ro6.54.2.

10 INDUSTRIAL USE OF ENZYMES

Matti Leisola !o"#i !o$ela Ossi %asti#e# Ossi T"r"#e#
Laboratory of Bioprocess Engineering, Helsinki University of Technology, Finland, and
&a#s S'(oe)a$er DSM esearch, MD !eleen, The "etherlands
*e+,or-s.
#nd$strial en%y&es, speciality en%y&es, protein engineering, en%y&e technology, en%y&e
prod$ction, biocatalysis, fine che&icals
/o#te#ts
'( Historical backgro$nd
)( En%y&e classification
*( En%y&e prod$ction
*('( Microbial prod$ction strains
*()( En%y&e prod$ction by &icrobial fer&entation
+( ,rotein engineering
-( En%y&e technology
.( Large scale en%y&e applications
.('( Detergents
.()( Starch
.(*( Drinks
.(+( Te/tiles
.(-( 0ni&al feed
.(.( Baking
.(1( ,$lp and paper
.(2( Leather
1( Speciality en%y&es
1('( En%y&es in analytics
1()( En%y&es in personal care prod$cts
1(*( En%y&es in D"03technology
2( En%y&es in fine che&ical prod$ction
2('( 4hirally p$re a&ino acids and asparta&e
2()( are s$gars
2(*( Se&i synthetic penicillins
2(+( Lipase based reactions
2(-( 0sy&&etric synthesis
2(.( En%y&atic oligosaccharide synthesis
5( F$t$re trends in ind$strial en%y&ology
0lossar+
Al$ali#e 1(os1(atase. 0n en%y&e that degrades ester bonds in alkaline conditions(
A)i#o a'i- a)i-ase. 0n en%y&e that is $sed in &an$fact$ring optically p$re a&ino acids(
#t hydrolyses an a&ide bond in nat$ral a&ino acid a&ides(
A)+lase. 0 gro$p of en%y&es that hydrolyse che&ical bonds bet6een gl$cose
&olec$les present in starch( This gro$p incl$des alpha3, beta3 and
gl$coa&ylase(
As1arta)e. 0 lo6 calorie high intensive s6eetener(
2eta34l"'a#ase. 0n en%y&e that degrades beta3gl$can co&&only fo$nd e(g( in barley(
2io'atal+st. #solated en%y&e or a 6hole cell 7living or dead8
2ro)elai#. 0 protein3degrading en%y&e fro& plants(
/atalase. 0n en%y&e that degrades hydrogen pero/ide to o/ygen and 6ater(
/ell"lases. 0 gro$p of en%y&es that synergistically degrade cell$lose fibers to
gl$cose(
/LE/. En%y&e crystal that has been &ade insol$ble by che&ical cross3
linking9 a &ethod to i&&obilise and stabilise en%y&es(
/(irall+ 1"re. Many organic &olec$les can have t6o che&ically identical b$t
str$ct$rally &irror i&age for&s( 4hirally p$re &eans that only one of
the for&s is present(
De5tra# s"'rase. 0n en%y&e, present in so&e lactic acid bacteria, that for&s a gl$cose(
poly&er and fr$ctose fro& the disaccharide s$crose(
De5tra#. !l$cose containing branched poly&er $sed e(g( in blood
replace&ents(
DNA31ol+)erases. 0n en%y&e that synthesi%es D"0 poly&ers(
Fer)e#tor. 0 biological reactor for c$ltivation of &icroorganis&s(
Fi'i#. 0 protein3degrading en%y&e fro& plants(
For)ate -e(+-ro4e#ase. 0n en%y&e that o/idises for&ate to carbon dio/ide and "0D(
0l"'oa)+lase. 0n en%y&e that splits gl$cose &olec$les fro& starch(
0l"'ose o5i-ase. 0n en%y&e that $ses o/ygen to o/idise gl$cose to gl$conic acid and
hydrogen pero/ide(
0l+'os+ltra#s6erases. 4atalyse the transfer of &onosaccharides fro& a donor to saccharide
acceptors(
0RAS3stat"s. is given to an organis& that is !enerally egarded as Safe(
&+-rolases. En%y&es that break che&ical bonds by adding 6ater( They can be
$sed to for& che&ical bonds in the absence of 6ater(
&+-ro5+#itrile l+ase. 0n en%y&e that catalyses the addition of H4" to aldehydes and
ketones(
I))"#oassa+. This is an analytical &ethod in 6hich antibodies are $sed to detect
specific &olec$les(
Iso)erases. En%y&es that catalyse intra&olec$lar reactions(
La''ase. 0 polyphenol o/idase fro& f$ngi( This en%y&e can $se o/ygen to
o/idise different types of aro&atic &olec$les and to for& lignin type
of aro&atic poly&ers fro& phenolic co&po$nds(
La'tase. his en%y&es degrade &ilk3s$gar lactose to gl$cose and galactose(
Lactose intolerant people can cons$&e s$ch &ilk(
Li4ases. En%y&es that synthesi%e che&ical bonds(
Li1o5+4e#ase. 0 lipid o/idising en%y&e e/tracted $s$ally fro& soybeans(
L+ases. En%y&es that re&ove che&ical gro$ps fro& their s$bstrates 6itho$t
addition of 6ater
Nitrile (+-ratases. En%y&es that catalyse addition of 6ater to nitrales res$lting in a&ide
for&ation
)
O5i-ore-"'tases. En%y&es that o/idise or red$ce che&ical co&po$nds(
%a1ai#. 0 protein degrading en%y&e fro& ani&al g$t(
%e#i'illi#. 0n antibiotic s$bstance e/tracted fro& &olds(
%e1si#. 0n en%y&e that degrades proteins and is isolated fro& ani&als(
%ero5i-ase. 0n o/idative he&e3containing en%y&e that $ses hydrogen pero/ide to
o/idise aro&atic co&po$nds( #t is responsible for lignin biosynthesis
in plants and initiates lignin biodegradation by certain rot3f$ngi(
%(+tase. 0 phosphatase en%y&e that hydrolyses phosphoester bonds in phytic
acid( #s 6idely $sed in ani&al feeds(
%rotei# e#4i#eeri#4. #&prove&ent of en%y&e protein by genetic &ethods(
Rare s"4ar. 0 s$gar that is rare in nat$re(
Re##i#. 0n aspartic protease 6hich coag$lates &ilk protein( #t is $sed in
cheese &an$fact$ring and isolated fro& calf sto&ach or prod$ced by
reco&binant f$ngi(
Restri'tio# e#7+)es. En%y&es that recognise specific +32 n$cleotides long se:$encies fro&
D"0( They are i&portant tools in gene technology(
Tra#s6erases.
