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Integrated Purification
Purification suite: Clarification, Chromatography and Ultrafiltration.
www.millipore.com/PurificationSuite

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1062 ENZYMES,IMMOBILIZATION METHODS
ENZYMES, IMMOBILIZATION METHODS
T ! S "
T " ! # & T !
TanabeSeiyaku Co.,Ltd.
Osaka, Ja pan
KEY WORDS
Carrierbinding
Cross-linking
Ent rappingenzyme
Immobilizationmethod
Immobilized enzyme
OUTLINE
Bibliography
In 1916, Nelson and Grifn (1) found that an enzyme in
water-insoluble form showed catalytic activity. They re-
ported tha t inverta seextractedfromyeast wasa dsorbed
on char coal, and the adsorbed enzyme showed th e same
activityasna tiveenzyme.In1948,Sumn er(2)foundt ha t
ur easefromjackbeanbecamewat erinsolubleonsta nding
in30%alcoholandsodium chloridefor1to2daysa tr oom
temperat ure a nd tha t th e water-insoluble urease was ac-
tive.Thus,ithasbeenknownforalongtimethatenzymes
in water-insoluble form show catalytic activity. However,
the rst att empt toimmobilizean enzymet oimproveits
propertiesforapa rt iculara pplicat ionwasnotma deuntil
1953, when Grubhofer and Schleith (3) immobilized en-
zymessuchascarboxypeptidase,diast ase,pepsin,andri-
bonuclease by using diazotized polyaminopolystyrene
resin. On th e other ha nd, before t his (1949), immobiliza-
tion of physiologically active protein was carried out by
Micheel and Ewers (4). Further, in 1951, Campbell et al.
(5) prepared immobilized antigen by binding albumin to
diazotized p-aminobenzylcellulose. Subsequent ly,a num-
ber of reports on the prepara tion and application ofim-
mobilizedant igensanda nt ibodiesappear ed.Suchstu dies
may also be applicable to immobilized enzymes.
Int he1960s,ma nypapersa ppear ed;inpart icular,Pro-
fessor Katzir-Katchalski and his co-workers at the Weiz-
mann Institute of Science, Israel, carried out extensive
stu diesonn ewimmobilizat iont echn iques,andontheen -
zymatic, physical, and chemical properties of immobilized
enzymes.
In1969,Dr.Chibataetal.atTanabeSeiyakuCo.,Ltd.,
Some books on immobilized enzymes have been pub-
lished(7,8)aswell.
Immobilized enzymes are dened as enzymes physi-
cally conned or localized in a certain dened region of
spacewithret entionofth eircata lyticactivities,andwhich
canbeus edrepea tedlyan dcontinuously. Accordin gly,en-
zymesmodiedtoawater -insolubleformbysuit abletech-
niques sat isfy th is denition of immobilized enzymes. In
addition, when an enzyme reaction using a substrate of
highmolecular weight iscarriedoutina rea ctorequipped
with a semipermeable ultraltration membrane without
leakageofthe enzyme fromth er eactor, thiscan alsobe
considered as a kind of immobilized enzyme system.
Theterm imm obilizedenzyme wasrecommendedatthe
Fir stEn zymeEngineer ingConferencein1971.Beforetha t
t i me, vari ous t erms such as wat er-i nsol ubl e enzyme,
trapped enzyme, xed enzyme, and matrix-supported en-
zymehadbeenused.
As functional groups involved in enzyme immobiliza-
tion,free,amin oan dcarboxylgroups,th esulfhydrylgroup
of cystein, the imidazole group of histidine, phenolic
groups,andh ydroxylgroupsofserinea ndt hr eoninema y
be considered. For immobilization of an enzyme, it is nec-
essaryt hat functional groupsin the activecenter should
notbe involvedint he rea ction leadingtoimmobilization
oftheenzyme. Furth er, because thet ertiar ystructureof
enzyme protein is maintained by relatively weak binding
forces,su chas hydr ogen,h ydrophobic,andionicbonds,it
isnecessar ytocar ryoutt heimmobilizationreact ionunder
mild conditions.
Methods for enzyme immobilization can be classied
into three basic categories as follows.
1. Carrier-binding method: the binding of enzymes to
water-insoluble carriers such as polysaccharide de-
rivatives,synth eticpolymers,an dporous glass,etc.
2. Cross-linking method: intermolecular cross-linking
of enzymes by means of bifunctional or multifunc-
tionalreagent ssuchasgluta ra ldehyde,bisdiazoben-
zidine,an dhexamethylenediisocyan at e,etc.
3. Ent ra ppingmet hod:incorporat ingenzymes int oth e
lattice of a semipermeable gel on enclosing the en-
zymesinasemipermeablepolymermembr an e,such
as collagen, gelatin cellulose t riaceta te polyacryl-
amide, and j-carrageenan, etc.
Thesemet hodsaresh ownschemat icallyinFigure1.
