiesmost appropriatelbr a
par t ic ularenz y m e .
Of the other n-raterials
commonly consideredas potentialsupportslbr largc'sc aler eac t or son
, l y th e c o n tro l l e d -p o re
recentl yi ntroducedby C orni ng
c e ra m i cs
seem likely to be useful [60] . Controlled-poreglassesfrom the samesourcc
e b e u s e fu l[6 0 . 6 1 ].
se emt oo ex pens i v to
A L T E R N A T IV E S A N D P R O S P E C V
T IE S
E x p e r i m e n t a lp r o c e d u r e sf b r e n z y n r ei s o l a t i o n .s t a b i l i z a t i o na, n d i n r n r o b i l i z u t i o n a r e s u f f i c i e n t l y a d v a n c e dt h a t i t i s p r a c t i c a lt o c o n s i d e rc a t a l y s i sb y c e l l - t ' r e e
e n z y m e s a s a n i n t e g r a l , i f r e l a t i v e l y u n e x p l o i t e d . t e c h n i q u ei n i n t e r m e d i a t e -a n d
large-scaleorganic synthesis. Given the availability of the technique, it is interesting to ask in what types of reactions enzymic catalysis rnight be useful. This
a r e a r e q u i r e s a s o p h i s t i c a t e db l e n d i n g o f t e c h n i q u e s f r o r r i o r g a n i c c h e m i s t r y ,
rricrobiology, enzymology, and polymer chemistry. Its technical cornplexity is
sucir that it will only be useful when cornpetitive"standard" synthetic tech-
4 A L T E R N A T I V EASN D P R O S P E C T I V E S9 2 1
niques, conventional organic Synthesis and fermentation, are unsatisfactory'
It is, however,
Organic synthetic procedure has the virtue of great flexibility.
s
e
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h
e
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e
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i
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n o t a b l y u n s u c c e s s f u lw h e n a p p l i e d t o
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that
materials
for
Lrlatio' of sintilar functionalities, and particularly
of
carbohysynthesis
and
in water and related solvents. Thus, the modification
accomeasily
is
seldom
drates. nucleotides, and certain types of polypeptides
for
developed
plished by organic synthetic techniques. Further, the procedures
and
synthesizing
easily
low-molecular-weight substances are not adequate for
macromolecules'
characterizing proteins, nucleic acids, and other biological
of problems'
types
Femrentation often offers a very successfulapproach to these
FermentaIt is difficult to improve on a successfulfermentation for simplicity'
slow and
a
tiol suff-ers.however, from disadvantagesof its own. It is intrinsically
f-ermentainflexible technique: the starting materials that are acceptable for a
the tnetabtion. ancl tl'reproducts of the fermentation,are relativelyfixed by
particular
o l i s r r - ro f t h e m i c r o o r g a n i s r n .l t i s s o m e t i m e s d i f f i c u l t t o i s o l a t e a
transformation of interest from the rnyriad other reactions usually taking place in
the cell. and. in consequence,improving a f'ennentation yield nray be a very
difficult task. Fermentation is also intrinsically inefficient. in the setrscthat the
particular material of interest in a fermentation is usually only a small fraction
of the cell's synthetic output. Particularly when fermentation yields are low.
the disposal of fermentation resicluesmay present a significant problertr. and
other aspectsof fermentation processespresent environmental problerns.
Enzymic synthesis offers a colnpromise between fermentation and chenlical
synthesis having distinct potential advantages.It affords higher selectivity than
chemical synthesis, but is lnore amenable to control and modification than
f e r m e n t a t i o n . I t p e r m i t s s i n g l e r e a c t i o n so r r e a c t i o ns e q u e n c e si n c o t l t p l e x n t e t a b o l i c p a t h w a y s t o b e i s o l a t e d a n d u t i l i z e d . a r - r dp r o n r i s e sh i g h e r y i e l d s . h i g h e r
p u r i t y . a n d g r e a t e r e a s e o f i s o l a t i o n t h a n e i t h e r ' 1 - e r t t t e n t a t i ool rl c l i e r t r i c a sl y n thesis. Elzyrnic reactions are run at roollt teulperature in water: etlzyntic
c a t a l y s i s t h u s c o m b i n e s t h e a t t r a c t i v e p r o c e s sc h a r a c t e r i s t i c so f f e r t t t e t t t a t i o t t
w i t h t h e e f f i c i e n c y o f c h e r n i c a ls y n t h e s i s .a l t h o u g h a t t h e c o s t o f h i g h t e c h n r c a l
complexity.