Tr+1si#. 0n en%y&e that degrades proteins and is isolated fro& ani&als(
8+la#ase. 0 gro$p of en%y&es that degrade plant fibers &ade of /ylose3s$gars
to /ylose &ono&ers(
8+litol. 0 tooth3friendly s$gar alcohol $sed in che6ing g$&s(
S"))ar+
En%y&es have been $sed since the da6n of &ankind in cheese &an$fact$ring and indirectly via
yeasts and bacteria in food &an$fact$ring( #solated en%y&es 6ere first $sed in detergents in the year
'5'+, their protein nat$re proven in '5). and their large3scale &icrobial prod$ction started in
'5.;s( #nd$strial en%y&e b$siness is steadily gro6ing d$e to i&proved prod$ction technologies,
engineered en%y&e properties and ne6 application fields( The &a<or part of en%y&es is prod$ced
by 6ith !0S3stat$s &icroorganis&s in large biological reactors called fer&entors( Us$ally the
prod$ction organis& and often also the individ$al en%y&e have been genetically engineered for
&a/i&al prod$ctivity and opti&ised en%y&e properties( Large vol$&e ind$strial en%y&es are
$s$ally not p$rified b$t sold as concentrated li:$ids or gran$lated dry prod$cts( En%y&es $sed in
special applications like diagnostics or D"03technology need to be highly p$rified( #solated
en%y&es have fo$nd several applications in fine che&ical ind$stry( En%y&es are $sed in prod$ction
of chirally p$re a&ino acids and rare s$gars( They are also $sed in prod$ction of fr$ctose and
penicillin derivatives as 6ell as several other che&icals( En%y&es sho$ld be considered as a part of
a rapidly gro6ing biocatalyst ind$stry also involving genetically opti&ised living cells as che&ical
prod$ction factories(
1. &istori'al 9a'$4ro"#-
Most of the reactions in living organis&s are catalysed by protein &olec$les called en%y&es(
En%y&es can rightly be called the catalytic &achinery of living syste&s( Man has indirectly $sed
en%y&es al&ost since the beginning of h$&an history( En%y&es are responsible for the biocatalytic
fer&entation of s$gar to ethanol by yeasts, a reaction that for&s the bases of beer and 6ine
&an$fact$ring( En%y&es o/idise ethanol to acetic acid( This reaction has been $sed in vinegar
prod$ction for tho$sands of years( Si&ilar &icrobial en%y&e reactions of acid for&ing bacteria and
*
yeasts are responsible for aro&a for&ing activities in bread &aking and in preserving activities in
sa$erkra$t preparation(
The fer&entative activity of &icroorganis&s 6as discovered only in '2
th
cent$ry and finally proved
by the French scientist Lo$is ,aste$r( The ter& =en%y&e> co&es fro& Latin 6ords, 6hich literally
&ean =in yeast>( This na&e 6as given since en%y&es 6here closely associated 6ith yeast activity(
The st$dy of en%y&es is a fairly recent activity( Scientists 6ho fo$nd o$t that an alcohol precipitate
of &alt e/tract contained a ther&o labile s$bstance, 6hich converted starch into s$gar, &ade the
first clear recognition of en%y&es in '2**( They called the s$bstance diastase( ?e kno6 no6 that it
6as an en%y&e no6adays called a&ylase( S$&ner finally proved the protein nat$re of en%y&es in
'5). 6hen he 6as able to crystalli%e $rease en%y&e fro& <ack bean(
,robably the first application of cell free en%y&es 6as the $se of rennin isolated fro& calf or la&b
sto&ach in cheese &aking( ennin is an aspartic protease 7see Me'(a#is)s o6 E#7+)e A'tio#:
6hich coag$lates &ilk protein and has been $sed for h$ndreds of years by cheese &akers( @h& in
!er&any prepared the first co&&ercial en%y&e preparation in '5'+( This trypsin en%y&e isolated
fro& ani&als degraded proteins and 6as $sed as a detergent( #t proved to be so po6erf$l co&pared
to traditional 6ashing po6ders that the original s&all package si%e &ade the !er&an ho$se6ives
s$spicio$s so that the prod$ct had to be refor&$lated and sold in larger packages( The real
breakthro$gh of en%y&es occ$rred 6ith the introd$ction of &icrobial proteases into 6ashing
po6ders( The first co&&ercial bacterial Bacillus protease 6as &arketed in '5-5 and beca&e big
b$siness 6hen "ovo%y&es in Den&ark started to &an$fact$re it and &a<or detergent &an$fact$res
started to $se it aro$nd '5.-(
#n food ind$stry 3 in addition to cheese &an$fact$ring 3 en%y&es 6ere $sed already in '5*; in fr$it
<$ice &an$fact$ring( These en%y&es clarify the <$ice( They are called pectinases, 6hich contain
n$&ero$s different en%y&e activities( The &a<or $sage of &icrobial en%y&es in food ind$stry
started in '5.;s in starch ind$stry( The traditional acid hydrolysis of starch 6as co&pletely replaced
by alpha3a&ylases and gl$coa&ylases, 6hich co$ld convert starch 6ith over 5-A, yield to gl$cose(
Starch ind$stry beca&e the second largest $ser of en%y&es after detergent ind$stry(
,resently the ind$strial en%y&e co&panies sell en%y&es for a 6ide variety of applications( The
esti&ated val$e of 6orld en%y&e &arket is presently abo$t US B '(* billion and it has been
forecasted to gro6 to al&ost US B ) billion by );;-( Detergents 7*1A8, te/tiles 7')A8, starch
7''A8, baking 72A8 and ani&al feed 7.A8 are the &ain ind$stries, 6hich $se abo$t 1-A of
ind$strially, prod$ced en%y&es( En%y&es are also indirectly $sed in biocatalytic processes
involving living or dead and per&eabilised &icroorganis&s( This revie6 concentrates on the $se of
isolated en%y&e preparations in large scale and speciality applications and che&ical &an$fact$ring(
The $se of &icroorganis&s as biocatalysts in che&ical prod$ction is, ho6ever, an interesting and
gro6ing field( The techni:$es of genetic, protein and path6ay engineering are &aking che&ical
prod$ction by living cells an interesting green alternative to replace traditional che&ical processes(
2. E#7+)e 'lassi6i'atio#
,resently &ore than );;; different en%y&e activities have been isolated and characteri%ed ;see
E#7+)olo4+< /o#'e1t a#- S'o1e o6 E#7+)e A'tio#=. The se:$ence infor&ation of a gro6ing
n$&ber of organis&s opens the possibility to characterise all the en%y&es of an organis& on a
geno&ic level( The s&allest kno6n organis&, Mycoplasma genitalium, contains +1; genes of 6hich
'+- are related to gene replication and transcription( BakerCs yeast has 1;;; genes coding for abo$t
*;;; en%y&es( Tho$sands of different variants of the nat$ral en%y&es are kno6n( The n$&ber of
reported *3di&ensional en%y&e str$ct$res is rapidly increasing( #n the year );;; the str$ct$re of
+
abo$t '*;; different proteins 6ere kno6n( The en%y&es are classified into si/ &a<or categories
based on the nat$re of the che&ical reaction they catalyseD
'( O5i-ore-"'tases catalyse o/idation or red$ction of their s$bstrates
)( Tra#s6erases catalyse gro$p transfer
*( &+-rolases catalyse bond breakage 6ith the addition of 6ater
+( L+ases re&ove gro$ps fro& their s$bstrates
-( Iso)erases catalyse intra&olec$lar rearrange&ents
.( Li4ases catalyse the <oining of t6o &olec$les at the e/pense of che&ical energy
Enly a li&ited n$&ber of all the kno6n en%y&es are co&&ercially available and even s&aller
a&o$nt is $sed in large :$antities( More than 1-A of ind$strial en%y&es are hydrolases( ,rotein3
degrading en%y&es constit$te abo$t +;A of all en%y&e sales( ,roteinases have fo$nd ne6
applications b$t their $se in detergents is the &a<or &arket( More than fifty co&&ercial ind$strial
en%y&es are available and their n$&ber increases steadily(
>. E#7+)e 1ro-"'tio#
So&e en%y&es are still e/tracted fro& ani&al or plant tiss$es( ,lant derived co&&ercial en%y&es