Thecarr ier-bindingmet hodistheoldestimmobilizat ion
met hod for enzymes, and many papers ha ve been pub-
lishedonthismethod.Whenenzymesa reimmobilizedin
th isway,car eisrequiredregar dingtheselectionof carr iers
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In1969,Dr.Chibataetal.atTanabeSeiyakuCo.,Ltd.,
inJa pansu cceededintheindust rializat ionof acontinuous
process for the optical resolution of -aminoacidsusing
immobilizedaminoacylase(6).Thiswastherstindustrial
applicationofimmobilizedenzymesintheworld.
In1971,therst En zymeEngineeringConferencewas
heldatHenniker, NewHampshire, andt hepredominant
th emeofthisconferen cewasimmobilizedenzymes.Adef-
init ionan dclas sicationof immobilizedenzymeswer epro-
poseda t th econference.Thisconferenceisbiann ua l,and
th ema intopicshavecontinuedto beimmobilizedenzymes .
th isway,car eisrequiredregar dingtheselectionof carr iers
as wel l as i n binding t echni ques. The carri er-binding
method can be further divided into three categories ac-
cordingtoth ebindingmodeof th eenzyme,tha tis,physical
adsorption, ionic binding, and covalent binding.
Thecross-linkingmet hodisbasedont heformat ionof
chemicalbonds,butwa ter-insolublecar riersa renotused
in this method. The immobilization of enzymes is per-
formedbyt he format ion ofinter molecular cross-linka ges
between th eenzymemoleculesbymea nsofbifunctionalor
multifunctional reagents.

ENZYMES,IMMOBILIZATION METHODS 1063
Water-
insoluble
carrier
Enzyme
Carrier-bindi ngmethod Cross-linking method
Latticetype Microcapsuletype
Entrappingmethod
Fi gure 1. Schematic diagrams of immobilized
enzymes.
Tabl e 1. Preparati on and Characteri sti cs of Immobi l i zed Enzyme
Carrier-binding method
Cha ra ct er is tic Phys ica l a ds or pt ion I onic binding Cova lent binding Cr os s-linkingmet hod Entr a ppingmet hod
Pr epar at ion Easy Easy Difcult Difcult Difcult
Enzyme act ivit y Low High High Moder at e High
Subst r at e specicit y Unchangeable Unchangeable Changeable Changeable Unchangeable
Binding for ce Weak Moder at e St r ong St r ong St rong
Regenerat ion Possible Possible Impossible Impossible Impossible
Gener al applicabilit y Low Moder at e Moder at e Low High
Theent ra ppingmeth odisbasedonconningenzymes
int helat ticeofapolymermat rixoren closingenzymesin
semipermeable membranes, and it can be classied into
the lattice and microcapsule types. This method differs
fromthecovalentbindingan dcross-linkingmet hodintha t
th eenzymeitselfdoesnotbindtothegelmat rixormem-
brane. Thus, this method may have wide applicability.
However,ifachemicalpolymer izat ionr eact ionisus edfor
entrapping, relatively severe conditions are required and
loseofenzymea ctivityoccursin somecases .Ther efore,it
isnecessaryt oselectt hemost suita bleconditionsfort he
immobilization of various enzymes.
Prepar ation methods and char acteristics of immobi-
lizedenzymesa resu mma rizedbroadlyinTable1,th ough
there are man yexceptions.
Immobilizationofenzymesbycovalentbindingorcross-
linkingiscarr iedout under relat ivelysevereconditionsin
compa risonwitht hoseofphysicaladsorptionorionic bind-
ing. Thus, in the former cases, conformational change of
th eenzymestr uctur eandpar tialdestr uctionoftheactive
center ma yoccur.Accordin gly,un lessimmobilizationofan
enzyme by covalent binding is carr ied out under well-
cont rolledcondit ions,immobilizedenzymeha vinghighac-
tivitycannotbeobtained.However,thebindingforcesbe-
tweentheenzymeand carr ierarest rong, andtheenzyme
cannotea silybelostfromcarr ierseveninth epresenceof
subst ra tesorsaltsa thighconcentr at ions.However,when
th e a ctivity of enzymes immobilized by covalent binding
decreases on long-term operation, regeneration is impos-
sible.
On the other hand, immobilization of enzymes by the
ionic-bindingmet hodcan bea chievedsimplyun dermild
conditions.Accordingly,in th iscase, prepa ra tionsh aving
relativelyhighactivityareobtained.However,thebinding
forcesbetweenenzymean dcar riera reweakincompa rison
with those in the covalent-binding methods. Therefore,
leakage of the enzyme from the carrier may occur after
changesinth eionicstrength ,pHofth esubstr at e,orprod-
ucts olution.Thist ypeofimmobilizedenzymecanbe re-
generat edwhen the enzymea ctivitydecreasesafterpro-
l onged operat i on. Thus, t he i oni c-binding met hod i s
advanta geous in compar ison with the covalent-binding
method, particularly when expensive carriers or enzymes
areused.
