P P GP inhighyieldwasdeveloped. Pennsylvania State Univ., University Author Owusu-Apenten iswithDept. ofFood Science, Borland Laboratory, iswithPrince ofSongklaUniv.,thor Adulyatham Songkhla, Thailand. MS 20030352Submitted 6/24/03,Revised 7/31/03, 12/15/04.Accepted Au- G P ( of aProteasefromGingerRhizome Stabilization andPartialPurification author Owusu-Apenten (E-mail: acetone powder (GP additives toGPsolutions, and(3)preparation ofgingerprotease simple extractionofGPfrom fresh ginger, (2)additionofchemical strategies forGPwere investigatedusing3enzymetreatments: (1) forGP.ported others (1973),recoveryandpurificationresultshavenotbeen re- and/or degreeofpurification.BesidesthereportbyIchikawa and lation techniquesforGPhavealsonotbeenoptimizedyield lain, ficin,andactinidin(Caygill 1979;Storey and Wagner 1986).Iso- least studiedoftheplantthiolproteasesincludingpapain,brome- tions dealingwithGPnumberedlessthanadozen.isoneof the Nevertheless, whenthisresearchbegan,thenumberofpublica- ray crystallography(Choiandothers1999;ChoiLaursen2000). others 1995),crystallized,andthe3-DstructuredeterminedbyX- use forcommercialapplication. of protease forindustry. The low storage stabilityofGPmaylimitits 1990). Gingerisofconsiderableinterestasapotentialnewsource for $17million(US)salesor8%marketshare(OuttrupandBoyce million (US). Two plantproteases(papainandbromelain) account The presentmarketforindustrialproteasesamountstoabout$242 andothers1973). and otherconnective tissueproteins(Thompson 1986; Megaandothers1987).GPishighlyactiveagainstcollagen 1973;Sakasai andothers1980;Lee gibain (Thompson componentisgingerprotease(GP)orzin- The meattenderizing P P IT IT IT IT IT Zingiber The objectivesofthisresearchwere2-fold.Firststabilization Zingibain waspurified(Ichikawaandothers1973;Ohtsuki as acommercialproduct.SomepossibleroutesofGPdeactivationandstabilizationarediscussed. as acommercialproduct.SomepossibleroutesofGPdeactivationandstabilizationarediscussed. 25.1 kDaforpapain.ThisworkshowsthatthestabilityofGPcanbegreatlyimproved,increasingitsattractiveness 25.1 kDaforpapain.ThisworkshowsthatthestabilityofGPcanbegreatlyimproved,increasingitsattractiveness tially purified252-foldwitharecoveryof61%.ThenomimalmolecularweightGPwas34.8kDacompared tially purified252-foldwitharecoveryof61%.ThenomimalmolecularweightGPwas34.8kDacompared Addition ofascorbate(0.2%w/v)increasedthet Addition ofascorbate(0.2%w/v)increasedthet E E ABSTRA ABSTRA Crude GPextractedfromfreshgingerhadahalf-life(t Crude GPextractedfromfreshgingerhadahalf-life(t research wastoinvestigatefood-compatiblemethodsforstabilizingGPduringstorageorenzymefractionation. research wastoinvestigatefood-compatiblemethodsforstabilizingGPduringstorageorenzymefractionation. fr fr yielded GPwitht yielded GPwitht as acommercialproduct.SomepossibleroutesofGPdeactivationandstabilizationarediscussed. 25.1 kDaforpapain.ThisworkshowsthatthestabilityofGPcanbegreatlyimproved,increasingitsattractiveness tially purified252-foldwitharecoveryof61%.ThenomimalmolecularweightGPwas34.8kDacompared Addition ofascorbate(0.2%w/v)increasedthet E ABSTRA Crude GPextractedfromfreshgingerhadahalf-life(t research wastoinvestigatefood-compatiblemethodsforstabilizingGPduringstorageorenzymefractionation. fr yielded GPwitht as acommercialproduct.SomepossibleroutesofGPdeactivationandstabilizationarediscussed. as acommercialproduct.SomepossibleroutesofGPdeactivationandstabilizationarediscussed. 25.1 kDaforpapain.ThisworkshowsthatthestabilityofGPcanbegreatlyimproved,increasingitsattractiveness 25.1 kDaforpapain.ThisworkshowsthatthestabilityofGPcanbegreatlyimproved,increasingitsattractiveness tially purified252-foldwitharecoveryof61%.ThenomimalmolecularweightGPwas34.8kDacompared tially purified252-foldwitharecoveryof61%.ThenomimalmolecularweightGPwas34.8kDacompared Addition ofascorbate(0.2%w/v)increasedthet Addition ofascorbate(0.2%w/v)increasedthet E E ABSTRA ABSTRA Crude GPextractedfromfreshgingerhadahalf-life(t Crude GPextractedfromfreshgingerhadahalf-life(t research wastoinvestigatefood-compatiblemethodsforstabilizingGPduringstorageorenzymefractionation. research wastoinvestigatefood-compatiblemethodsforstabilizingGPduringstorageorenzymefractionation. fr fr yielded GPwitht yielded GPwitht A A A A A thylenediaminetetr thylenediaminetetr thylenediaminetetr Y Y Y thylenediaminetetr thylenediaminetetr Y Y with bothcomminutedgingerrhizomeorjuicebeinguseable. inger is a meat tenderizing agent from antiquity (Pruthi 1980) inger isameattenderizingagentfromantiquity(Pruthi actionation b actionation b actionation b actionation b actionation b A A A A A K K K K K A A A A A eywor eywor eywor eywor eywor DUL DUL DUL DUL DUL CT CT CT CT CT Y Y Y Y Y ds: gingerpr ds: gingerpr ds: gingerpr ds: gingerpr ds: gingerpr A A A A A : G : G : G : G : G THAM THAM THAM THAM THAM inger pr inger pr inger pr inger pr inger pr y ionex y ionex AC y ionex y ionex y ionex