incl$de proteolytic en%y&es papain, bro&elain and ficin and so&e other speciality en%y&es like
lipo/ygenase fro& soybeans( 0ni&al derived en%y&es incl$de proteinases like pepsin and rennin(
Most of the en%y&es are, ho6ever, prod$ced by &icroorganis&s in s$b&erged c$lt$res in large
reactors called fer&entors( The en%y&e prod$ction process can be divided into follo6ing phasesD
'( Selection of an en%y&e
)( Selection of a prod$ction strain
*( 4onstr$ction of an overprod$cing strain by genetic engineering
+( Epti&isation of c$lt$re &edi$& and prod$ction conditions
-( Epti&isation of recovery process 7and p$rification if needed8
.( For&$lation of a stable en%y&e prod$ct
4riteria $sed in the selection of an ind$strial en%y&e incl$de specificity, reaction rate, pH and
te&perat$re opti&a and stability, effect of inhibitors and affinity to s$bstrates( En%y&es $sed in
paper ind$stry sho$ld not contain cell$lose3degrading activity as a side activity beca$se this activity
6o$ld da&age the cell$lose fibres( En%y&es $sed in ani&al feed ind$stry &$st be ther&o tolerant
to s$rvive in the hot e/tr$sion process $sed in ani&al feed &an$fact$ring( The sa&e en%y&es &$st
have &a/i&al activity at the body te&perat$re of the ani&al( En%y&es $sed in ind$strial
applications &$st $s$ally be tolerant against vario$s heavy &etals and have no need for cofactors(
They sho$ld be &a/i&ally active already in the presence of lo6 s$bstrate concentration so that the
desired reaction proceeds to co&pletion in a realistic ti&e fra&e(
>.1. Mi'ro9ial 1ro-"'tio# strai#s
#n choosing the prod$ction strain several aspects have to be considered( #deally the en%y&e is
secreted fro& the cell( This &akes the recovery and p$rification process &$ch si&pler co&pared to
prod$ction of intracell$lar en%y&es, 6hich &$st be p$rified fro& tho$sands of different cell
proteins and other co&ponents( Secondly, the prod$ction host sho$ld have a !0S3stat$s, 6hich
&eans that it is 0enerally Regarded As Safe( This is especially i&portant 6hen the en%y&e
prod$ced by the organis& is $sed in food processes( Thirdly, the organis& sho$ld be able to
prod$ce high a&o$nt of the desired en%y&e in a reasonable ti&e fra&e( The ind$strial strains
typically prod$ce over -;3gFl e/tracell$lar en%y&e proteins( Most of the ind$strial en%y&es are
-
prod$ced by a relatively fe6 &icrobial hosts like Aspergillus and Trichoderma f$ngi, Streptomyces
f$ngi i&perfecti and Bacillus bacteria( Geasts are not good prod$ces of e/tracell$lar en%y&es and
are rarely $sed for this p$rpose( Most of the ind$strially $sed &icroorganis&s have been genetically
&odified to overprod$ce the desired activity and not to prod$ce $ndesired side activities(
>.2. E#7+)e 1ro-"'tio# 9+ )i'ro9ial 6er)e#tatio#
Ence the biological prod$ction organis& has been genetically engineered to overprod$ce the
desired prod$cts, a prod$ction process has to be developed( The opti&isation of a fer&entation
process incl$des &edia co&position, c$ltivation type and process conditions( This is a de&anding
task and often involves as &$ch effort as the intracell$lar engineering of the cell( The bioprocess
engineer asks :$estions likeD is the organis& in :$estion safe or are e/tra preca$tions needed, 6hat
kind of n$trients the organis& needs and 6hat is their opti&alF econo&ical concentration, ho6 the
n$trients sho$ld be sterilised, 6hat kind of a reactor is needed 7&ass transfer, aeration, cooling,
foa& control, sa&pling8, 6hat needs to be &eas$red and ho6 is the process controlled, ho6 is the
organis& c$ltivated 7batch, fed3batch or contin$o$s c$ltivation8, 6hat are the opti&al gro6th
conditions, 6hat is the specific gro6th and prod$ct for&ation rate, 6hat is the yield and vol$&etric
prod$ctivity, ho6 to &a/i&ise cell concentration in the reactor, is the prod$ct secreted o$t fro& the
cells, ho6 to degrade the cell if the prod$ct is intracell$lar, does so&e of the ra6 &aterials or
prod$cts inhibit the organis& and finally, ho6 to recover, p$rify and preserve the prod$ct( 0 typical
en%y&e prod$ction sche&e is sho6n in Fig$re '(
The large vol$&e ind$strial en%y&es are prod$ced in -; H -;; &
*
fer&entors( The e/tracell$lar
en%y&es are often recovered after cell re&oval 7by vac$$& dr$& filtration, separators or
&icrofiltration8 by $ltrafiltration( #f needed the p$rification is carried o$t by ion e/change or gel
filtration( The final prod$ct is either a concentrated li:$id 6ith necessary preservatives like salts or
polyols or alternatively gran$lated to a non3d$sty dry prod$ct( En%y&es are proteins, 6hich like any
protein can ca$se and have ca$sed in the past allergic reactions( Therefore protective &eas$res are
necessary in their prod$ction and application(
4. %rotei# e#4i#eeri#4
Eften en%y&es do not have the desired properties for an ind$strial application( Ene can in s$ch a
case try to find a better en%y&e fro& nat$re( E/tensive search for ne6 en%y&e variants in
organis&s that gro6 in e/tre&e conditions has been going on for &ore than ); years b$t has
res$lted in relatively fe6 s$ccesses( So&eti&es a desired property, like e/tre&e ther&o stability,
has been fo$nd b$t other proble&s have s$rfaced( The en%y&e &ay not be f$nctional in the desired
te&perat$re( #t &ay also prove very diffic$lt to overprod$ce the en%y&e in a s$itable host( 0nother
option is to engineer a co&&ercially available en%y&e to be a better ind$strial catalyst( T6o
different approaches are presently availableD a rando& &ethod called directed evol$tion and a
protein engineering &ethod called rational design( Table ' s$&&arises so&e of the reasons 6hy
ind$strial en%y&es need to be &odified and table ) describes so&e of the re:$ired tools $sed in
&odification 6ork(
Several en%y&es have already been engineered to f$nction better in ind$strial processes( These
incl$de proteinases, lipases, cell$lases, 3a&ylases and gl$coa&ylases( Iylanase is a good e/a&ple
of an ind$strial en%y&e, 6hich needs to be stable in high te&perat$re and active in physiological
te&perat$res and pHs 6hen $sed as feed additive and in alkaline conditions 6hen it is $sed in
bleaching in p$lp and paper ind$stry( Ene of the ind$strial prod$ction organis&s of /ylanases is
Trichoderma f$ng$s( #ts /ylanase has been p$rified and crystalli%ed( By designed &$tagenesis its
ther&al stability has been increased abo$t );;; ti&es at 1;
o
4 and its pH3opti&$& shifted to6ards
.
alkaline region by one pH3$nit( The three di&ensional str$ct$re of a /ylanase en%y&e is sho6n in
Fig$re )( The kno6n str$ct$re of an en%y&e is $sed to design and si&$late &$tations( The &ost
s$ccessf$l strategies to i&prove the stability of the Trichoderma /ylanase incl$de the stabili%ation
of the alpha3heli/ region and the "3ter&in$s(
5. E#7+)e te'(#olo4+
Ho6 are the en%y&es $sed and applied in practical processesJ This is the field of en%y&e
technology( The si&plest 6ay to $se en%y&es is to add the& into a process strea& 6here they
catalyse the desired reaction and are grad$ally inactivated d$ring the process( This happens in &any
b$lk en%y&e applications like li:$efaction of starch 6ith a&ylases, bleaching of cell$lose p$lp 6ith
/ylanases or $se of en%y&es in ani&al feed( #n these applications the price of the en%y&es &$st be
lo6 to &ake their $se econo&ical( E/tracell$larly prod$ced b$lk en%y&e concentrates cost only US
B ';3);F kg protein(