Unlike th e ionic-binding,covalent-binding,and cross-
l inki ng met hods i n t he ent rapping met hod, no binding
Gener al applicabilit y Low Moder at e Moder at e Low High
Cost of immobilizat ion Low Low High Moder at e Low

1064 ENZYMES,IMMOBILIZED, REACTORS
betweenenzymeandcarriershouldoccurintheory.There-
fore,inma nycases,prepar at ionsh avinghighactivityare
obta ined.However,inthismeth od,regenera tionof activity
lossesisimpossible,asinthecaseofthecovalent-binding
method. Amajor disadvant ageoftheent rappingmethod
is th at its a pplication is limited to sma ll molecular sub-
st rat e and product mol ecul es; t he ent rapped enzyme
shows little or no activity toward macromolecular sub-
st rat es.
Although a number of enzyme immobilization methods
ha vebeenstudied,noidealgener almet hodsapplicablefor
th eimmobilizationofma nyenzymesh aveyetbeendevel-
oped.Eachmethodha sspecicdisadvan ta ges.Therefore,
inpra ctice, itisnecessarytonda suitablemethodand
condit ionsfort heimmobilizat ionofapa rt icularen zymein
th elightoftheinten dedapplicat ion.
Becau seoft hespecialcha ra cteristicsof immobilizeden-
zymes,aconsider ableamountofworkhasbeencar riedout
on application ofimmobilizedenzymesa ss olidcata lysts
ha vinghighsubs tr at especicity.Immobilizedenzymesar e
usedinvar iouselds,suchaschemicalprocesses, an alysis,
medicaltr eat ment ,foodprocessing,afnitychr omatogra-
phy,an dsofort h.For the efcient ut ilizationofsuchim-
mobilizedsystems ,chemicalengineerin gstudiesa ren ec-
essary.
BIBLIOGRAPHY
1. J .M.NelsonandE.G.Grifn, J.Am .Chem.Soc. 38,11091115
(1916).
2. J.B. Sumner, Science 108,410413(1948).
3. N. Grubhofer and L. Schleith, Naturwissenschaften 40, 508
(1953).
4. F.MicheelandJ .Ewers, Makromol.Chem. 3,200209(1949).
5. D.H.Camp bell,E.Luescher,andL.S.Lerma n, Proc.Nat.Acad.
Sci. 37,575581(1951).
6. I.Chiba ta ,T.Tosa,T.Sa to,T.Mori,an dY.Mat uo, Proc. of the
4th Int. Fermentation Symp.: Fermentation Technology Today,
1972,p.383389.
7. L.B.WingardJ r.ed., Enzyme Engineering, Wiley, New York,
1972.
8. O.R. Zaborsky, Imm obilized En zymes, Chemical Rubber Co.
Pr ess,Ohio,1973.
SeealsoB ! , ;B " ! , - "
" ! ;B " ! , # ` - ;
B " .
ENZYMES, IMMOBILIZED, REACTORS
U K
L ! ! G
C ! " W &
Forschun gszentru mJ ulichGmbH
J u lich,German y
KEY WORDS
Aminoacylase
Enzyme, immobilized
Glucose-fructose isomerase
Glycerol kinase
Lipase
Masstr ansportlimitation
Membrane
Nitrilehydrata se
PenGacylase
Reactor
OUTLINE
Introduction
Reactors for Immobilized Enzymes
Char acterizat ionofImmobilizedEnzymes
Selected Examples of Reactions Using Immobilized
Enzymes
Pr oductionof -AminoAcidsUsingAminoAcylase
Pr oductionof6-AminoPenicillanicAcidand 7-
AminoCephalosporanicAcidUsingAmidases
Pr oductionofHigh-Fr uctoseCorn Syrup Using
GlucoseI somerase
Pr oductionofAcrylamideUsingNitr ileHydrat ase
Pr oductionofGlycerol-3-Ph osphateUsingGlycerol
Kinase
Pr oductionof(2R,3S)-Meth yl-p-
Methoxyphenylglycidat eUsinga Lipase
Miscellaneous Processes Using Immobilized
Enzymes
Nomenclature
Bibliography
INTRODUCTION
The use of enzymes in food as well as in pharmaceutical
and chemical industries has increased st eadily since the
ear ly1970s.Applicat ionsr an gefromsynt hesisint hemil-
ligra mscaleforn at ur alornonnat ur aloligosaccharidesup
toindust rialsynt hesisfort heproductionof bulkchemicals
sucha sacrylamide.Someexamplesar elistedinTable1.
Thisha sbeenwelldocument edbyalargenumberoftext-
books,reviews,an doriginalpa pers (1,520).This ar ticle
highlight ssomeofthemostimporta nta spectsfort heap-
plicationofimmobilizedenzymesan dpresen tsselectedex-
amples.
The most common denition for immobilized enzymes
is that proposed by Katchalski-Katzir in the 1960s (16):
Enzymes physically conned or localized in a certain de-
nedr egionofspa cewithr eten tionofth eircata lyticactiv-
ities,whichcanbeu sedrepea tedlyand cont inuously. Ac-
cording to this denition, thr ee types of immobilized
enzymescanbedistinguished: