of anti

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Recovery ofDige st iveEnzymesFrom AtlanticCod (Gadus morhua)and The irUtilization in FoodProcessi ng

BY

@Xiao. Q ing Han

A thesis sub m itt ed tothe SchoolofGra dua te St udies in partialfulfil m e nt oftherequ ir e ment s for the

degreeofMasterof Scienc e

Depa rtme ntof Biochemistry MemorialUnivers it y ofNe wfound land

1993

St.John's Newfo undla nd Canada

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.+.

Nalionallibraty dcaMda

== ~8fanch

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~or.;o

:~nationale

CrrectJ::ndesaetJlisihonset desservicesbitlliog raphiQues 39S._WelnglCn OII-aCO-;OI Il;IA CfM

The author has granted an irrevoca ble non-exclusivelicence aIJowlngthe Nallona'Ubraryof Canada to reproduce, loan.

distr ibute or sell copies of his/herthesis hy any meansand In any form or formal,making thisthesisavailabletoInterested persons.

Theauthor retainsownershipof lhecopyright inhis/her thesis.

Neitherthe thesisnor substantial extract sfrom it may beprint edor otherwise reproduced without his/herpermission.

Canada

L'auteuraaccords unancenee irrevocable et non exclusive permellan t Il la BibIJolhllqu e

natlonale du Canada de

reproduire,preter ,dlstr lbuerau vendre des copies de sathese de quelquo mantere et SQUS quelqueforme que ce solt pour mettredes exemplalres do celte these

a

18 disposition des personn es lnteressees.

L'aut eur conserve la propriatadu droil d'aut eur qui prol ege sa these.Nila lhesenides extralis substantlels de celle-cl ne dolvsnt etre lmprlmes ou autrement reprodults sans son autorisatlon.

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,,\

4\

42 3.4 Experimenta lDesignandOptimizat ion of Co-extract ion Conditions. 43

3.4.1 3.4.2

Experimentaldesign

Statistical analysis andlocation of opt imumprocessconditions 46

4 Result s and Discussion

4.1 Optimizationof ProcessingConditions for Co-extr action ofProteolytic EnzymesFromViscera of Cod(Gadu3 morhua.)

4.1.1 ModelFitt ing.

4.1.2 Overalleffect of procen variables 4.1.3 Locationofoptimumprocessingcondit ions,.

".

"

50 52 51 4.2 Co-extractionofCrudeAcidand AlkalineProteases and TheirProperties61 4.2.1 Co-extrac tionofcrude acid andalkaline proteases,. 61 4.2.2 Properties ofcrude codacidand alkalineprcteaaes 63 4.3 PartialPurificationand Characterization ofAcid Protease!From At·

lar.tic Cod.

4.3.1 Multipleformsof gastricproteases

11 12

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4.3.2 Effect of NaC !on activit iesofgastricproteeses 77

4.3.3 Estim ationof relativemolecularmass. 78

4.3.4 pHprofilesofpartiallypurified codgastricproteascs 81 4.3.5 Catalyticpropert ies andspecificactivity 82 4.3.6 Inhi bition ofproteolyti cactiv iti es by pepstati n. 85 4.3.1 Conclusions oncharacterizationofacid prcteeeee 89 4.4 Part ialPurificationandChara ct eriza.tion of Alkaline Proteeses From

Atla ntic Cod

4.4.1 Partialpurifica tionandgeneralchara cte ristics .

4.4.3 Influenceof inhi bitorsonproteo lytic adivit;es. 4.4.4 Conclusionson alkalineprcteases co-ext racted. 4.5 UseofFishProteaseslnFood Processing .

4.5.1 Proteolyticenzymesasrennetmbst itu tes. 4.5.2 Prepar a.tionoffish proteinhydrcl ysates . 4.6 Conclusions

4.4.2 Cat alyt ic propert ies .

91 91 93

93 98 99 99 103 113

5 Appendi x

6 REFER ENC ES

tv

114

12 1

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ABSTRACT

Anext ract ion process forthe recoveryof proteolytic enzymesfromthe digest ive tractofcod(Gadus morha)is described.Processingvariableswereopri m lzed by em- ploying arot at ableexperimentaldesignwith computer graphics-assisted responsesurface methodology(RSM). A simple extractionprocedurewas developedwhich effec- tivelyisolate d both acidand alkalin eprct eas esfrom codviscerawithrecoveryyields of 52% and 30%,respectively.Furtherpurificationand characterizationrevealed that thecrudeacidproteasesconsistedof thr eetypesof gastric enzymes designa t edas acid proteaseaA, B, and C.Acid proteas eB was classifiedasfish peps inII. Acid proteases A and C possessed propertiessimilar to chymosin and gastricsin,respectively.The partially purifiedalkaline protease!possessedpropertiesof trypsin- like enzymes and actedon Ncbeeacy l-Layroal neethylester(BT E E), a syntheticsubstrate for chymotrypsin.

Utilizat ionof thecrudeisolatedprcteases was testedin milk-dottingforcheese makin g,as well as prepa rat ionof proteinhydrolysatesfromunder-utili zed fishspecies.

Cod pepsin was capableofclott ingmilk efficiently at low temp era tures,whichshows itspotent ial useincold rennetingof milk.Capelin proteinhydrolysat ewitha.recovery yield of55.8% was obtainedwhen crude cod gastricprcteaeeswere used over a 4 h hydrolysis periodat ambienttem pe rat ures.

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ACKNOWLE DGEMENTS

Ma ny thanks to my eupervisor ,Dr. Fereidcon Shahidi,forfinancial supportand superv isionof thist}lesis;to Dr.D.Heeley and Dr.J.Sanoub, members of mysuo pervisorycommittee;to Dr.RyseerdAmarowicz for his continuedinterest in enzyme purificationandelectrophoresis;to Mr.RonPegg for his assistancein the prepare- tionofexperimental materials,to Dr.MingHu(Depart mentof Computer Science, Univers ityof Toronto)for hisessisteccein performingRSM methodology, and tomy wife Mindy Liu forhersupportduringthe completionof this thesis.Iam particu- larlyindebtedto Dr.E.Barn sleyfor hiscrit ical reviewand recommendatio nson this thesis.

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List of Tables

2.1 Differe nt ia t io nof fishpepsins.

3.1 Proc ess va ria blesandthe ir le velsinrotata bleexpe rime ntde-

4.1 Exp erim en tal dataforthethree-Iact or-three-le ve!respon se sur face an alysisfo rmodel co-extrac tio nsyst em . 4.2 Regress io ncoefficients for secondorde r poly nomi a lsin mo d el

co-e xtrac tion system.

4.3 Analysisof va rian c eforres p on sevariable s and overall effect dprocess va riables .

4.4 Predict edle velsof processvari ablesand resul t sofconfirmed ex p e rime nts..

4.5 Co-e xtration and pu rifica t ion sche me for acidandalkaline prot ea sesfromAtlantic codviscera..

L2

47

50

53

ss

61 4.6 Su m ma ryofcha ra c te ris t icsofthe three gastricproreeses... 74

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4.7 Conclus ionsin characte rizat ionof cod gastric prcteases. 89 4.8 Characte risticsof the isolat ed alka lineproteases. 91 4.9 Protei nrecoverybyvar ious proteolyt icenzymesandcompo-

sitionsof cape linprotein hyd rol ysa t es . 103

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List of Figures

3.1 Flow sheet (orco-extraction ofacidandalkaline prot eas es from Atlanticcodvisce ra.

3.2 Flowsh ee tforpreparation ofproteinhydroly sate,

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55 4.i Re sp on sesurfaceof enzymerecover y at differen tpHandpoly-

acrylicacid(PAA)co ncent ra tion.

4.2 Cont our plotofinflue nce ofpHandpolyacrylicacid(PAA) concentrationon theco-e xtra c tionof acidandalka linepro-

te ases.. 58

4.3 pH pr ofilesofcr udecod acidandalkaline proreas es . 63 4.4 UVsp ect r um ofacidand alkaline proteasesafterco-ext rac tion .65 4.5 Optimalreactiontem pe raturesofcrudecodacidand alkaline

pecte eees. 66

4.6 Influenceof temperat ure on the sta bilityofcrude enzym es.. 68 4.7 Sephadex G-75gelfilt ra t ion chro ma to gr a phy ofcr udeacid

proteases teem Atla nticcodviscera.. 72

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73 4.8 DEAE Sephad exA·50ion-exchange ch romatog rap hyof At -

la ntic codgast ricpro teases.

·

1.9 Estimat ionof relati vemolecularmas s(:\1.)of enzymesby gel

permeat ion chro mat ograp h)'. 78

4.tOEst ima t io nofrelativemolecularma ss(:\'1,) bySDS-PAGE 79 4.ltpH profilesofthre egast r ic proleases.. 82 4.12Substrateinhibitio nof three cod gastric pro teases. 85 4.(3Inhibition ofpepsta ti nonpro te olyt ic act ivities. 87 4.14Hydr olysis ofcod alkaline pro t ea se onsynt heticsubs trate,

benzoyl-L -a rginineet hylest e r, BAEE,and benzoyl-Lvtyrosi ne

et hylester,BTEE.. 93

4.15In hibition ofalkaline prot easesbysoybean trypsininhibitor. 94 4 16In fluenceof2-mercap toe tha no!(ME)ontheactivityofcod

tryp sin andbovinetry psin. 96

4.17 InfluenceofpH valuesonmilk-clotting time at25°C. 99 4.18Influence of enzym e conce nr retlononthemilk-clott ingtime . 101 4.19 5D S-PAG Eofcope lin proteins and theirhydrolyaa tes 106 4.20Hyd r olys is ofca pe linprote inswit hdiffe rentenzyme s. 108

vi

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AE

APDT BAEE

Listof Abbreviations and Symbols Acidicproteolyticenzymes Acety l-L-phenylalanyl.L-diiodotyrosine Beneoyl-Lerginine ethylester BAP NA

BE BTEE

BU CPR CPU

DFP DMSO FPH RPU MCU ME M.