0n alternative 6ay to $se en%y&es is to i&&obilise the& so that they can be re$sed( The largest
application of an i&&obilised en%y&e is the conversion of gl$cose syr$p to high fr$ctose syr$p for
food applications( #n the early applications the gl$cose iso&erase en%y&e containing cells 6ere
per&eabilised and i&&obili%ed on a solid s$pport( The en%y&e containing s$pport &aterial 6as
packed into a col$&n thro$gh 6hich the gl$cose sol$tion 6as passed( Finnish S$gar 4o&pany
developed in early 2;s an alternative &ethod 6here the intracell$lar gl$cose iso&erase fro&
Streptomyces rubiginosus 6as p$rified by crystalli%ation and the p$re en%y&e 6as bo$nd to an
anion e/change resin, 6hich can be regenerated 6ith fresh en%y&e after the previo$s one is
inactivated( 0nother 6ay to i&&obilise en%y&es is to $se $ltrafiltration &e&branes in the reactor
syste&( The large en%y&e &olec$les cannot pass the &e&brane b$t the s&all &olec$lar reaction
prod$cts can( Therefore en%y&es are retained in a reaction syste& and the prod$cts leave the syste&
contin$o$sly( This &ethod has been $sed in prod$ction of chirally p$re a&ino acids fro& race&ic
&i/t$res of a&ino acid derivatives( En%y&es have also been i&&obili%ed on &e&branes for
analytical p$rposes( The best3kno6n e/a&ple is gl$cose o/idase, 6hich is $sed to &eas$re gl$cose
concentrations in biological sa&ples( Many different laboratory &ethods for en%y&e
i&&obili%ation based on che&ical reaction, entrap&ent, specific binding or absorption have been
developed(
0 novel approach to $se en%y&es 6as introd$ced by Finnish S$gar 4o&pany in the late 2;s( #t 6as
based on the $se of cross3linked crystalline gl$cose iso&erase 7Fig$re *8( En%y&e crystals contain
$s$ally *;32;A free 6ater and the en%y&e is active even in the cross3linked insol$ble for&( The
di&ensions of an en%y&e reactor, packed 6ith this kind of a &aterial, are considerably s&aller
co&pared to traditional i&&obili%ed syste&s beca$se the carrier &atri/ can be co&pletely o&itted(
The concept, originally developed in Finland, 6as later applied to other en%y&es by 0lt$s Ltd in
US0, 6hich has developed novel applications for the 4LE4s, 6hich is the trade&ark for /ross3
Linked En%y&e /rystals( These applications incl$de chiral separations, controlled release of
che&icals, specific separations and recently even cofactor entrap&ent into the crystal str$ct$re( 0ll
this is possible beca$se an en%y&e crystal contains 6ater, pores, active centre, hydrophobic areas
and ionic properties(
6. Lar4e s'ale e#7+)e a11li'atio#s
Table * s$&&arises &a<or large3scale en%y&e applications( Each of the& is disc$ssed in the te/t in
so&e detail( #nd$strial En%y&ology is reco&&ended as a good reso$rce te/t for those 6ho need a
&ore co&prehensive treat&ent of an individ$al s$b<ect(
1
6.1. Deter4e#ts
Detergents 6ere the first large scale application for &icrobial en%y&es( Bacterial proteinases are
still the &ost i&portant detergent en%y&es( So&e prod$cts have been genetically engineered to be
&ore stable in the hostile environ&ent of 6ashing &achines 6ith several different che&icals
present( These hostile agents incl$de anionic detergents, o/idising agents and high pH(
Late 2;s lipid degrading en%y&es 6ere introd$ced in po6der and li:$id detergents( Lipases
deco&pose fats into &ore 6ater3sol$ble co&po$nds by hydrolysing the ester bonds bet6een the
glycerol backbone and fatty acid( The &ost i&portant lipase in the &arket 6as originally obtained
fro& Humicola lanuginose. #t is prod$ced in large scale by Aspergillus oryzae host after cloning the
Humicola gene into this organis&(
0&ylases are $sed in detergents to re&ove starch based stains( 0&ylases hydrolyse gelatinised
starch, 6hich tends to stick on te/tile fibres and bind other stain co&ponents( 4ell$lases have been
part of detergents since early 5;s( 4ell$lase is act$ally an en%y&e co&ple/ capable of degrading
crystalline cell$lose to gl$cose( #n te/tile 6ashing cell$lases re&ove cell$lose &icrofibrils, 6hich
are for&ed d$ring 6ashing and the $se of cotton based cloths( This can be seen as colo$r
brightening and softening of the &aterial( 0lkaline cell$lases are prod$ced by Bacillus strains and
ne$tral and acidic cell$lases by Trichoderma and Humicola f$ngi(
6.2. Star'( (+-rol+sis a#- 6r"'tose 1ro-"'tio#
The $se of starch degrading en%y&es 6as the first large3scale application of &icrobial en%y&es in
food ind$stry( Mainly t6o en%y&es carry o$t conversion of starch to gl$coseD alpha3a&ylase c$ts
rapidly the large alpha3',+3linked gl$cose poly&ers into shorter oligo&ers in high te&perat$re( This
phase is called li:$efaction and is carried o$t by bacterial en%y&es( #n the ne/t phase called
saccharification, gl$coa&ylase hydrolyses the oligo&ers into gl$cose( This is done by f$ngal
en%y&es, 6hich operate in lo6er pH and te&perat$re than alpha3a&ylase( So&eti&es additional
debranching en%y&es like p$ll$lanase are added to i&prove the gl$cose yield( Beta3a&ylase is
co&&ercially prod$ced fro& barley grains and $sed for the prod$ction of the disaccharide &altose(
#n the United States large vol$&es of gl$cose syr$ps are converted by gl$cose iso&erase after 4a
)K
7alpha3a&ylase needs 4a
)K
for activity b$t it inhibits gl$cose iso&erase8 re&oval to fr$ctose
containing syr$p( This is done by bacterial en%y&es, 6hich need Mg
)K
ions for activity( Fr$ctose is
separated fro& gl$cose by large3scale chro&atographic separation and crystalli%ed( 0lternatively,
fr$ctose is concentrated to --A and $sed as a high fr$ctose corn syr$p in soft drink ind$stry(
0n alternative &ethod to prod$ce fr$ctose is sho6n in Fig$re +( This &ethod is $sed in E$rope and
$ses s$crose as a starting &aterial( S$crose is split by invertase into gl$cose and fr$ctose, fr$ctose
separated and crystalli%ed and then the gl$cose circ$lated back to the process(
6.>. Dri#$s
En%y&es have &any applications in drink ind$stry( The $se of chy&osin in cheese &aking to
coag$late &ilk protein 6as already disc$ssed( 0nother en%y&e $sed in &ilk ind$stry is beta3
galactosidase or lactase, 6hich splits &ilk3s$gar lactose into gl$cose and galactose( This process is
$sed for &ilk prod$cts that are cons$&ed by lactose intolerant cons$&ers(
En%y&es are $sed also in fr$it <$ice &an$fact$ring( Fr$it cell 6all needs to be broken do6n to
i&prove <$ice liberation( ,ectins are poly&eric s$bstances in fr$it la&ella and cell 6alls( They are
2
closely related to polysaccharides( The cell 6all contains also he&icell$loses and cell$lose(
0ddition of pectinase, /ylanase and cell$lase i&prove the liberation of the <$ice fro& the p$lp(
,ectinases and a&ylases are $sed in <$ice clarification(
Bre6ing is an en%y&atic process( Malting is a process, 6hich increases the en%y&e levels in the
grain( #n the &ashing process the en%y&es are liberated and they hydrolyse the starch into sol$ble
fer&entable s$gars like &altose, 6hich is a gl$cose disaccharide( 0dditional en%y&es can be $sed
to help the starch hydrolysis 7typically alpha3a&ylases8, solve filtration proble&s ca$sed by beta3
gl$cans present in &alt 7beta3gl$canases8, hydrolyse proteins 7ne$tral proteinase8, and control ha%e
d$ring &at$ration, filtration and storage 7papain, alpha3a&ylase and beta3gl$canase8(
Si&ilarly en%y&es are 6idely $sed in 6ine prod$ction to obtain a better e/traction of the necessary