L a r g e - s c a l ee n z y r n i c s y n t h e s i s h o l d s p r o r n i s e i n a n u m b e r o f a r e a so 1 - t i n c
c 6 e r r r i c a l s - V p t h e s i sp,a r t i c u l a r l y i n a r e a s r e l e l a n t t o b i c l l o g i c a l l ya c t i v c ' c t r t l t poulds. Specitic areas in which researclt cotrld produce useful svllthetic
i n e t h o d s i n c l u d e t h e s e:
S y n t h e s i so f n u c l e o t i d e sa n d n u c l e o s i d e sf r o t n s u g a r sa n d p u r i n e s .p y r i r t r i tlines. or analogslright be carried out efficiently using enzynles.The availability'
of enzymes frorn the so-called "salvage patl-rways" that accept the prelortrtcd
l i t r o g e l h e t e r o c y c l e s b y p a s s e sm a n y o f t l t e s t e p s i n t h e m a i n b i o s y n t h e t i c
pathways [62] , and would capitalize on the ability of cell-freeenzyrnic synthesis
to select one, minor. enzyrnic pathway fiom atnongthe largenulnber of paths
l.
922
L A R G ES C A L EE N Z Y M I C
SYNTHESIS
competing in vivo fbr the same substrate. Unlike chemical syntheses.l'lo protective groups would be involved in the enzyrnic syntheses.
2. Steroid nrodiflcation, particularly oxidative functionalization,will proba b l y p r e s e n t u n u s u a l a n d d i f f i c u l t p r o b l e r n si n i s o l a t i o n a n d s t a b i l i z a t i o no f t h e
e n z y t l t e si n v o l v c d[ 6 3 l . T l t e a r e ai s n o n e t h e l e s os n e o f g r e a tp r a c t i c a li r n p o r t a n c e ,
a n d f e r m e n t a t i o n m e t h o d s . a l t h o u g h e x t e n s i v e l yd e v e l o p e d [ 6 a l . d o n o t a l w a y s
produce the desiredproducts in high yield or purity.
3 . A s y n m e t r i c a n d s t e r e o s e l e c t i v es y n t h e s e sc x p l o i t t h e a b i l i t y o f en z y r n c s
to catalyze the formation of optically active products. This area is fully
described in Chapter IV of Part I of this book.
4 . T h e s y n t h e s i so f n o n r i b o s o m a lp e p t i d e sa p p e a r sp o s s i b l e[ 6 5 ] A n e x a r n p l e
o f a n i m p o r t a n t m e m b e r o f t h i s c l a s so f c o m p o u n d s t h a t i s w i t h i n r e a c h o f
p r e s e n t t e c l t n i q L r e si s t h e a n i n r a l f i r o d a n t i b i o t i c b a c i t r a c i n [ 6 6 ] T h e b i o s y n t h e s e s o f p e n i c i l l i n a n d c e p h a l o s p o r i na r e n o t p r e s e n t l y u n d e r s t o o d , a n d t h e
large-scalecell-free enzymic synthesis of these types of nraterials lies in the
future. Studies directed toward the simpler but related antibiotic Grarnicidirr
S h a v e b e e n d e s c r i b e d[ 6 5 ]
5. Intermediates useful in the synthesis of semisynthetic antibiotics are
p r e s e n t l y o b t a i n e d b y c h e r r r i c a lo r e n z y r n i c d e g r a d a t i o no f f ' e r n t e n t a t i o l tp r o d u c t s . I n p r i n c i p l e ,i t s h c l u l da l s o b e p o s s i b l et o p r o d u c et h e s co r c o n t p l e n t e n t a r y
materials by cell-free enzyrnic synthesis. Such reactions would require a flrrn
k n o w l e d g e o f t h e b i o s y n t h e s i so f t h e t a r g e t a n t i b i o t i c . a n d w o u l d i n t e r r u p t t h i s
b i o s y n t h e s i sb y u s i n g o n l y t h o s e e n z y m e s r e q u i r e d t o s y n t h e s i z ei n t e r n r e c l i a t e s
a l o n g t h e p a t h t o t h e p r o d u c t o b t a i n e d b y f e r r n e n t a t i o n 1 6 7 1 .T h e a v a i l a b i l i t y
o f i n t e r m e d i a t e st o p e n i c i l l i n s ,c e p h a l o s p o r i n so, r t h e a m i n o g l y c o s i d ea n t i b i o t i c s
w o u l d b e p a r t i c u l a r l y u s e f u l , a n d t h e e n z y r n i cs y n t h e s i so r s u b s e q u e n tm o d i f i c a t i o n o f t h e s e i n t e r r n e d i a t e sm i g h t p r e s e n t a n e a s i e rp r o b l e r n t h a n t h e t o t a l s y n t h e s i so f t h e c o m p l e t e a n t i b i o t i c .
6. Carbohydrates are particularly difficult to modify chernically.A large
n u m b e r o f e n z y r n i c t r a n s l b r m a t i o n sh a v e r e c e n t l y b e e n c a t a l o g u e d [ 6 8 ] . b u t
most have not been exploited for synthesis.