PAA pI PMSF

a-benzoy1-0L-arginine-p-nitroanilide Alkalineproteolyticenzymes N-benzoyl-L-tyrosine ethy lester UnitofBTEEact ivity Capelinproteinhydrolysate Caseinproteolysisunit Dalt on(Unit ofmolecular weight) Diisopropylfluorophosphate Dimethylsulphoxide Fishprotein hyd rolysate Haemoglobin proteolysisunit Milkclottingun it 2-Mercaptoet ha.nol Relativemolecula r mass Polyacrylicacid Isoelectricpoint Phenylmetb ylsulp bonylHuorlde

vii

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RA RSM SAS SST!

SDS SDS·PAGE sop

TOA Tri!

~.

lEI

K~.

R' [51 V•••

Relativeactivity Responsesurface methodology Statisticalanalysissystem Soybean trypsininhibitor Sodium dodecy lsulphate 5DS-polyacrylamidegelelectrophoresis Seal gastric proteasea

Trichloroaceticacid Tris{hydroxymethyi]amino methane Regression coefficients Enzyme concentration(w/ v) The average hydrophobic.ityofaprotein Michaelis-Menten constant Protein turnover constant Correlationcoefficient Substrateconcentration Maximum velocity of enzyme reaction

viii

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Chapter 1 Introduction

The managementofthe fishery resourcesand new productdevelopmentusing underutili zedspeciesarcmajorconcern sfor the worldleading fish-prod ucingand fish-exporting countri es. Oneof thepotential problemsassociated with increased fishingisincreasedproduction ofprocessing discards and fish offal.Approximately 100million metric tons offish,invertebr ates,andmolluscsare la nded annually.The ut ilization of thecatch isIarfrom opt im al,as onlyabout70%of thetotallandingsare usedfor human consumption(Sikorski and Pan,1993).Ofthis,theaverageprocess yield is15-30%.Thus, thereisa large amountofprocessingby-products available.

In Canada, approximately 50% of the fishprocessing discards areusedinproduc- tion offishmeal,while theremainderrepresents a disposal and pollution problem (Simpson andHurd,1981). Althoughtherearemanyapprceehes tofurt her utilize fishprocessing wastes , interest hasbeenexpressed inisolating added-valuecompo-

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nents fromsuch raw materia l. Digestivepreteases are apossiblegroup ofcompounds which couldbeeffectivelyisola ted and characterized.

Enzymes play animportant rolein industrialmuufacturingandprocessing, par- ticularlyin the food industry(Knorr andSinskey,1985).Enzyme productioniscur- rentlyexperiencing un precedentedgrowt handexpansion(Wasserm an,1990).The valueof theworldenzymemarkethas increased fromS 220 Min.1960,S400M in 1970, S 550 Min 1980,S1,.000 M in1985toanapproximat e value of $ 2,000 Min 1990 (ChaplinandBueke,1990). Thi!rapidincreasei. due to theavailabil·

ity ofalargenumberoi enaymesatre:ativelylow cost,andtheirpotentialusein a varietyofapplications.Of theenzyrnCllinUI!e,proteasesconstit uteanimportant groupwithglobal ,.desrepresentin gnearly 60% of thetotalenzyme mar ketvalue (Godfrey andReichelt,1983).Utilization of proteases intheiood indust ry includes cheesema.nufactu ring(Ville r,1981;Law and Goodenough,1991), mea t tenderization.

(Fawcett andMcDvwell,1987;Etherington, 1991),beerchill·p roofing{Lea,1991), flavourdevelopmentinprocen ing(ln, 1990),modification ofviscoelasticity ofbread dough(Hamer,1991), as wellas productionto!thefunctio nal proteins (Feeney,1977;

Whitaker, 1980; Han,1989; Shahidi et41.,1993). Prot eolyt icenzymeshaveclini- calimportan ce intheproduefic n of protein.hydrolysatesfortherap euticdiet!(Free, 1980; Adler-Nissen, 1986),and are alsovaluableaslaundr y detergent additivesorin enayrne-based cleanersfor ultrafiltrationsystems (Smith end Bradley,1987).

Proteases fromvarious sourcesdiffergreatlyintheir catalyticandphysicalprop-

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erties;suitabilityof a particular enzymefor a specific industrialapplicat iondepends on severalfactorsincluding specificity, reactio n conditions,response toinhibitorsand activators,costand theavailabilityoftheenzyme andtechnical service support.

Earlierinvestigations onfish proteases werefocused onthe influenceof enzymes onthe propertiesand quality of fish duringprocessing andstorage. Onlya few studies have been carried outtoexplore the possibilityofenzyme utilizationin food production,such as processing of fishproducts.Inrecent yearssome intere9ting new applications of enzymes in fishproceeeinghaveemerged.1 his is,in part ,dueto an increasedknowledgeof enzymes and their uses,but also because manyenzymes, including fish enzymes, have becomecommercially available.

Theapplicationof marine enzymesin a varietyof epplicatic ns is ofinterest. Prete- olytic enzymesfrom cold-ada ptedfish visceraare moreact iveatrelativelylowtemper- aturesand are lessstable thermallythan theircounterpar tsfrom warm -bloodedland- based animalsandplant sources (Simpsonand Haard,1987).Theselow-tempera t ure- activeand thermallyunstableenzymesareexpectedto havesufficientlyuniqueprop- erties tojustify theirapplicationin certainfood processingoperation s.

Codviscera(stomach, pyloric caeca and intestines)constit uteapproximately 7%

of thefishweight. Therefore, a large quantityof viscerais available forrecoveryof proteolytic enzymes. Recently,crude codpepsinpreparationshave beenproduced commerciallyby a NorwegianCompany(Marine Biochemicals,Tromso) (Stefann on and Steingrimsdotti r,1990).InIceland,extensiveresearch and developmentprojects

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in the area of enzymeproductionand ut ili:::ation have been conducted.The main emphasisison the productio n ofenzymesfrom thermophilic and psychrophilicmi- croorganismsand fromthe viscera of cod,Gadusmorhua,(Stefansson,1988). This latterresearch groupisinterested inotherproteolytic enzymes such astrypsin and chymotrypsin(Stefensscnand Sieingrimsdott ir,1990).

The currently establishedextract ionprocedurescan onlyisolate one ofthe diverse variety of digestiveenzymesin eodviscera . Thereare many unresolvedconcerns as- sociatedwith enzyme cheractenaationandtheir catalyticmechanisms. Furthermore, establi shinganisolationtechnologythat can co-extract most ofthe enzymes presented andsuitablefor scaling-upis wanted. The present studyhas focused onpresenting a simple andeffective procedureforthe isolationof bothacid and alkalineproteases from cod viscera. Theextracts couldthen be used directlyfor food processingor may be further purifiedforspecific applications.Use of the isolatedproteolyticenzymes in food processingwas nlsoconsidered.

The objectivesto the present studywere: (1) toestablish anisolationprocess that can co-extract acidandalkaline protease!fromcod viscera,adaptableto large scale production;(2)toop timize processingconditionsfor theco-extractionprocedure established;(3) to purify (or partiallypurify)and cbaracterize theenzymes isolated in orderto evaluatethe co-extractionprocedure;and (4) to apply isolatedproteases for milk.c1ottingin cheesemakingas wellasproduction offishprotein hydrolysates.

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Chapter 2 Literature Review

There: isa.renewed inte rest inthestudy

or

proteolyticenzymes, mainlydue to the recognitionthat proteolysisplaysanessential role inmany cellularprocessessuch as digestion,translocation ,proteinturnover, secret ionof proteins,and the activationof many tcxina ofimportanceto foodscientist , such as the neurotoxinssynthesizedby Clostridium botulinum(Loffler,1986).Enzyme technologyhasevolved to becomean integralpart ofthefood industry,andenzyme productionis currentlyexperiencing unprecedentedgrowthandexpansion.Recent technologicaladvancesin enzymology suggestthatthere:maybe no limitto the kinds of reactions enzymescan catalyze (Wasserm&.J1, 1990).

Amongcommer ciallyavailable enzymes, proteaeesconstituteapp roximatelybalf ofthe worldenzymeproduct ion. The term proteolyticenzymeor proteaseissyn- onymouswithpeptidaseandthese terms refer to a.ll enzymeswhichca.talyzethe

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hydrolysisof peptid e bonds inpolypep tidesandproteins.All prcteeses are'intra·

cellular'at some stagein theirexist ence(Bondand Butler, 1987).Some proteases aresynt hesizedforexport to extracellularspaces,and exert theirbiologicatattionas discreteentitiesoutsidecells.

Fromananalysisoftheir in-vitroproperties,proteaseshave beenclassified ina numberof ways.Onthe basis of~h.epHrangeo'lerwhichtheyareactive, prcteasesare cleaelfiedasacidic,neutral andalkaline.Based on theirability to hydrolyzespecific proteins, theyare classifiedaskeratinase, collagenase, elastase,etc.,Accordingto their similaritiestowell-characterized enzymes, protease! areclassified as trypsin, chymotr ypsin, cathepsin, chymosin,et c..However,the mostsati sfactory classification isbasedontheir mechanism of adion(Hart ley,1960).Thisclassificat ionwhichis usedby the Enzyme Commissionconsistsof four distinctclassesof serine,cystei ne, aspartyland meta llcproteaees.