co&ponents and th$s i&proving the yield( En%y&es hydrolyse the high &olec$lar 6eight
s$bstances like pectin(
6.4. Te5tiles
The $se of en%y&es in te/tile ind$stry is one of the &ost rapidly gro6ing fields in ind$strial
en%y&ology( Starch has for a long ti&e been $sed as a protective gl$e of fibres in 6eaving of
fabrics( This is called si%ing( En%y&es are $sed to re&ove the starch in a process called desi%ing(
0&ylases are $sed in this process since they do not har& the te/tile fibres(
En%y&es have replaced the $se of volcanic lava stones in the preparation of Deni& 7special soft
cotton based fibre 6here the dye has been partially faded a6ay8 fro& an indigo3dyed cotton fibre to
achieve a high degree of dye fading( The stones ca$sed considerable da&age to fibres and
&achines( The sa&e effect can be obtained 6ith cell$lase en%y&es( The effect is a res$lt of
alternating cycles of desi%ing and bleaching en%y&es and che&icals in 6ashing &achines(
ecently, hydrogen pero/ides have been tested as bleaching agents to replace chlorine3based
che&icals( 4atalase en%y&e, 6hich destroys hydrogen pero/ide, &ay then be $sed to degrade
e/cess pero/ide( 0nother recent approach is to $se o/idative en%y&es directly to bleach te/tiles(
Laccase H a polyphenol o/idase fro& f$ngi 3 is a ne6 candidate in this field(
Laccases are prod$ced by 6hite3rot f$ngi, 6hich $se the& to degrade lignin 3 the aro&atic poly&er
fo$nd in all plant &aterials( Laccase is a copper3containing en%y&e, 6hich is o/idised by o/ygen,
and 6hich in an o/idised state can o/idatively degrade &any different types of &olec$les like dye
pig&ents(
Ether en%y&es, 6hich interact 6ith te/tiles, are often added to 6ashing po6ders( These e/a&ples
6ere disc$ssed $nder detergent en%y&es(
6.5. A#i)al 6ee-
#ntensive st$dy to $se en%y&es in ani&al feed started in early 2;s( The first co&&ercial s$ccess
6as addition of beta3gl$canase into barley based feed diets( Barley contains beta3gl$can, 6hich
ca$ses high viscosity in the chicken g$t( The net effect of en%y&e $sage in feed has been increased
ani&al 6eight gain 6ith the sa&e a&o$nt of barley res$lting in increased feed conversion ratio(
Finnfeeds #nternational 6as the pioneer in ani&al feed en%y&es(
En%y&es 6ere tested later also in 6heat3based diets( Iylanase en%y&es 6ere fo$nd to be the &ost
effective ones in this case( 0ddition of /ylanase to 6heat3based broiler feed has increased the
available &etaboli%able energy 13';A in vario$s st$dies( Iylanases are no6adays ro$tinely $sed in
feed for&$lations( Fig$re ) sho6s the three3di&ensional str$ct$re of a Trichoderma /ylanase(
5
Us$ally a feed3en%y&e preparation is a &$ltien%y&e cocktail containing gl$canases, /ylanases,
proteinases and a&ylases( En%y&e addition red$ces viscosity, 6hich increases absorbtion of
n$trients, liberatates n$trients either by hydrolysis of non3degradable fibres or by liberating
n$trients blocked by these fibres, and red$ces the a&o$nt of faeces(
0nother type of i&portant feed en%y&e is phytase &arketed e(g( by DSM in the "etherlands(
,hytase is a phosphoesterase 6hich liberates phosphate fro& phytic acid 6hich is a co&&on
co&po$nd in plant based feed &aterials( The net effect is red$ced phosphoro$s in faeces res$lting
in red$ced environ&ental poll$tion( The $se of phytase red$ces the need to add phosphor$s to the
feed diet(
En%y&es have beco&e an i&portant aspect of ani&al feed ind$stry( #n addition to po$ltry, en%y&es
are $sed in pig feeds and t$rkey feeds( They are added as en%y&e pre&i/es 7en%y&e3flo$r &i/t$re8
d$ring the feed &an$fact$ring process, 6hich involves e/tr$sion of 6et feed &ass in high
te&perat$re 72;35;
E
48( Therefore the feed en%y&es need to be ther&o tolerant d$ring the feed
&an$fact$ring and operative in the ani&al body te&perat$re(
6.6. 2a$i#4
Si&ilar fibre &aterials are $sed in baking than in ani&al feed( #t is therefore conceivable that
en%y&es also affect the baking process( 0lpha3a&ylases have been &ost 6idely st$died in
connection 6ith i&proved bread :$ality and increased shelf life( Both f$ngal and bacterial a&ylases
are $sed( Everdosage &ay lead to sticky do$gh so the added a&o$nt needs to be caref$lly
controlled(
Ene of the &otivations to st$dy the effect of en%y&es on do$gh and bread :$alities co&es fro& the
press$re to red$ce other additives( #n addition to starch, flo$r typically contains &inor a&o$nts of
cell$lose, gl$cans and he&icell$loses like arabino/ylan and arabinogalactan( There is evidence that
the $se of /ylanases decreases the 6ater absorption and th$s red$ces the a&o$nt of added 6ater
needed in baking( This leads to &ore stable do$gh( Especially /ylanases are $sed in 6hole &eal rye
baking and dry crisps co&&on in Scandinavia(
,roteinases can be added to i&prove do$gh3handling properties9 gl$cose o/idase has been $sed to
replace che&ical o/idants and lipases to strengthen gl$ten, 6hich leads to &ore stable do$gh and
better bread :$ality(
6.?. %"l1 a#- %a1er
#ntensive st$dies have been carried o$t d$ring the last t6enty years to apply &any different
en%y&es in p$lp and paper ind$stry( 0 real e/cite&ent started 6ith the discovery of lignin degrading
pero/idases in the early 2;s( #n spite of e/tensive research no o/idative en%y&es are applied in p$lp
and paper ind$stry( The &a<or application is the $se of /ylanases in p$lp bleaching( Iylanases
liberate lignin frag&ents by hydrolysing resid$al /ylan( This red$ces considerably the need for
chlorine based bleaching che&icals( Ether &inor en%y&e applications in p$lp prod$ction incl$de
the $se of en%y&es to re&ove fine particles fro& p$lp( This facilitates 6ater re&oval(
#n the $se of secondary 7recycled8 cell$lose fibre the re&oval of ink is i&portant( The fibre is
dil$ted to 'A concentration 6ith 6ater, flocc$lating s$rfactants and ink solvents added and the
&i/t$re is aerated( The ink particles float to the s$rface( There are reports that this process is
facilitated by addition of cell$lase en%y&es(
';
#n paper &aking en%y&es are $sed especially in &odification of starch, 6hich is $sed as an
i&portant additive( Starch i&proves the strength, stiffness and erasability of paper( The starch
s$spension &$st have a certain viscosity, 6hich is achieved by adding a&ylase en%y&es in a
controlled process(
,itch is a sticky s$bstance present &ainly in soft6oods( #t is co&posed of lipids( #t is a special
proble& 6hen &echanical p$lps of red pine are $sed as a ra6 &aterial( ,itch ca$ses proble&s in
paper &achines and can be re&oved by lipases(
6.@. Leat(er
Leather ind$stry $ses proteolytic and lipolytic en%y&es in leather processing( The $se of these
en%y&es is associated 6ith the str$ct$re of ani&al skin as a ra6 &aterial( En%y&es are $sed to
re&ove $n6anted parts( 0lkaline proteases are added in the soaking phase( This i&proves 6ater
$ptake by the dry skins, re&oval and degradation of protein, dirt and fats and red$ces the processing
ti&e( #n so&e cases pancreatic trypsin is also $sed in this phase(
#n dehairing and de6ooling phases en%y&es are $sed to assist the alkaline che&ical process( This
res$lts in a &ore environ&entally friendly process and i&proves the :$ality of the leather 7cleaner
and stronger s$rface, softer leather, less spots8( The $sed en%y&es are typically alkaline bacterial
proteases( Lipases are $sed in this phase or in bating phase to specifically re&ove grease( The $se of
lipases is a fairly ne6 develop&ent in leather ind$stry(
The ne/t phase is bating 6hich ai&s at deli&ing and des6elling of collagen( #n this phase the
protein is partly degraded to &ake the leather soft and easier to dye( ,ancreatic trypsins 6ere
originally $sed b$t they are being partly replaced by bacterial and f$ngal en%y&es(
?. S1e'ialit+ e#7+)es
#n addition to large vol$&e en%y&e applications, there are a large n$&ber of speciality applications
for en%y&es( These incl$de $se of en%y&es in analytical applications, flavo$r prod$ction, protein
&odification, and personal care prod$cts, D"03technology and in fine che&ical prod$ction( The
latter application 6ill be separately disc$ssed beca$se of its i&portance( Here 6e disc$ss the other
aspects of speciality en%y&es(
?.1. E#7+)es i# a#al+ti's
En%y&es are 6idely $sed in the clinical analytical &ethodology( 4ontrary to b$lk ind$strial
en%y&es these en%y&es need to be free fro& side activities( This &eans that elaborate p$rification
processes are needed( Table + s$&&arises so&e of the &ain analytes &eas$red en%y&atically(
"or&ally a$to&atic analysers carry o$t these &eas$re&ents( The reactions nor&ally involve either
changes in "0D7,8F"0D7,8H proportions, 6hich can be detected spectrophoto&etrically or
prod$ction of H
)
E
)
6hich can be detected in pero/idase catalysed reactions leading to colo$red
prod$cts, 6hich can be easily :$antified spectrophoto&etrically(
#&&$noassays are based on detection of target &olec$les by specific antibodies( The detection of
the antibody3antigen co&ple/ is $s$ally based on en%y&es linked to the antibodies( This en%y&e is
either an alkaline phosphatase, 6hich can be detected in colo$r for&ing reaction by p3nitrophenyl
phosphate or pero/idase, 6hich is detected in the presence of H
)
E
)
6ith a colo$r for&ing s$bstrate(
''
0n i&portant develop&ent in analytical che&istry is biosensors( They are based on H
)
E
)
prod$cing
o/idative en%y&es( T6o different types of electrodes, one based on pero/ide detection and the other
based on o/ygen cons$&ption, can be $sed to :$antify the analyte in :$estion( The &ost 6idely
$sed application is a gl$cose biosensor involving gl$cose o/idase catalysed reactionD
gl$cose K E
)
K H
)
E

gl$conic acid K H
)
E
)
Several co&&ercial instr$&ents are available 6hich apply this principle for &eas$re&ent of
&olec$les like gl$cose, lactate, lactose, s$crose, ethanol, &ethanol, cholesterol and so&e a&ino
acids(
?.2. E#7+)es i# 1erso#al 'are 1ro-"'ts
,ersonal care prod$cts are a relatively ne6 area for en%y&es and the a&o$nts $sed are s&all b$t
6orth to &ention as a f$t$re gro6th area( Ene application is contact lens cleaning( ,roteinase and
lipase containing en%y&e sol$tions are $sed for this p$rpose( Hydrogen pero/ide is $sed in
disinfections of contact lenses( The resid$al hydrogen pero/ide after disinfections can be re&oved
by a he&e containing catalase en%y&e, 6hich degrades hydrogen pero/ide(
So&e toothpaste contains gl$coa&ylase and gl$cose o/idase( The reasoning behind this practise is
that gl$coa&ylase liberates gl$cose fro& starch3based oligo&ers prod$ced by alpha3a&ylase and
gl$cose o/idase converts gl$cose to gl$conic acid and hydrogen pero/ide 6hich both f$nction as
disinfectants(
Dent$res can be cleaned 6ith protein degrading en%y&e sol$tions( En%y&es are st$died also for
applications in skin and hair care prod$cts(
?.>. E#7+)es i# DNA3te'(#olo4+
D"03technology has revol$tionised both traditional biotechnology and opened totally ne6 fields
for scientific st$dy( #t is also an i&portant tool in en%y&e ind$stry( Most traditional en%y&es are
prod$ced by organis&s, 6hich have been genetically &odified to overprod$ce the desired en%y&e(
eco&binant D"03technology allo6s one to prod$ce ne6 en%y&es in traditional overprod$cing
and safe organis&s( ,rotein engineering is $sed to &odify and i&prove e/isting en%y&es as
disc$ssed $nder Protein engineering. En%y&es are the tools needed in genetic engineering and are
shortly disc$ssed here( For &ore infor&ation the reader is referred to specific te/ts dealing 6ith
genetic engineering(
D"0 is basically a long chain of deo/yribose s$gars linked together by phosphodiester bonds(
Erganic bases, adenine, thy&ine, g$anine and cytosine are linked to the s$gars and for& the
alphabet of genes( The specific order of the organic bases in the chain constit$tes the genetic
lang$age( !enetic engineering &eans reading and &odifying this lang$age( En%y&es are cr$cial
tools in this process( The D"0 &odifying en%y&es can be divided into t6o classesD
'( estriction en%y&es recognise specific D"0 se:$ences and c$t the chain at these recognition
sites(
)( D"0 &odifying en%y&es synthesi%e n$cleic acids, degrade the&, <oin pieces together and
re&ove parts of the D"0(
estriction en%y&es recognise a specific code se:$ence in the D"0( This is $s$ally +32 n$cleotides
long se:$ence( Their role in nat$re is to c$t foreign D"0 &aterial( These en%y&es do not c$t the
cellCs o6n D"0 beca$se its recognition sites are protected( More than '-; different restriction
')
en%y&es have been isolated fro& several bacterial species and they are $sed in c$tting the D"0 in
:$estion at specific points( These en%y&es are essential in gene technology(
D"03poly&erases synthesi%e ne6 D"03chains( Many of the& need a &odel te&plate, 6hich they
copy( "$cleases hydrolyse the phosphodiester bonds bet6een D"0 s$gars( Linases add phosphate
gro$ps and phosphatases re&ove the& fro& the end of D"0 chain( Ligases <oin ad<acent
n$cleotides together by for&ing fosfodiester bonds bet6een the&(
#n the cell these en%y&es are involved in D"0 replication, degradation of foreign D"0, repairing
of &$tated D"0 and in reco&bining different D"0 &olec$les( The en%y&es $sed in gene
technology are prod$ced like any other en%y&e b$t their p$rification needs e/tra attention( Many
restriction en%y&es fro& different so$rces are prod$ced in Eshcerichia coli by reco&binant D"0
technology( They are often labile and therefore preserved at H);
E
4 in b$ffered glycerol sol$tion(
@. E#7+)es i# 6i#e '(e)i'al 1ro-"'tio#
Biocatalysis has been $sed in fine che&ical prod$ction for a long ti&e( Us$ally the catalyst has
been a living organis&( Ethanol, acetic acid, antibiotics, vita&ins, pig&ents, solvents are b$t a fe6
e/a&ples of biotechnical prod$cts( Ene of the reasons to $se 6hole cell catalysts lies in the need to
co&bine che&ical energy so$rce 7in the for& of 0T,8 or red$cingFo/idising po6er 7in the for& of
"0D7,8H8 to the prod$ction process( This is elegantly done in a living cell( Candida yeasts can
red$ce the -3carbon s$gar /ylose to a tooth3friendly polyol called /ylitol by a /ylose red$ctase
en%y&eD
/ylose K "0DH /ylitol K "0D
The en%y&e can be isolated and the reaction proceeds easily in a test t$be( Ho6ever, the red$cing
po6er of "0DH has to be regenerated for the reaction to proceed( This is done in a living cell by
other reactions, 6hich red$ce "0D back to "0DH( Ene can isolate another en%y&e, 6hich does
the sa&e and co$ples t6o reactions together( Ene s$itable en%y&e is for&ate dehydrogenaseD
/ylose K "0DH /ylitol K "0D
for&ate K "0D 4E
)
K "0DH
4o$pled en%y&atic reactions have been e/tensively st$died b$t only fe6 co&&ercial e/a&ples are
kno6n( Le$cine dehydrogenase is $sed co&&ercially to prod$ce L3tert3 le$cine 6ith a conco&itant
cofactor recycling $sing the for&ate red$ction for cofactor regeneration( #n spite of so&e s$ccesses,
co&&ercial prod$ction of che&icals by living cells $sing path6ay engineering is still in &any cases
the best alternative to apply biocatalysis( #solated en%y&es have, ho6ever, been s$ccessf$lly $sed in
fine che&ical synthesis( ?e disc$ss here so&e of the &ost i&portant e/a&ples(
@.1. /(irall+ 1"re a)i#o a'i-s a#- as1arta)e
"at$ral as 6ell as synthetic a&ino acids are 6idely $sed in the food, feed, agroche&ical and
phar&ace$tical ind$stries( Many proteinogenic a&ino acids are $sed in inf$sion sol$tions and
essential a&ino acids as ani&al feed additives( 0spartic acid and phenyl alanine &ethyl ester are
co&bined to for& the lo6 calorie s6eetener asparta&e( #n addition to nat$ral a&ino acids also
synthetic ones are inter&ediates in the prod$ction of phar&ace$ticals and agroche&icals( For
e/a&ple several tho$sand tons of D3phenylglycine and D3p3hydro/yphenylglycine are prod$ced
'*
ann$ally for the synthesis of the broad3spectr$& antibiotics a&picillin, a&o/icillin, cefale/in and
others(
"at$ral a&ino acids are $s$ally prod$ced by &icrobial fer&entation( "ovel en%y&atic resol$tion
&ethods have been developed for the prod$ction of L3 as 6ell as for D3a&ino acids( The concept is
based on the specificity of en%y&es to detect only one of the t6o chiral &olec$les of a&ino acid
derivatives( Ene approach is described in sche&e '( ace&ic &i/t$re of a&ino acid a&ides is
synthesi%ed by Strecker synthesis( ,er&eabilised cells of Pseudomonas putida containing a&ino
acid a&idase en%y&e are $sed to specifically hydrolyse the nat$ral for&( L3for& of the a&ino acid
is prod$ced and separated( The D3for& can then be che&ically for&ed or recycled after
race&i%ation(
0sparta&e, the intensive non3calorie s6eetener, is synthesi%ed in non3a:$eo$s conditions by
ther&olysin, a proteolytic en%y&e, fro& "3protected aspartic acid and phenylalanine &ethyl ester
7Sche&e )8( The en%y&e catalyses not only a typical condensation reaction in the absence of 6ater
b$t sho6s re&arkable selectivity in for&ing the correct bond to for& asparta&e( 0fter the
condensation reaction the protective gro$p is re&oved(
@.2. Rare s"4ars
"on3nat$ral &onosaccharides are needed as starting &aterials for ne6 che&icals and
phar&ace$ticals( E/a&ples are L3ribose, D3psicose, L3/ylose, D3tagatose and others( So&e of the
s$gars are presently prod$ced by che&ical iso&eri%ation or epi&erisation( ecently en%y&atic
&ethods have been developed to &an$fact$re practically all D3 and L3for&s of si&ple s$gars(
Fig$re + gives an e/a&ple ho6 en%y&es can be $sed to convert s$crose into vario$s nat$ral s$gars
and a rare s$gar psicose(
!l$cose iso&erase is one of the i&portant ind$strial en%y&es $sed in fr$ctose &an$fact$ring(
ecently it has been sho6n that it can catalyse previo$sly $nkno6n conversions( For e/a&ple L3
arabinose is iso&erised to L3rib$lose and slo6ly also to L3ribose( D3/ylose is iso&erised to D3
/yl$lose and slo6ly to D3ly/ose( 0lso +3carbon s$gars are good s$bstrates( En%y&atic &ethods are
an i&portant tool in prod$ction of rare s$gars(
@.>. Se)is+#t(eti' 1e#i'illi#s
,enicillin is prod$ced by genetically &odified strains of Penicillium strains( Most of the penicillin
is converted by i&&obilised acylase en%y&e to .3a&inopenicillanic acid, 6hich serves as a
backbone for &any se&isynthetic penicillins( These can be synthesi%ed by che&ical or en%y&atic
&ethods(
@.4. Li1ase 9ase- rea'tio#s
#n addition to detergent applications lipases can be $sed in versatile che&ical reactions since they
are active in organic solvents( Th$s 6ater can be replaced by other n$cleophiles like alcohols( The
transferase activity of lipases is $sed to convert lo6 val$e fats into &ore val$able ones in
transesterification reactions( This occ$rs 6hen lo6 val$e fats are inc$bated in the presence of
lipases and fatty acids( Lipases have also been $sed to for& aro&atic and aliphatic poly&ers( The
en%y&e can be $sed for enantio&eric separation of alcohols( #n place of alcohols also a&ines can be
$sed as the n$cleophile( This &akes it possible to separate rase&ic a&ine &i/t$res( 4hirally p$re
a&ines can be $sed as b$ilding blocks for bioactive &olec$les( Several other intensively st$died
synthetic reactions are possible in lipase3catalysed reactions(
'+
@.5. As+))etri' s+#t(esis
,roteases and lipases are $sed in biocatalytic chiral hydrolytic resol$tions as sho6n in sche&e '(
4hiral co&po$nds can alternatively be prod$ced in biocatalytic asy&&etric syntheses in 6hich a
prochiral prec$rsor is converted to a chiral &olec$le by enantioselective addition reaction( Lyases
catalyse the addition of a s$bstance to a do$ble bond or the eli&ination of a gro$p res$lting in an
$nsat$rated bond( 0 chiral co&po$nd is for&ed in s$ch a reaction( 0&&onia lyases are $sed to
prod$ce a&ino acids fro& alpha3keto acid prec$rsors( E/a&ple is L3aspartate a&&onia lyase in
prod$ction of L3aspartic acid(
0 novel lyase application involves hydro/ynitrile lyase, 6hich catalyses the addition of H4" to
aldehydes and ketones( The en%y&e fro& r$bber tree has been cloned and overe/pressed in
&icroorganis&s( This en%y&e prod$ces val$able che&ical inter&ediates(
0 third i&portant biocatalytic en%y&e gro$p is nitrile hydratases( They catalyse the addition of
6ater to nitriles res$lting in the for&ation of a&ides( They are $sed for e/a&ple in the prod$ction
of acryla&ide fro& acrylonitrile and nicotine a&ide(
@.6. E#7+)ati' oli4osa''(ari-e s+#t(esis
The che&ical synthesis of oligosaccharides is a co&plicated &$lti3step effort( The saccharide
b$ilding blocks &$st be selectively protected then co$pled and finally deprotected to obtain desired
stereoche&istry and regioche&istry( Biocatalytic synthesis 6ith isolated en%y&es like
glycosyltransferases and glycosidases or engineered 6hole cells are po6erf$l alternatives to
che&ical &ethods(
!lycosyltransferases catalyse the transfer of &onosaccharides fro& a donor to saccharide acceptors(
Typically the donor is a n$cleotide( The type of donor that the en%y&e $tilises and the position and
stereoche&istry of the transfer to the acceptor classify these en%y&es( These en%y&es can also be
e/tracell$lar( Leuconostoc lactic acid bacteria prod$ce an en%y&e called de/tran s$crase( #t converts
s$crose into fr$ctose and a gl$cose poly&er called de/tran 7Fig$re +8( De/tran is $sed in bio&edical
applications and as a &atri/ in separation processes( The en%y&e can $se other &olec$les than
gl$cose as acceptor and th$s novel oligo&ers 6ith e(g( antibacterial properties can be prod$ced(
!lycosidases are hydrolytic en%y&es, 6hich can be $sed for synthetic reactions in a si&ilar &anner
than ther&olysin is $sed for asparta&e synthesis( Eligosaccharides have fo$nd applications in
cos&etics, &edicines and as f$nctional foods(
A. F"t"re tre#-s i# i#-"strial e#7+)olo4+
#nd$strial en%y&e &arket gro6s steadily( The reason for this lies in i&proved prod$ction efficiency
res$lting in cheaper en%y&es, in ne6 application fields and in ne6 en%y&es fro& screening
progra&&es or in engineered properties of traditional en%y&es( "e6 applications are to be e/pected
in the field of te/tiles, ne6 ani&al diets like r$&inant and fish feed( #t can be e/pected that
breakthro$ghs in p$lp and paper 6ill &aterialise( The $se of cell$lases to convert 6aste cell$lose