7 . A l e s s s p e c i l i c a p p l i c a t i o n o f e n z y m i c s y n t h e s i sL i e si n t h e a r e a o f d r u g
metabolism. Tlte usual approach to the isolation of drug metabolites is to f'eecl
the drug to an appropriate animal, and to isolate metabolites from blood or
u r i n e . T h e s e i s o l a t i o n so f t e n p r e s e n t s e v e r ee x p e r i m e n t a l d i f f i c u l t i e s . A s i r r i p l e r
(although not entirely equivalent) preliminary approach is to examine transf o r m a t i o n o f t h e d r u g o f i n t e r e s t b y e n z y n e s o r e n z y r n e m i x t u r e s b e l i e v e c tl o
b e i n v o l v e d i n m e t a b o l i s m ( e . g . , c r u d e o x i d a s e p r e p a r a t i o n sf r o m l i v e r ) . T h e
m a t e r i a l sg e n e r a t e db y s u c h a p r o c e d u r e w o u l d p r o b a b l y , a t l e a s t ,b e r e l a t e dt o
those formed in vivo, and their isolation would be lessdifficult.
8. It may prove possible to use enzymes ordinarily involved in hydrolysis tcr
form peptide, phosphodiester, or related bonds by changing solvent or other
4 A L T E R N A T I V EASN D P R O S P E C T I V E S9 2 3
environmental pararneters.These hydrolyses are reversible, and the practical
problems to be solved in using enzymes Lo catalyze the reverse reactions are
those of stabilizing the enzyme under the conditions necessary to run the
reaction, dealing with the substrate specificity of the enzymes, and preventing
multiple additions through suitable blocking groups. Several examples of
r e a c t i o n so f t h e s et y p e s h a v e b e e n r e p o r t e d [ 9 , 6 9 ] g. Ribosomal peptide synthesis of proteins representsa possibility for the
distant future. Elegant studies by Schechterhave pointed to a general procedure
tor isolating specific in-RNA's from eukaryotic cells, by precipitating the ir'tRNA, together with associatedribosomesand growing polypeptide ohains,using
a n t i b o d i e sa g a i n s tt h e s e p o l y p e p t i d e s [ 7 0 ] . T h e s e m - R N A ' s c a n t h e n . i n p r i n c i p l e . b e u s e d i n c o m b i n a t i o n w i t h m i x t u r e s o f r i b o s o m e s ,/ - R N A ' s , a t n i n o a c i d s .
and nucleoside triphosphates to generate proteins. This system would clearly be
a difflcult one to develop for practical synthesis, but for sufficiently valuable
p r o t e i n s ( e . g . ,h u m a n g r o w t h h o r m o n e ) , i t m i g h t p r o v e v e r y u s e f u l '
' These and similar subjects all are described in terms of cell-free enzymes.
P r o g r e s si n l a r g e - s c a l ee n z y m i c s y n t h e s i sw i l l a l s o d e p e n d o n t w o r e l a t e d a n d
. h e f l r s t i s t h e d e v e l o p r n e n to f n t o r e g e n e r a lt e c l t n i q t r e s
c o m p l e r n e n t a r ys u b j e c t s T
i
n
t
m
o
b i l i z e d w h o l e c e l l s . A s p r e s e n t l y p r a c t i c e d .t h i s t c c h u
s
i
n
g
for synthesis
l
i
r
n i t e d i n i t s a p p l i c a t i o n t o r e l a t i v e l y s i r n p l er e a c t i o t t s .
lique will probably be
C o m b i n e d w i t h t h e p r o c e d u r e sd e v e l o p e dt o p r o t e c t . i m m o b i l i z e . a n d s t a b i l i z e
cell-free enzylnes, it might have very great usefulnessby providing amethod of
circumventing the labor required to isolate and (possibly) stabilize cell-free
enzymes. Alternatively, a compromise between the use of immobilized enzymes
and immobilized whole cells-synthesis using, S?y, irnmobilized intact mitochondria or membrane fragments-rnight offer important advantages.The second
is the refinement of microbiological techniques for isolating andior mutating
rrew strains of organismscapable of carrying out new types of transfbrmations.
One important advantage of synthesis using immobilized enzymes relative to
synthesis by ferrnentation is the ability of the former to take advantageof
relatively unimportant metabolic pathways. Thus, if microorganisms can be
developed that show detectable activity of a particular type, isolation and
stabilization of the enzymes responsibleoft'ers a possiblesynthetic route even if
t h e e n z y m e sa r e p r e s e n ta t s u c h l o w c o n c e n t r a t i o n st h a t f e r m e n t a t i o nw o u l d n o t
be practical.
Acknowledgment
M a n y o f t h e i d e a s i n c o r p o r a t e di n t o t h i s c h a p t e rw e r e g e n e r a t e di n d i s c u s s i o n s
w i t h r n y c o l l e a g u e si n t h e M . l . T . E n z y m e T e c h n o l o g y G r o u p , p a r t i c u l a r l y
P r o f e s s o r sD . I . C . W a n g , C . K . C o l t o n , A . D e m a i n , a n d A . S i n s k y , a n d w i t h
Drs. A. Ramel and A. H. Nishikawa, of the Biopolymers Laboratory of
Hoffmann-LaRoche.
924
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