Protease!may alsobe subdivided into exopeptidases,whoseaction isdirected by the amino- orcarboxy-terminus ofthepepti de(EC3.4.11.19), and endopeptidases, whichcan cleavepeptide bondsinte melly and usually can not accommodate the chargedamino- orcarboxy-ter minal amino acids near the activesite (EC3.4.21-24, 99).ftbasbeensuggested that the termendopeptidasebe usedsynonymouslywith protease,and this recommendation isadoptedthroughout in thisthesis.

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2.1 Prot eolytic E n zymes Fr om Dige st ive Tract s o f Marine F ish

Stu diesonproteaseactivityof enzymes present in fishviscerabegan in thenineteenth centu ry. Stirling (1884)showed tha t extr acts from cod andherringstomachwere ableto digestfibrin inthe presenceof dilute hydrochloricacid. Vonk(L929)found that thepepsincont ent of pike stomach washigherthan tha tinthe stomach of mammalsinvestigat ed.Hesuggestedthatfish producedmorepepsinto compensate for reducedactivitydue to low secretionofgastricacid(Vonk, 1929).Sincesalmo n pepsin wasfirst crystal1izedin 1939(~orri5andElam, 1940), gastric enzymes from severalfish species havebeen purifiedand studi ed (Norris and Methies, 1953;Kubot a andOhnuma,1970; Nodaand Murakami1981). Proteolyticenzymes wh.ich have been widelyinvestigated include serine(e.g. trypsin-like)prcteasee,and aspartic proteases (e.g.pepsin,cathepsinDeec.),alth.ough othertypes or prcteas essuch as pancreaticmetal1oproteinase(Yoshinakae~al.,1984; 1985a andb)haveoccasionally beenexamined.

There have beenseveralexcellent reviews on the proteolyticenzymesmentioned above(Barrett , 1979;Foltmann,1981;Bond and Butler,1987;Gildberg,1988;Hurd, 1992).Sincethepresent studyincludes aspartyl(pepsin , orpepsin-like enzy me)and serineprcteeeee(t rypsin,ortrypsin-likeenzymes),thefollowingreview focuseson these two classesof prcteeeee.

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2.1.1 Aspartylprotease s

Aspartylprcteases (EC 3.4.23) constituteone of the fourmain classes of enzymes present ineukaryotes thatact on interior peptidebonds of proteinsand oligopep tides underacidcondition'.Becauseoftheir optimalactionunderacidiccondition" as·

partylproteases arereferredto&IIacid peoteesee.Sinceit was found thatthetwo functional groups of catalyt icsite of theseenzymesare alwaysaspart icacid ca rboxyl groups,thenewname aspartyl proteasehasbeen adopted.The term'aspartyl protease' is moreappropriatethan'acid protease'because someenzymes suchas rennin, now known tobean aspartyl protease,donotnecessarily belongtoacidproteue groupand their optimum pHis around6-8(Fruton,1981).

The aspartyl protease.ofgastricjuice are all secreted&lIinactive precursor- zymogenl. These zymogen. areirreversi blyconvertedinto active enzymelby reo leasingpeptidechains (&Cti vationsegments){rom NHrtmninal.egments(Folt man andPedersen,1971). No zymogen'formicrobial aspartylproteases havebeenfound.

A.partylprotcues include pepsin, renin, cathep.in. 0andE,aswellatsome microbialproteases. Membersof the pepsingroupinclude pepsinsA,B,C,and0 (Foltmann,1981). Chromatographicseparationofthecomponents hasshewnthat the predominantpepsinA(usuallydenoted pepsin) of adultmammalsisaccom panied by pepsinC(thecurrently-preferred name isgastricsin),aswell as by the minor components denotedpepsinBandpepsi n0(Ryle,1970).Thesevariousaspartyl proteaseshaveincommontheprop~rtyofcleavingproteinsandsuitable oligopep tides

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at pH1.5-5.5. A widely used diagnostic test is theirinhibitionby the natu rally occurringpeptidepepstatinand by active-site-directeddiazo compounds.Available structuraldat aindicatethatallaspartylproteesesbelong to one family (Huang et c.l.,1980).Theenzymes genera lly have molecularweights inthe range of 30,000to 40,000D, andpossess bilcbalstru cturescomprisedof mainly ,a-sheet with acleftthat contains theactivesite (Blundell dai.,1980).

It is difficultto obtain a highlypurifiedgastric proteases due to the occurrenceof autodigestion. The most homogeneouspigpepsinis obtainedby a shortactivatio n of purified pepsinogen, followed by column chromatography at 4° C.Underthese conditions, minimalautodigestionofthe active pepsin occurs (Foltmann,1981). At present,the preferredmethod for thepreparationof apparentlyhomogeneous pepsin israpidacti vat ion of crystallized pepsinogen, and passageofthe mixture throug h eulpboethylSephadex C·25 to remove peptides and then through Sephadex0·25 to remove saJts(Fruton, 1987).However , accept a ble results havealsobeenobtained from startingmaterialsconta iningactive enzymes(Foltmann,1981).

Scientists fromdifferent research areas have different interestsinthese enzymes.

Biochemists are mainly interestedinthepepsin and pepsin-likeenzymesfromthe stomach ofverte brates ,includingmammals. However,both pepsin-likeenzymes and cathe psin Dhavebeen subjectto considerable investigationloy marine biologistsand other resear chers;cath epsinsareconsidered to beimport ant inkeeping quality of fish duringproce eemgand storage. Recently,foodsciet'.tists haveengagedin therecover y

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10 of marineenzymesfortheirsubsequentutilizationin the food industry(Haard, 1992).

Cast ric digestion is consideredto be theonlyphysiologicalrole of fish peps ins (G ildberg, 1988). Therefore, fish species which lack a stomach or have a stomach

withoutsecretionof gastricadds do nothave pepsin (Kapoore!aI.,1975).In such fish, both trypsin and cathepsin!take part in digestionat close toneut ral pHs(Jany, 1976).Itis nowwidelyacceptedthatcathepsinsplayan important rolein digestio nin many invertebrates (VcnkandWestern, 1984). SincecathepsinD isone of themajor tissueprotease!(Barrett , 1977)and possiblythe most abundantacid proteasesin vertebrates (Ikedaetal.,1986),theact ion ofth isproteolyticenzymein fish hall been studied for several decades.

Most fishbelongingto the OsteichthyesandSelachiicla.sses possessetcmachs whichsecrete HCIandpepsinogen(Mernettetal.,1969;Twining, etai.,1983;Aru n- chalam andHaard, 1985),and ithas been characterizedfromcod(Breweret11.1., 1984), salmon(Norris and Elam,1940),dogfish(MernettetaI.,1969),hake(Sanchez·Chi ang andPonce,1981), andtrout(Owenand Wiggs,1971).Asparticprcteaeesfrom the gastricmucosaofharpseal(pagophilw groenla ndicus)have alsobeenpurified and characterized.FoureymogeneofacidicproteasesA, B, C,and0wereisolated(Sham- suz zamanandHaar d ,1983). Threezymogensofgastricproteaseshave beenisolat ed from the sto machmucosa.of Greenlandcod (Gadus 09ac)by exclusion chromatography and chrc rnatofocuslng (Squiresd11.1.,1986a).

Researchhallproventhatmany fish speci essecreteatleast two pepsins with

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I!

differentpHopt ima(Noda and Murakami,1981;Gildbergand Rlla,1983; Martinez and Olsen ,1986). Apart from theconfusioninthe mammaliangastric protein a, these pepsinsare usuallyreferredtoasfishpepsinIand pepsin II(Gilclberg, 1988). Fish pepsinswhich havebeeninvestigated and classifiedintothese two groupsare summarizedinTabl e 2.1.

2.1.2 Serine protease s

Theserineprotease!comprisea large group of enzyme swhichis distingu ishedby the reactivi tyofaserineresidue inthe activesite (Hartley,1960). Most members of thegroupare endopep tidases.Theseri neprotease!also exhibitstrongesterolyt ic activitytowardestersanalogoustothespecific peptidesubereere. a fact withlittle physiologicalimportance, butone used by biochemi sts inkineticstudies(Mathews andvanHolde,1990). Acommontest forthese enzym esis the inhibitionoftheir hydrolase activit y by th ereactionof serineresiduewithdilsdpropylflucrophcep hate (OFP) (Walsh and Wilcox,1970).

Comp arisonsofrelative rates ofcatalysisusing avarietyofsyntheticsubstrates haveleadtothedefinit ionofthe specificitiesofrepr esent a tive members of the class.

These ran ge fromthena rrow specificity ofthe trypsins,which isdirectedtowardthe bondon the carboxyl sideof arginine andlysine, to therather broad specificity of the subtilisin s whichwill attackbondsbetweenawidevarietyof aminoacids.Ser- ineprcteaeeethathave beenchara cterized in prokaryot esandeukaryotesbelong to

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12

Table2.1: Differ ent ia tion of fish pe psi ns ITEM S Pensin I Pe usinII Re fer en ce s Molecularweight (D)" >35x10

s

35X10³ GHdberg,(1988) Relativeamount:^b

inSardine major mino r Noda and Murakami(1981)

in Capelin major minor CildbergandRaa(1983)

lseelecee!cpoint(pW 6.5-7.0 closeto4.0 Cildberg(1988) pH optimum :" (3-4) (2- 3) Gildb erg(1988)

Cod 3.6·3.7 2.5 - 2.8 BjellanddaL(1988)

Sardine 4.0 2.0 NoclaandMurakami 1981

Ci\pelin 3.7 2.5 Gildberg and Raa.,1983

pH sta bil ityinstrong'\,:id

andneutral conditions less stable stable Gildberg(1988) Temperature optimum:

Capelin low high CildbergandRaa(1983)

Cod low high Martinezand Olsen(1986)

Specificactivity(U/mg):"

Atlanticcod 3,000 2,000 Gildbergand Almas(1986) Capella 1,100 2,000 Gildhert> andRaa(1983) InfluenceofNaCl':

Americansalmon activated unaffected Sanchez-Chianp;et al.(1987) .. AccordingtoSDS.polyacrylamid:::gel electrophoresis.Thesymbol0refersdalton.