into s$gars and f$rther to ethanol by fer&entative organis&s has been a &a<or st$dy topic for years(
#ncreasing environ&ental press$res and energy prices 6ill &ake this application a real possibility
one day(
Tailoring en%y&es for specific applications 6ill be a f$t$re trend 6ith contin$o$sly i&proving tools
and $nderstanding of str$ct$re3f$nction relationships and increased search for en%y&es fro& e/otic
'-
environ&ents( This &eans that there 6ill be a specifically tailored /ylanase for baking, another for
feed and a third one for p$lp bleaching(
"e6 technical tools to $se en%y&es as crystalline catalysts, ability to recycle cofactors, and
engineering en%y&es to f$nction in vario$s solvents 6ith &$ltiple activities are i&portant
technological develop&ents, 6hich 6ill steadily create ne6 applications(
En%y&es sho$ld, ho6ever, not be considered alone b$t rather as a part of a biocatalyst technology(
?hole cell catalysts, increased ability to engineer &etabolic path6ays and a co&bination of specific
biocatalytic reactions 6ith organic che&istry for& a basis to develop ne6 technologies for che&ical
prod$ction(
2i9lio4ra1(+
Bhat M 7);;;8 4ell$lases and related en%y&es in biotechnology( Biotechnology Adances 1@, *--3
*2*( MThis revie6 paper disc$sses present and possible f$t$re trends9 /ylanases 6ere first s$ggested
for p$lp bleaching by NTT3Biotechnology in Finland, @h& En%y&e is the leader in the field see
httpDFF666(roeh&en%y&e(co&FO
Biochimica et Biophysica Acta 7);;;8 154>, );*3)-) M4ontains several revie6s of engineered
en%y&es of ind$strial i&portanceO
4hotani !(, Dodge T(, Hs$ 0(, L$&ar M(, LaD$ca (, Tri&b$r D(, ?eyler ?( and Sandford L(
7);;;8 The co&&ercial prod$ction of che&icals $sing path6ay engineering( Biochimica et
Biophysica Acta 154>, +*+3+--( MThis revie6 article disc$sses the latest trends in $sing engineered
organis&s in che&ical prod$ctionO
Doran ,(M( 7'5558 Bioprocess Engineering Principles( +*5 pp( 0cade&ic ,ress( M0 te/tbook, 6hich
describes engineering aspects of bioprocessesO
Flickinger M(4( and Dre6 S(?( 7eds(, '5558 Encyclopedia o! Bioprocess Technology" !ermentation#
biocatalysis# and bioseparation 7- vol$&es8, Pohn ?iley Q Sons M!ood reso$rce book on all
aspects of &odern bioprocess technologiesO
!odfrey T( and ?est S( 7eds, '55.8 $ndustrial Enzymology, Mac&illan MThis is a basic te/tbook on
ind$strial en%y&es, a$thored by ind$strial e/perts9 666(novo%y&es(co& is a 6ebpage of 6orldRs
largest en%y&e co&pany, second largest is !enencor #nt( #nc(, 666(genencor(co&O9
,alcic M(M( 7'5558 Biocatalytic synthesis of oligosaccharides( Current %pinion in Biotechnology
10, .'.3.)+ M0 good revie6 6ith several references abo$t the topicO
,astinen E, Nis$ri L, Schoe&aker H and Leisola M 7'5558 "ovel reactions of /ylose iso&erase
fro& Streptomyces rubiginosus( Enzyme and Microbial Technology 25, .5-31;;( M!l$cose
iso&erase is a traditional na&e9 /ylose iso&erase 6o$ld be a &ore correct na&e altho$gh it has
recently been sho6n to catalyse &any &onosaccharide iso&eri%ationsO
Sch&id 0(, Dordick P(S(, Ha$er B(, Liener 0(, ?$bbolts M( and ?itholt B( 7);;'8 #nd$strial
biocatalysis today and to&orro6( &ature 40A, )-23).2 M0 good revie6 article on develop&ents of
en%y&atic and 6hole cell biocatalytic applications and trends in che&ical ind$stry9
httpDFF666(isrs(kaga6a3$(ac(<pF is a page of a recently for&ed #nternational Society of are
S$garsO
Nis$ri, L( 7'5218 Stable gl$cose iso&erase concentrate and a process for the preparation thereof(
'S Patent (#)**#++, MThis patent describes the first large scale crystalli%ation process of an
intracell$lar ind$strial en%y&eO and Nis$ri, L( 7'55-8 ,reparation of cross3linked gl$cose iso&erase
crystals( 'S Pat. -(./**.( MThis patent describes the first preparation of a cross3linked en%y&e
crystal catalystO
'.
Fi4"re 1. 0 typical en%y&e prod$ction sche&e( Large vol$&e ind$strial en%y&es are $s$ally
not p$rified( Their recovery is often finalised by an $ltrafiltration step( Speciality
en%y&es need &ore p$rification(
'1
Fi4"re 2. Three3di&ensional str$ct$re of a Trichoderma /ylanase ##( This en%y&e is $sed in
baking to i&prove bread :$ality, in ani&al feed to i&prove digestibility of feed, in
cell$lose p$lp bleaching to red$ce the $se of chlorine che&icals and in fr$it <$ice
&an$fact$ring to facilitate <$ice e/traction and clarification( The t6o active centre
gl$ta&ates and the one alpha heli/ are sho6n in a green colo$r(
'2
Fi4"re >. 4ross3linked gl$cose iso&erase crystals( The average crystal si%e of these crystals is
2. &( They can be $sed in chiral separations and as an i&&obili%ed en%y&e in a
backed3bed or fl$idised bed col$&n( 4ross3linking &akes the en%y&e insol$ble b$t
it retains its activity as 6ater containing poro$s &aterial(
'5
Strecker reacton: HCN
NH
3
H
3
O+
Amidase
P. putida
O. antropii
M. neoaurum
rac-1
1
L-2 D-1 D-2
O
R
NH
2
R
N
NH
2
R
NH
2
O
NH
2
R
NH
2
O
NH
2
R
OH
O
NH
2
R
NH
2
O
NH
2
R
OH
O
S'(e)e 1. For&ation of a race&ic a&ino acid a&ide 7'8 synthetically by Strecker reaction and
en%y&atic resol$tion of the race&ic a&ide &i/t$re by a&idase to for& L3a&ino acid
7L3)8 and D3a&ide 7D3'8 6hich can be hydrolysed to D3a&ino acid 7D3)8(
);
Aspartase PhCH
2
OCOCl
3
Z-3
Z-3
rac-4 Z-5 5
Thermolysin deprot.
HO
OH
O
O
OH
O
NH
2
HO
O
O N
H
O
HO O
HO O
H
2
N
O
O
O
H
N
O
N
H
O
O
OH O
O H
2
N
N
H
O
O
O
O
HO
S'(e)e 2. L3aspartic acid 7*8 is for&ed fro& f$&aric acid by aspartase 6hich catalyses the
addition of a&&onia to f$&aric acid( 0 protective gro$p is added to for& S3aspartic
acic 7S3*8 6hich is co&bined $sing a race&ic &i/t$re of DFL3phenylalanine &ethyl
ester 7rac3+8 by ther&olysin to give S3asparta&e 7S3-8( By re&oving the protective
gro$p by catalytic hydrogenation, asparta&e 7-8 is obtained(
)'
Table 1. Change in enzyme characteristics by protein
engineering
Enzyme Indstry !eed
"ylanase feed temperature stabilit
acid acti!it
pulp and
paper
temperature and alkali stabilit
hi"her pH#optimum
glcoamylase starch hi"her pH#optimum

glcose isomerase fructose substrate specificit
acid stabilit
thermo stabilit
proteinase deter"ent thermostabilit
alkali stabilit
o$idati!e stabilit
))
Table 2. Tools in protein engineering
Target #ethod
protein structure crstalli%ation
$#ra crstallo"raph
N&R
modellin" and
simulation
computational methods
"ene plasmids
e$pression sstems
tar"eted muta"enesis
PCR
'N( shufflin"
random muta"enesis
)*
Table 3. $arge scale enzyme applications
Indstry E%%ect
&etergent
proteinase protein de"radation
lipase
cellulase
fat remo!al
color bri"htenin"
Te"tile
cellulase microfibril remo!al
laccase color bri"htenin"
Animal %eed
$lanase fiber solubilit
phtase release of phosphate
'tarch
amlases "lucose formation
"lucose isomerase fructose formation
(lp and paper
$lanase biobleachin"
)rit *ice
pectinase
cellulase)
$lanase
*uice clarification)
*uice e$traction
+a,ing
$lanase dou"h conditionin"
alpha#amlase
"lucose o$idase
loaf !olume+ shelf#life
dou"h ,ualit
&airy
rennin protein coa"ulation
+re-ing
lactase
"lucanase
papain
lactose hdrolsis
filter aid
ha%e control
)+
Table 4. #a*or enzymatically measred analytes
Analyte Enzyme
alcohol alcohol dehdro"enase
ammonia -#"lutamate dehdro"enase
carbon dio$ide phosphoenolpru!ate carbo$lase
cholesterol cholesterol o$idase
"lucose "lucose o$idase
o$alate o$alate o$idase
urea urease
uric acid uricase

)-