6 The seasonal variationsinrelativeamounts may besubstan tial (Squireset al., 1986a );

CMammalianpepsinshave much[ower plthan fish pepsins,and valuesas [ow as 1.0 havebeen reported(1'iseliusetal., 1938);

<IpH optimu mW'\.Smeasuredusinghaemoglobin as asubst rate;

• Theacti vitiesweredetermined by 111incubati on atpH 3.0and 25⁰C using haemoglobinasa substrate.

'Influence of NaCIon fish gastri cprcteaseswerealsoreportedby Squiresetal(1986b) andSaochee-ChiengandPonce (1982)using different classificationsystems.

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L3

thechymot rypsin andthesubtilisinfamilies(Barrett,1986).Onlythe chymotrypsin familyhasbeenfoundin eukaryot es. The ch.ymotry psinfamily includesmanyex- tracelLular prote ases such.astrypsin, elastase,thrombin,plasmakallikrein ,plasma coagulationprcteases, andcellul~rprotease (BondandButler,1987).

Serineprot easesarewidelydist rib utedinnature. Asurvey of prot eolyticen- zymes in variousspecies of fish digesti vetractshas revealedthatse rineproteasesate distributedi,1fish.intestinewith a highactivity at alkalineratherthan neulralpH.

In teleostfishes, whichdo nothave a distinctpancreas, serineproteases havebeen foundin thepyloric caeca,butonlyintheactiveform(Walsh andWilcox,1970).

Proteolyticenzymes distributed in theintestinalorgansofmarine verte brateswere generallysimilarto those of mammals,such as trypsin-orchym"c.rypsin-likeenzymes intheintestine and pancreas ,and pepsin-like enzymes inthe stom ach.

Trypsin-andchymot rypsin-likeenzymeshave amolecularweightof appr oximately 25,000D (Prahl andNeurath, 1966;Reek andNeurat h,1972; CohenandGertler, 1981),a high iscelectricpoint (pI), andtheir actionisinhibited byphenylmet hyl- sulpbonylB.uoride(PMSF)(Fahrney andGold, 1963;Jany,1976),soybean trypsin inhibitor(SBT I),andaminoacidderivativesof chloromethylketone (Gatesand Travis, 1969;Camachoet4l.,1970;BondandButler,1987).Itisbelieved thatthestruct ure of theactive site offish pancreaschymot rypsin is simila rto that of other verteb rate formsofthe enzyme (Barnard andHo pe, 1969).Chymot rypsinfromthe pancreas of carphas been foundtobe similarto mammalianenzymesinbot h physical andki-

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14 netic properties,including inhibition patterns(Cohen and Gertler,1981;Cohenet al., (98 1). However,chymotrypsin,isolatedfrom the pyloric caeca of herring and capelin (Kalac,1978)and from mackerel(Ooshriro, 1968) werefoundto have greater relati ve activitieson peptide and caseinsubstrates than bovineo-chymctr ypein.Theactivity of dogfish chymotrypsinwastwotothreetimes higbeethan bovine chymotrypsinin the hydrolysisof collagen and otherproteins(Ramakrishna andHultin ,1987& and b).

Trypsin has been found in pancreatic tissues of all the~speciesl^frominverte brate s to rnemmals (Walsh, 1970; Kapooret aJ.,1975). Trypsin-like proteolyticenzymes have been purified and characterized in several fishspecies including African lungfish (Reeck and Neurath, 1972; deHaenet:al.,1977),mackerel(Pyeun and Kim, 1986), berring(Kalac,1978),carp(Coben and Gertler, 1981;Cohenetal.,1981),sardine (Murakami and Noda., 1981),cepelln [Hjelmelaadand Raa, 1982),catfish (Yoshinaka et al.,1984, 1985a), chum salmon (Uchidaee al., 1984a and b),eel (Yoshinakad aI., 1985c),Greenlandcod(Sim pson and Heard, 1984a and b),Atlanticcod[Ovemell, 1973; Simpsond al.,1990),andanchovy (Martinez et at., 1988).A commercially Available procedure for the recovery of crude trypsin or trypsin-likeenzymesfrom cod Gadus fflOThuQhas been reported to be in progress (Stefanssonand Stei!l.grimsdottir, 1990).

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IS

2.2 Cold-adaptedFish Proteases

Poikilothermicorganisms from temperateregions usually undergoanesthesiawhen encounteringenvironm ental te mperat ures near 0⁰C.However,certainpoikilotherms remainactiveatsuch lowtemperatur es (Simp so n andHaard,(987).Theprocess bywhichd.speciesadapts toaspecific ther ma l environmentove rmany genera t ions hasbeendescribed as'evoluti onarytemperat urecompensation' (Hazeland Prosser, 1974).Asa consequenceof cold adaptation, digest iveproteolyti cenzymesfrom ccld- adaptedfishare moreactiveat low tempe ra t uresthan theircounterparts derived from vertebrat eand thermophilic organisms (Hultin, 1978;Haardet al.,1982; Arunchalam and Haard,1985;Simpsonand Heard, 1987).

Trypsin from cod(GadulJmorhua) was more heatlabile witha.rela tivelyhigher activityatlow reactiontemperaturesthan bovine trypsin,which was considered to be bett ersuit edto producefishproteinhydrolyeatesat lowtemperatures and for pre- venting copper-inducedoxidat ionof milk (Simpsonand Hurd,1984a). Incomparison with mammalian pepsins,pepsins from coldandtemperate waterfishhavea higher activityatlow temperatures (Kitamilc.do and Tachino,1960; Gildberg and Raa, 1983),and express a much lower temperaturecoefficient (Haa rdelal.,1981;Brewer etai.,1984). Thisparticulartempe-ctureproperty of proteolyt ic enzymesfromcold- adaptedfish has been exploitedin certain food processing operat ions(Haard ,1992).

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16 2.2.1 Enzy m icadaptabilityon genetic basis

Attemptshave beenmadetoelucidatethebiochemicalpropertiesof cold-ada pt ed fishprotease! aswet!as thenatureofenzymicadaptationtothe coldenvironment.

Poikilorher meadaptedto cold temperatures exhi bit enzymicadaptabilitybyincreas- ingthelevels ofenzymes present in the system ,orbyevoluti onaryadaptab ilityof preexistin genzymes(Somer a,1969;Somero,1971). Theconservationof abasic physiological function ,together with theachievementof coldadaptab ilitymeybethe resul t of appropriate alterat ionsinthe prim arystructureof the enzymebecau se the primaryst ructu re determin es thedistribut ion of possible tertiarystructuresin certain environments.A modelof mutation-adsorp tionha.,beenintroduced toelucidate the principlethatgradual treasformeficnofshap e and functionresults from changes inprimarystructure(Conrad,1979). The main conclusioninthis model is that a subsystem isembodiedinthe moleculewhichservesas abuffer,absorbing mutation orotherformsof geneticvariati ons and expressing these asvariationsinfeatu res of the shape crit icalforfunct ion. The condit ion for aneffecti ve evolutionary response to selectionisgradueliamof functionchange inresponsetoprimar y str ucturechanges (Conrad, 1979).

Proteinengineeringhasrevealedthat thefunction of enzymes,includingther- mostability,tempera tureoptimumAndkineticpropert ies could becont rolledin a predictablefashion byapplying site-directed mutag enesis(Ulmer, 1983).Sit e-directed mutagenesispermits a lingle or a fewselectedaminoacidresidues in a specificenzyme

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17 to be preciselyaltered(Nosoh and Sekiguchi,1991).

2.2.2 Re lat io nshipsbe twe enmolecularstruct ure,stab ility and catalytic efficiency ofenzym es

Underconditions of low temp eratu res,the catalytic efficiency ofmostcold -adapted poikilothermic enaymesishigherthanthatof theirhomologsderivedfrom warm- tem perat ure-adaptedorganisms(Low and Somere,1976;Simpsonand Haard,t984b).

Thegreatercatalyticefficiency of enzyme sfrom cold-adaptedpoikilot hermsat low reactiontemperatureshas beenattributed to theirrelat ivelymoreflexiblestr uct ures whichperm itthem to undergoconformationalchanges thatfavoura hig herreact ion rate (Somera,1915).

Thegreate rst ability of proteinsfromthermo philicorganismshas beenattributed tothepresenceofgreater hydrogenbonding(Koffler,1957),moreextensivehydro pho- bicinteractio n(SingletonandAmelun xen, 1973),and morepronoun ceddisulphide linka ges(Komats uand Feeney,1970).The foldingofa polypeptidechainisdriven by thestrongtendency ofsequesteringhydrophobicsidechains fromsolvent,which arefollowed oraccompaniedby variousnon-covalentinteractions such asHcbcndi ng, elect rosta ticandhydrophobicinteractions,and van derWaalscontacts,betweenside chainsand betweenmainchainand sidechains of polypeptidesand protei ns[Noeoh and Seikiguchi, 1991).The three-dimensionalstr uctureisfinally formedwitha deli- catebalance between the stabil izingfactora drive nbytheeenon-covalent interactions

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18 andthedestabi lizing effectoftherma lenergy.Heat, orden atu rant ssuchasurea and guan idinehydrochlorid ecause coope rati veunfoldi ng ofthe protein (Tan ford, 1968).

Nogeneralizedmechanism for protein sta bility hasyet beenpresented {Noeob and Sek iguchi,199 1).

Adetailed investigat ionon comparativepropertiesof Green lan dcod and bovine

t~ypsin9indi cat edthat Greenlandcodtrypsin had a less-ordered struct ure than

bovinetrypsin (Simpsonand Haard,1984b). Furthermore, itwas noted that 1) at 0.2 to 35.3° C,Greenland codtrypsincont ained 7.3·7.8% a- he lix and92.2·92 .7%

random coil, whereasbovine trypsin contained 11.5-12.0%a-helixand 88.0·89.5 % random coil; 2) Greenlandcodtrypsin had only8 cysteineresidues as opposedto 12in bovine trypsin ; and 3)theaverag ehydrophob icity (H1J..uc)of Greenlandcod trypsin(0.86 kCal/residue)was lessthan that of bovine trypsin(1.04kCal/residue).

Enzyme-catalyzed reactions occur only afterthe contactof enzyme on an appropriate position of thesubstratemolecule.Ithubeenreportedtbat subst ratebind ing affinity of enzymes,bothintracellularand extrace llularprcteese e,is temperatur e- dependent(Hazel and Prosser, 1974). Hultin (1978)observedfivedifferenttypes of temperaturedependent affinityrela tionships for enzymes from different sources.

Digestive enzymes withtemperature-dependentKm valuesnave been discussed and reviewedby Simpson andllaard (1987).

Thedifferent bindinga.ffinityof substratefor enzymeshas been explained interm s 01enzyme-substrateinteractionswhich arestabilizedby aaet of weak bends which

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19 exhi bit opposing stabilitycharact eristics as afuncti on oftempe rat ure(Sim p sonand Haard, 1987). Itisconsideredthattheformat ionof hydro gen bonds andelectro- st atic interaction sproceed exothe rm ically andwilltherefore bemoreimport antat lowtem pe ratu res.In contrast , hydrophob icbondsform endotherm icallyandpre- vailatelevated tem peratu res.Aminoacid composition and averagehydrop hobicity dat ahave demonstra ted thatproteins from thermophilic organisms havedistinctly higherhydrophobicitiesthantheir mesophili c counterparts(Bigelow,1967; Hazeland Prosser ,1974).

2.2 .3 Multipleforms ofenzy mesan d thermal com pe nsatio n

It has been a common view that thermal compensation mechanismsin poikilotherm, allow their metabolicprocessesto continued rates that arerelativ elyindependent of temperature.Poikilothermsadaptedto cold environments exhibitenzymicadapt.

abilityby: (1)increasingtheconcentratio ns of enzymespresentin the sys te m,(2) changingthetype of enzyme presenti~thesystem,and (3) evolutio naryada ptability of preexisting enzymes(Somero,1969; HochackaandSomero,1911;Owen and Wiggs, 1971).Poikilothermsexhibit these differentthermalcompensation mechanismsin reo sponee toenvironmentalchanges.

Themeetimmediateresponse of a poikilothermto a cold environmentis to gen- cratemoreenzymesfor keeping itsmetabolicratesrelativelyconstant.Quantitati ve differenceshavebeen observedwhenmeasuringthe same fishspeciesat differenttern-

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20 peratures(Smit,1961;Olll'enand Wiggs, 1971).The increased pepsinogenquantit ies observed asaresultofcold acclimationinthese stu diesprovideatlea.st putial com- pensationfor lowered temperat ures.Since pepsioo!cnsecretingcells dischargeall accumulatedzymogen granulesuponst imulatio n(Hirschowit z, 1957),itisconsidered that tbeincreased amount ofpepsinogen wouldenhancedigestionratesin cold acclimated fish species.

Existence of mult iple forms of fish pepsinogen! is also rela tedto temperature ccmpenaatic n. ArunchalamandHaa.rd(1985) observedthat Arcticcod pepsinhad two isozymeforms havi ngmar ked differencesinKm•One isozyme form of Arcticcod pepsinhad aK...similarto that

or

^{porcin e}^{pepsin and}^the^other^one^had^{a very}^high

K...withacorrespondinglyhighV...The dige.tiveenzymes andtheir corresponding geneshave been modified durin gacclim ationand mutation,from "..,hichtheirtherm al and kinetic properties have changed a.ad quanti t ieshavebeee altered to adapt to the new environment.Theoccurrenc eof multiple form.ofgut rie proteaseewhich hu beenrepor ted for othermari ne species include:nrdine[Ncdeand Murrablni, 1981),harpaeal (Sha.m.unamanand Hurd,1984), Atlanticcod.(Bre weretal.,1984), Greenland cod (Sqiure.doi.,19861.),and dogfish (Gue rardand Gal,1987).

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21

2.3 Utilization of Proteases From Marine Fish

Theuse of enzymesis wellestablishedand widespreadwithin the foo d industry, The propert iesofen...ymesmakethemideal toolsforthe manipulationofbiological materia ls.However,of thethousand sofenzymes described sofarby biochemist s,only a mere handful of enzymes areactuallyusedcommer ciallyin the food industry.This is dueto awide rangeofreasonsincludingunsu it able reactionconditions,instability oftheenzyme duringprocessing,orthe prohibitivecost involved in obtaininglarge amountsof sufficiently pureenzymes.

Traditionally, enzymes havebeen used for variousfood processingapplicat ions suchasmeattenderizat ion (Fawcet tand McDowell,1987;Etherington ,1991),baking (Hamer,1991),and cheese production(Visser, 1981; Law and Goodenough,199t). However, enzymes can theoret ically be used in almostall food processing eperariona in which a biochemicalor chemicalreaction takes place.Among theenzymes employed in the food industry,proteesesare most exte nsively used for improvingthe quality, stability,andfunctionalitiesof foodproducts. Thetopic on proteolytic enzymes from marine organisms andtheir applicationin food processing has beenreviewed by Mohr(1980), Simpson andHaard (1987),Stefll.nssonand Steingrimsdo ttir(1990), andHa.a.rd (1992).Utilizationof protease!discussedherearelimited to the use of isolatedproteaaesas rennet substitu t es formilk-clottingandfor preparationoffish proteinhyd rolysates.

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22 2.3.1 Utilizationoffishproteases as ren net su bst i tutes

Theuseof biocat.1l.yst. intbefoodindus tr)',esp cci.J.' yinprepar ationof dair y products, is oneof theoldestexamples of bioteehnology. A typical caseistheuseof proteolyti c enzymesinthe prod uctionof cheese. Oneof the key stepsin cheese mao' ufacturingisthe enzymat iccoagulat ion ofmilk.Additionofappropria te enzymesto milkleadstopar tial proteolysisof x-ceseinwhich des ta bilizesthe casein micelleand brings ahoutcoagulation of the milkproteinstoformthecurd.Theidealenzyme for thisconversion isrennet(m ainlychymosin)which is obtainedcomme rciallyfrom the fourthstomach(abomuum)ofunweaned calves.Chymosinisahighlyspec:ific endoproteinaee,which splitsthe e-ceseininto aglycoma.cropeptideand para./t.cuein by selectively cat alyzingthehydrolysis of thebondbetweenphenylalaninelOSand methionine 106(Berridge, 1951iFox,1969iDalgleish. 1982).Calfrennet,consisting mainlyof chymo,sinwitb a smallbutvariableproportionof pepsin, is arelatively expensiveenzyme for thecommercialproductionofcheese.

Attemptshave beenmadeto lindvarioussubsti t utesforcalf rennetdueto&

declinein the number ofslaughtered calves andanincrease inthedemandforcheese (deKoning,1978). Rennin substitutesobtained from microbialoriginhavenow been acceptedbythe indumt ry. However. alimita tion of microbialrennetisthe relativelybroadspecificityof theenzymespresent. This is oneofthe major problem.

usociatedwit htheuse ofproteasesforcoagulating milk.Sincemicrobial proteases

arenot&Ispecifica.rennetandbringaboutmore hydroly. is,it may producebitter

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23

peptide! orleadto destabi lizat ionofthe curdgeL Anotherproblemassociat edwith the development of microbialrcnneteis temperaturestability.Chymosinis arela tivel y unstable enzyme thatlosesmostofitsactivity during com plet ion ofitsfuncti onin milk-clotting.The enzymefrom microbia lsourcessuch as Mucorrniehei,however"

retains its activityafter clotting and isst ill act iveat the maturationstagesof cheese- making which mayprod uce bitteroff.flavours.Attemptshavebeenmade to clone chymosinintoEscherichia coliandSaccharomycescerevisia eandthe enzyme has been secretedin an activeformonlyfrom the latter(Chaplinand Becke,1990).

Chickenpepsin

nat

also beenemployedasa rennet substitute(Emmons et aI., 1976;Stanleyand Emmons,1977; GordinandRosenthal,1978).However, cheddar cheesepreparedwiththis enzyme may acquire intenseoff-fla.vours. Use of gastric proteasesfrom marine species as arennetsubstitute has also beenexamined.Fish stomach containssubstantialamounts of proteasesactiveatless acidconditionsthan mammalian pepsins.Morerecentresearch hasrevealed that many fishspeciessecrete at leasttwo pepsinswithdifferent pHoptima(Noda andMurakami,1981,Gildber g and Rae, 1983;Mart inezend Olsen,1986). Somegastr ic protease,from marine specieshave charact eristic ssimilar tothat ofchymosin in clotting milk at higherpHs (Shamsuzzam anandHu rd, 1983; Brewerel al.,1984).rthasbeen reportedthat dogfishpepsinIIcanclot milkeffectively evenat pH6.8 (Guerard and Carl,1987).

A crude preparationof gastr ic proteasesfromharpseal(PhlXBgN~nlBndiclI)was found tocoagulatemilk over awiderpH range than porcine pepsin(Shamsuzzama n

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24 andHurd, 1983).Simp legutriemucosalextrncts fromyoungharp seal contained four zymogen!of acidic proteaeesA,B,C, and D. ProteaseA was.a chymoeln-Iike ratherthana.pepsin-likeenzyme (Sbamsuzuman and Haard,1984).Cod pepsin, whichexhibits geetr icain-like properties,iswell suitable forthe cold rennetingofmilk because of its relativelylow temper atur ecoefficient(Brewereeal.,1984). However, the use of codpep sin immobilizedon Sepharos e[Ha ard 1986)and seal gastric protease immobilizedon chiti n(HanandShahidi,1993) toperform coldrennetingisnot feasiblesince the highmolecu laractivityof enzymes atlow temperatures appear to be lost asaconsequenceof immobilization.Itisconsidered thatimmobilizat ions inany waywillgreatlyinhib itthe struct ural flexibility of gast ric prct eases thusreduce their ac ti vi ~ : es.To date,ther ehasbee n no successfulattempt to use im mobilized prot eases formilk-clotting in cheese making.Thesearchfora low costrennetsubs tit ut e is conti nuing.

Veryrecently, using protein engineeri ngtechnology, a new chymosinsubstit ute name d 'chymcgen' hasbecome commerciallyavailable in Denmark (Brusgaar d,1992;

Halfhide,1992). Compared to chymosiD.fromothersour ces,the engineered enzyme producedfrom AJpergillU! nigerhas advant agesincluding vegeta rian improved,secure supplywith apurechymosin, and comparab le performanc ein cheesemakingto that ofcalf rennet (Halfhide,1992).

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25 2.3.2 Recovery of Fish Proteins by Enzymic Hydrolysis

Ther e has been muchinterest in developing newproducts Crom protein resources available fromthe sea. The effectiveutilizatio nof underutilizedspecies of fishand fish pro cessing was tewillhave an immediat e impact on the world'sanimalfeed and human food supplies.Therefore,moreemphasis should be placedon the'totalutilizatio n'of presentresou rces.

Enzymatic hydroly..is offoodproteinsisawidelyused method forimprovingtheir solubilityand solubility-depende ntfunctional prope rties(Adler.Niss en, \976,1986;

Hao,1 989;Shahidi etat.,1993 ).However, use ofenzymesin fish processingis mini mal comparedto otherfieldsoffood processing.Prepar ati on offishprotei nhydro lysates usingproteol yt ic enzymesisthe firstcasetobeconsidered.

Fishproteinconcent ratesmaybe prod uced by variousprocessesto afford products with differentpropert ies and com mercial valuesfor a variety ofapplications.Most ofthe existin g processi ng method scanbeclassifiedintochemical(addic or alkaline ext raction ), physico-chemical (aqueousextr act ionororganicsolvent extraction),and biological (enzym ati c and microbialhydrolyis). However,sever e conditions ofalkaline treetmeaeoffishproteins may in extr eme cases causechangesthatmayhe undesir able from anutri ti onal pointof view(Franzen and Kinsella,1976;Feeney, 1977;Sikors ki andNa.czk , 1981;RobbinsandBallew, 1982). Thelou of funct ionalproperties when emp loying organil: solvents andhigh lipidresiduesinthe aqueousextrac tionproce- duresareconsideredpracticaldifficulties in organic solvent ext racti on .

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26 Application ofenzy m e technology in fish processing has attracted considerable interest for convertingwastes and underutilizedspeciesof fishinto protein concentrates.

The treatment of fish or fish processingwasteswith proteolyt icenzymes representsan interestingalternativeto mechanicalmethods forseparating flesh (rom bonesusing deboning machines (Mohr,1977) and chemicalmethodsforthe preparationof fish protein concentrates.

The productionof fishproteinhydrolysate!using proteoly ti c enzymes has been investigatedsince the 1960's. ln 1961, the termfishprotein concentrates (FPC)was adopted by the Food andDrug Administ ration(FDA) to replacetheearlier name of"fieh flour"(Shenoud aand Pigott,1975).Since then,considerable researchon preparation offishprote in hydrolysate! usingdifferentproteolyticenzymes has been reported. Onedisadvan t age of proteinhydrolysat esrelates totheir bitternesscaused bythe presenceofbitter peptides.In the late1970's, Alcalese was foundto be useful for producingblandprotein hydrolysatesfromdeboned cod offaland fresh herring (LalASidiset0.1.,1978; Lalasidieand Sjoberg,1978), sardine (Sugiyamaetai.,1991)as

well&Sother fishIpecies( Heviaetat.,1976;Haleand Bauersfield,1978;Thankamma

etai.,1979).

Therearetwo typesof pre cesses for theproduction of fish protein hydrolysates namely autolytic process andacceleratedhydrolysis(Mohr, 1977). The autolytic process dependson theactionof the digestive enzymes ofthe fishitself which lasts from afewdays10seve ralmont h.,dependingon the process.Thereare no enzyme

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27

costsinvolvedfor auto lyticprocessanditis simpleto ope ra te.However,prolonged digestionnormall yresultsin problems related tothefunctional proper tiesof protein hydrolysate,and the time of theprocessingcycle.

Accelerated hydrolysis usingcommercialprotea se!offers far bett erpossibilities than autolysisdoes,sinceitallows controlof produc tproperties. Therelat ive ac- tivitiesofmorethan twentycom mercially available proteolyt icenzymeshave been

determined using washedand Ireeee-driedfishproteins(Ha le, 1969).Enzymatic hydrolysis of fishprotein:.using enzymes of animal,plant,or microbia l origins has been thoroughlystudied(Hale , 1969;Cheftel etal.1971jWesselsandAtkinson, 1973;

Hale,1974;Mackie,1974; Hevia.etaI.1976;Hale and Ba.uersfie1d, 1978; Sugiyamaet al.,1991) andexcellentreviewshave been written on this topic(Finch,1971;Mohr, 1977;Sikorskiand Naczk,1981).However ,acceleratedhydrolysisis generally a more complex process,and thecostofenzymes meyinfluence:the economicalaspectsend commercialviabilityof the process.

Althoughtheaccelera ted hydrolysisof fish proteinsusing commercialprcteascs has been studied, the useof proteolytic enzymes from the digestivetracts of marine specieshas not receivedadequate attention,Basedon theconsiderationth",t proteolyticenzymes presentinthe digestive tracts of marinespeciesare able to digest theirfeed(mai nly smallfish), the useof proteolyticenzymes isolatedfrom codand sealdigestivetractsfor preparationoffishprotein hydrolysates fromcapelin was investigatedin tbisstudy.

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Chapter 3 Materials and M ethods

3.1 Materials

3.1.1 Sa mp le collec tio n

Atlan t iccod(Gadusmorhua)were obtained IreshfromConcept ion Bay, Newfound- land.Theviscerawereremoved, split, cleaned,andthe liverand fat were discarded.

Thecleaned viscera were vacuum packagedand storedat•60° C untiluse.

Male and spentcape lin

Male and spentcapelin(Maliotulvilosus)werecollect ed ataayBulls, Newfoundland in June.Thewhole fishwas washed , vacuum packaged ,and storedat -600 C until

28

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29 3.1.2 Chemica ls

Caseinwas obtai ne dfro m BDH Chem icals(Toronto,ON).Polyacrylic acid was ob.

tainedfrom Aldrich ChemicalCompany(Milwaukee , WI).DE:AESephadex A·50 was obtainedfromPh ar maciaFine Chemicals(Uppsala,Sweden).Calf chymosin (E.C.3.4.23.4),bovineserum albumin(BSA),c-benzoyl-Df.-a rginine p-nitroenilide (BAPNA), haemoglobin(washedand dialyzed) ,tris(hydro:tymet hyl)·emincmet he ne, benzoyl-L-arginine ethylester(BAEE),Ncbenzoyl-Layroein ethylester (STEE), pe p- statin, porcinepepsin, and otherchemicals were purchasedfrom SigmaChemical Company (St. Louis,MO).

3.1.3 Equ ipment

Majorequipmentusedin this study were:Sorvallsupers peedRC2· B automat icreo frigeratedcentrifuge(IvanSorvallInc., Norwalk,U SA) j Fisher eccum enr pHmeter, Model 805 MPwithgel-filled poly mer.bodycombin ationelectrode(FisherScien.

tific Company,Ottawa, ON) ;HP8452Adiode-a rray UV/Visible spectrophotome ter withHP89531AUV/Visoper at ing softwareand GP·IOO Grap hics print er(Hewlett.

PackardCompa ny,San Francisco,CAliGelelect ro pho resisappar atus GE.2/ 4i and Elect rophoresisCODsta-at powersupplyECP S2000/300(PharmaciaFineChemicals, Uppsal a, Sweden ).

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30

3.2 Experimental Procedures

3.2.1 Co-e xt ractionofacid an dalkali ne pr ot ease sfrom cod viscera

Fi6u rc 3.1summarize sthe procedu reused for theco-extra ct ion of prot eolytic enz ymes (rom digestive tractsofAtlanticcod(Gadutl morhua).Frozenvisce ra were thawed at4°C overnigh t and mincedwith ice-cold distilledwate r at a 1:4ratio(wl'l)for 3 min.A 1.0 NsolutionofHelwas addedtothemince to adj ustthe pHto 6.0,and themixturewasthen blendedfor50minat room temperat ure. After blending,the slurry was centri fugedat13,200x gfor 20 minat4° C,and the aqueous phase was separatedto isola teacidand alkaline proteaeee.

A1.0%solu tionofpolyacrylicacidwesaddedto the aqueous extracttogivea 0.05 to0.15%solution, similarto theproccedureof Reece (1988). Themixturewas allowed tostaDdfor30 min,cent rifugeda.t 12,100xg for15 minat 4°Cand thesuper nat ant was decanted.Crude acidicprote esee could be obtainedbyasimple ultrafiltra tio n of the supernatanttc aspecific con cen tra t ion.In this stud y,thesupern atant was made 63%sat urated with solid ammoniumsulph ate at 4°C andcentrifugedat 13,200x. g for 30min.The precipitatewas dissolvedina minimumamountof 0.1M acetate bufferatpH 6.0,anddia lyzed against thesame bufferovernight to obtain crudeacid prcteeeea.

The polyecrylicacid precipitate was redissolvedin50 roM TrisbufferatpH7.8.

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31

MINCE D VISCER A

\Va

Polacrlieacid

~~=?=;::::C~= s ;

Cr udealkaline proteases Crudeacidic peotee ees Figure3.1:Flowsheet forco-extractionofaddandalkalineproteasesfrom Atla ntic cod villcera^ll•

•Abreviat ions are:5,sludge;SI, sludge that canhefurthertreatedforrecovering precipitate(polyacrylic acid);b, The supernatantcan alsobe concentratedby ultra- filtratioDor other meant to"blain crude acidprcteaees.Thedu hboxinclude. key step. forwholeprocedure of co-extractionprocedure.

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32 The precipita te formedwas removedbycentrifugatio n andthesupe rn at ant wasmade 63% satu rated withsolidammoniu m sulphateat4°C.The precip it ateformed was diuolved ina minimum of20 mM Trisbuffer,pH 8.2containi ng5mMcalcium chloride,and dialiaed overnightagainstthe samesolutio ntoobta incrude alkaline protease.

3.2.2 Purificat ion of proteol ytic enzy mes

Gelpermea tionchro mato grap hy

Crudeenzymepreparations,preparedasdescribedin Section3.2.1 (30 mL of crude acid oralkalineproteaeee],weresubject ed to gelpermeation chromatographyin a 3.2x100emSephad exG-75 col um n. For acid proteases, the columnwas equilibrated and elutedwith 0.1 M acetate buffer, pH 6.0.For alka.lineprotease"the colum nwas equilibratedand elut edwith50 mMTri sbuffer,pH 7.8.Theproteolyticactivity of the fractions was measuredas describedlater (Sectio n 3.3.3).Fractionscon tain ing proteol yticactivitywerecollectedforfurtheruse.A portionofthe partially purified enzymes was concentratedbyfreeze dryingor ultrafiltration.

Ion · e x changc chromatog r ap hY

DEA E-Sephadex A-50was packedintoa2.5x40 ern columnforthe purificat ion of acidpreteeees.The columnwasequ ilibrat ed with0.1M acetatebuffer,pH 6.0.

Enzyme collectedfrom Sephadex0-75gelchromatographywas appliedtothe column

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33 whichwasthen eluted withthesame bufferunt iltheA~80of the fractio ns and their proteol yticactivityapproachedzero.Thecolum n wastheneluted sequen tiall ywith 0.2,0.4, and 0.8M sod ium chloride in 0.1M ace tatebufferatpH 6.0,5.5,and 5.0, respectively. Aliqucta from alternatefra ct ions wereassayed forproteolytic activity.

Fract io nscontaining proteolyt icactivitywerecollectedIor charac t erizati on.

3.2.3 Prod uction of fish pro t ein hydro lysa tes

Productionofcapelin proteinhydrolysates(CPH)was carr ied outas summarized in Figure3.2.The ground frozenfis hsam p les werethawed,mixed wi thanequalweight of waterand homogenized in a Waring blenderfor appr oxima tel y2min.ThepH value ofthesuspensionWi\.!adj ustedto the optimum valueof theproteaseunder investi gation(pH 3.0 for crudecod acid proteascsand seal gastric proteeses; pH 8.5 forcodalkaline prcteee eeand Alcelese:pH7.0 for Neutrase.].Hydrolysiswas carried outfor 60 to 240 minat roomtemp er at ure wit hpH.stdcont rol. Theenzy m es were inactivated by heatingto 85°C for 5min.Thesludge was removed by suction filtration.ThepH oftheresultantfil~ratewas adj usted to 5.5 with4N NaOH orHOI.

Thehydrolysatewas dehydratedby Ireeae-dryin g. The ratio of tot al Kjeldah l nitrogen in thefinalproductto thatoriginallypresentinthe groundfishwascalcu la ted as the yield ofprotein.

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34

Com minute d fish sample Wae

Enz me

Cha e a1

Q.olWl~_~:;::::==:J===::::;--SIUdge

PROTEIN HYDROLYSATE

Figurr;3.2:Flow sheetfor pre parationorprotein hydr olys a t e.

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35

3.3 Analytical Methods

3.3.1 Proteindet ermination

Proteincontent duringextractionand purificationof enzymes was determ inedaccord.

ing tothe methodof Lowryet al (1951) usingbovineserumalbuminiUa standard (Appendix A).The reactionmixtur ewas com posedofl.Oml, protein solution and 4.0 ml,workingsolut ion.Theworkingsolution waspre paredby adding1mL1% sodium tart ratea.nd 0.5%CuSO~to 49 mL 2%Na2C03in0.1 M NaOH.The absorbanceat 660am wasreadafter theaddition of Folinreagentfor30 minatambientte m per- ature.Pro tein content during column chro matogra phywas measured and expressed

a!'absorb anceat 280 am using a0.1M ace ta te buffer,pH3.0,as a blank(Ap pendix B). Tolal crude protein (Nx6.25)content was determinedbytheKjeldahl method (AOAC, 1990).

3.3.2 Determination of lipid content

The total lipidinfish samples andprotein hydrolysetes wasextracted accordin gto the metho d ofBlighandDyer(1959) andWoyewodadill.(1986).Ten gramsoleclid sample (50mLfor liquidsam ples)washomogenized inchloroform/methanol/water (1:1:1,vfv/v).The mixturewasheldovernightfor separatio n.The chloroformlayer was separatedandthesolventwas removedbyevaporat ion(evaporatedat. room temperat ur e for two hours,and thenheated at95°C for30 min).

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36 3.3.3 Overallprote o lyt icact ivity

Prot e ol y ti c activityusingbovi ne ha e m oglobin substrate

Proteolyticactivityofacid proteases was det ermi nedusing bovine haem oglobi n sub- strateasdescribed byAnson(1938)andRyle(1970)withminor modificat ions as describedbelow. Thereactionmixture was comp osedof0.2 mLenzyme solution, 1.0 mL 0.2M acetatebuffer, pH 3.0,and 0.6mL1.5%(w/v)bovine haemoglobin solutio n.Thereactionwas terminatedby addition of2.5mL solutionof 5,0%(w/ v) trichloroaceticacid(TCA) after 10 min of reactionat25°C.Blan kswereobta.inedby addingTeAto the enzymepriorto the additionof substrate.After30 min standing at room temperatur e, thesolutionwas filteredthrough a Whatma nNo. 3filterpaper and theabsorba nceofthe TeA-solublematerialwas read at280 nm.Aline arrela- tionehip existed atA<0.45.One haemoglobinunit (HU) wasdefinedastheamount of protease whichincreasedthe absorbance at280nm ofTCA-solub lematerialby 0.001unit permin underthe aboveexperimentalconditions.

Prote o lyt icactivity using a caseinsubst ra t e

Proteolyticactivity using a caseinsubstratewas determinedundertheconditions described above for haemoglobinsubstrate except foremployingdifferent buffers (pH 3.0 foracid proteeses,and pH 8.2 for alkaline proteases]. One caseinunit (CU) was defined as the amountof proteasewhich increasedtheabsorbance at280nm of TeA-solublemateria lby 0.001unit perminundertheaboveexperimental conditions.

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37 3.3.4 Assay of pepsin activity using a synthetic substrate

A synthet icsubstrate,acetyl.L. phenylala nyl.L.d iiodotYfosine(APDT)was usedfor measurementof pepsinactivity accordingto the method describedby Ryle (1970).

A 0.5 MHe lsolutionwas added tothe enzymepreparationto bring the mixture topH2.0.Theacidified enzyme solution was incubatedatroom temperat urefor 10min beforeperformi ng the measurement ofenzymeactivity.Thereactionmixtu re contained0.5mL ofenzymesolut ion,0.25 ml,ofHeland 0.25ml, of APOT solutio n.

After20 minreactionat 25° C, 1.0 ml, ofninhyd rinreagent,prepared accord ingto the method describedby Hyle (1970), was addedtothe test mixture.All test tubes wereplacedina boiling water bath for exactly15 minand werethen cooledundera streamofcoldwater. Thereaction mixturesweredilutedwith 5mL60% ethanoland tubeswere then shaken thoroughly.Theabsorban ce of solutions at 570 nmwasread against awater blank.One APDTunit was defined as the quantity of theenzyme requiredtoin creasethe absorbanceat570nm by 0.001units permin underthe experimental conditions.

3.3 .5 Trypsin esterase activity

Trypsinesteras e activityofalkalineprcteases was measured usingbenzoyl-L-arginine ethylester(BAEE)iLSa substrate according to themethod describedbyRick (1965).

Toa3.0mLcuvette , 2.8 mL of 1 mM BAEE in50mMTris buffer,pH 8.0, contai nin g 20mM CaChwas added.A 0.2 mLaliquCI' ofproperly dilutedenzymesolution was

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38 thenadded at zero timeand mixedimmediat el y. Theabsorbanceat 254nmwas measuredcon t inuous ly foraperiod of 10 min.One unitof BAEEactivity was defined as theamoun tof tryps inor trypsin-like enzymeswhichincreasedthe absorbance a.t 254 nmby0.001permin. Specificactivitywas expressedas unitsofenzym a t ic activityper mgenzy me.

3.3.6 Chymotrypsin esteras eactivity

Chymot rypsinest ereseactivitywas measuredbythe methodof Walsh (1970)with minormodific ations.N·Benzoyl· L·tyrosineethylester(ST EE)was usedas substr a te.

Toa 3.0 mL cuvette,1.5 mL of 0.1MTris buffer,pH8.2,containing50mM CaCI_2, and 1.4 mLof1 mM BTEEin50% me thanol was adde d. 0.1mL of enzymesolution was then add e datzerotimeandmixedimmediately. The absorbanceat 255 nmwas mea sured at1minintervals. OneBTEEunit(BU)Walldefin edalltheamountof chymotrypsin -like enzymewhichincreasedtheabsorba nceat256 omby0.001unit permin.

3.3.7 Trypsinamidaseactivity

The amidaseactivity of trypsinwas measuredbythe method of Arnon(1970) with minor modification, usinge-beeeeyl-Db -erginin e p-nitrcanilide(BAPNA)asasub- ttrate.Onegram of BAPNAwas diss olvedin 50 mLdimethylsulphoxi de(DM SO) anddiluted tobOOmLwith water.Thereaction mix t ure cont ained0.2 mL ofpar-

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J9 tially purifiedcod alkalineproteasesolution,2.0mLof 0.1MTris-He lbuffer,pH8.2 containing 5 mM CaCIJ ,and 1.0 mL BAPNAsolut ion.Themixture was incubated for40 min a125°C.The reactionwas stoppedbythe additionof 1.0 mL30%(v/v ) aceticaci dsolutio nandthe amountof p-nitroanilid ereleasedwas measured at410 nrn against a blank(theblankwas preparedwithth e additionof1.0 M 30% acetic acid solu t ionbefore tha t of BAPN A solution].OneBAPN Aunit wasdefinedasthe amount of enzymewhich increasedtheabsor banceat 410nmby0.001per min under the aboveassay co n dition s.

3.3.8 Milk-clotting activity

Milk-clott ing act iv itywasassayedbythe procedureofMa n j iclat(1988)withminor modificationsasdescribe dbelow. Enzymesolution (0.2mL)atconcentratio nsofO.!

to 1.0mg/mLwa saddedto 30mLof reconstitutedmilkconsisting of12% Car nation instant skim milkpowderin0.01 M CaC1:l solutionatan appropriatepH(from 5.50 to 6.70,adjustedbyHelorNaO H).Themixturewas then gentl y and constantly swirledunt ilthefirstappearance ofa whiteprecipi tateat the bottom of thebeaker.

The timeforfleck sto ap p earwastaken asthe milk -clotti n gtime (MCT). Onemilk- clottingunit(MeU )was defined as theamountofenzyme thatdot tedLO mLof the reccnst itut ed mil kin 100sat25⁰C (Squiresetai., 198611.; Manji etaL, 1988).

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40 3.3.9 Mltk-clot ting unitto proteoly t icunitratio The ratio of milk-clottin g unittoproteolyt ic unit(~ I CU/ PU)was usedtoidentify suitableproteesesas milk-clott ingagents incheese-making(Puhan andlevine,1973;

de KoningetaI.,1978). Prcteaseswhichwere successfu l in cheese-mak ing had a relatively highmilk-clottin gto proteolyticactivityratio(Visser ,1981).

3.3.10 pH optimaandsta bi li ty

The effectof pH on the hydrolysis rate ofhaemoglobin andcasein was determined usingvarious buffers duringthe hydrolysisperiod.Theprocedur esfor measuringthe reteof proteolytichydrolysiswere the sameas those describedabove (Section 3.3.3).

TodeterminepH stability,the prot easeswere incub atedinvarious buffersat different pHconditione(pH 1.4 to 8.5foracid proteaseAnd 3.0to 11.0for alkalineprotease) for2 h at25° C,beforetheresid ua lactivi tieswere determinedusingthe methods describe din Section 3.3.3.Theuniversalbuffersemployedwerepreparedaccording tothe methoddescribedbyTecrelland Stenhagen(1938).

3.3 .11 Temperat ureoptimumand thermalsta bili ty The effectDf temperature on therate of hydrolysisof caseinand haemoglobinwas determinedat varioustemperaturesvaryingfrom 5to 80° C.Todetermine ther- mos tab ility,proteaseswere incubatedat varioustemperatures fordifferentperiods (from2 to60 min) before residual activit ies weredete rmined using standardmethods

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describedinSectio n3.3.3.

3.3.12 Inh ibi t io n ofenzy meactivity

Theenzymetobetestedwaspre-incu batedwith eachinhibitor in a total volumeof 0.40mL buffer(0.1MacetatepH3.0forad d proteases, 0.05MTrisbuffe r Ior alkaline pro teeses]for20 to60minat20⁰Cpriortothe addition ofsubst ratesoluti onforassay of the remainingactivity.Soyabeantrypsininhibitor(SBTl)wasdissolvedinenough deionizedwateratconcentra.tions of0.025, 0.050,and 0.100mg/ mLThealkaline protea se solutionswereadded separately to equalvolumes ofthe SBTIsolutionsand inc ubatedinanice bathfer 30min.Afterincubation ,residu altrypsinactivitywas determined using BAP NAas substrate under condit ionsdescribedabove.

Pepstatinwas dissolvedinDMSOanddilutedto 1.0)(io-'Massto ckpepsta tin soluti on. Acidproteaseswereincubat ed separately with differentconcentrationsof pepstarin solutionfor30minat roomtemperature.After in cubatio n,theresidua.l proteolyticactivitywas det ermi nedusinga 1.5%solutionofhaemoglobin assubstrate understand ard assay conditio nsas describedinSect ion3.3.3.

3.3.13 Estimati o n of rela t ivemol ecula r mass, M ..

Therelativemolecula rmass(M.)of the Atlan tic codgMtricprotease! was estima ted by polyacrylamidegelelectrophoresisinthe presenc e ofsodiumdodecylsul phate (50S),and by gel permeat ion chrom ato graphyusing a1.5x 100cm columnpacked

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42 with Sephade xG·75.

50S· polyacrylamideget electrophoresis (SDS· PAGE) was performedat pH 8.3 usinga10%sepera ring geland a 3.75% 'tacking gel in adiscontinuous buffersystem , according to the methodof Laemmli(197D).Thegelwasstainedby 0.1%(w/v) CoomassieBrilliantBlue R250. Proteinstandards usedfordet erminingthe relation- shipbetween mobilityon gels and M., arepresented in Figure 4.10.

The Sephaclex0-75gelfiltrat ionchromatography was performedaccordingto the methodofWhitaker(1963) andAndrews(1965).The colum n wasequilibrat ed andeluted with0.1M acetate bufferof pH 5.5. The proteinswerecolumnchro- matographed and the elutionvolume of each proteindeterm ined .The columnwas calibratedby usingbovinealbumin,egg albumin,chickenovalbumin,soybea ntrypsin inhibitor,equine myoglobin,and a-lacta lbumin.TheM~of aproteinWiUdetermined from a plot of logarithmof M.versus the distr ibut ioncoefficient(Kd).The valueof Kdfor a givensoluteisdefined by the relationship(P rice andStevens,1989):

where

v..

isthe elution volume ofthe moleculeestimated,Viisthe elut ionvolume of a small molecule (vit am in B_{l a )}whichis tot ally included by the column, YOisthe elutionvolumeof a molecule(dextran blue withaM~of2 X106 )which is completely excluded by the column.

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43

3.4 Experimental Design a nd Optimization of Co- extracti on Conditions

Optimumsolutionscanbeobtained by variousapproaches.Theclau icalapproach to optimiza tionis totes tonevariable at a time or ,-lternativelymodify the variables by so-called "back-and forth "method .Theseapprcacheerequirealargenumber of experimentsand often donotconsider theintera.ctions among thevariables.

Moresophisticate dtechniquesapplied in conjunctionwithresponse surface meth od- ology(RSM), arecanonical, ridge regression ,steepestascentordescentmet hods (Myen , 1971). Theprinciplesandfunctions of RSM werefirstdeveloped by Box andWilson (1951). RSM has beenmodifiedand expanded into a powerfultool for empericalmodeldevelopment and optimization(Khuri andC"rnell,1987). RSM can be definedas ast at ist ic,ll method thatuseaquantitativedata fromappropriate experimental designstodetermine and simultaneously solve multivariate equations (Giovanni, 1983).These equations can begraphicallyrepresentedas response surfaces which can be used to describehowthe testvariablesaffectthereaponee,todetermine theinterrelationshipsamong thetestvariables,and to describethecombined effect of all testvariableson theresponse.

Computergraphics-assistedoptimizat ion approach is considered to be more ad- vent-edin theinvestigat ion andopti mization ofcomplex systems . Inthe graphical approach,the predictivemodelsare used to creat contoursurfaces or contourlines

of anti

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a

Contents

,,\

".

"

List of Tables

ss

List of Figures

lEI

Chapter 1

Introduction

Chapter 2

Literature Review

or