). Secondly, purifying amethodforpartially

1/2 1/2 1/2 1/2 1/2 AND AND AND AND AND

of 18 mo at 5 °C. CrudeGPextractedfromacetonepowderwassufficientlystabilizedtoallow °C. CrudeGPextractedfromacetonepowderwassufficientlystabilizedtoallow of18moat5 of18moat5 of 18 mo at 5 °C. CrudeGPextractedfromacetonepowderwassufficientlystabilizedtoallow of18moat5 of 18 mo at 5 °C. CrudeGPextractedfromacetonepowderwassufficientlystabilizedtoallow °C. CrudeGPextractedfromacetonepowderwassufficientlystabilizedtoallow of18moat5 of18moat5 Introduction offinale aacetic acid(EDT aacetic acid(EDT aacetic acid(EDT aacetic acid(EDT aacetic acid(EDT R R R R R otease (GP)orzingibainisofinter otease (GP)orzingibainisofinter otease (GP)orzingibainisofinter otease (GP)orzingibainisofinter otease (GP)orzingibainisofinter otease otease otease otease otease change chr change chr change chr change chr change chr ICHARD ICHARD ICHARD ICHARD ICHARD [email protected]). Park, PA 16802. , zingibain,stabilization,pur , zingibain,stabilization,pur , zingibain,stabilization,pur , zingibain,stabilization,pur , zingibain,stabilization,pur O O O O O WUSU WUSU WUSU WUSU WUSU omatogr omatogr omatogr omatogr omatogr Roscoe) -A -A -A -A -A A) hadnoeffectonGPstability A) hadnoeffectonGPstability A) hadnoeffectonGPstability A) hadnoeffectonGPstability A) hadnoeffectonGPstability Direct inquiries to P P P P P ENTEN ENTEN ENTEN ENTEN ENTEN aphy withouttheadditionofto aphy withouttheadditionofto aphy withouttheadditionofto aphy withouttheadditionofto aphy withouttheadditionofto 1/2 1/2 1/2 1/2 1/2 1/2 1/2 1/2 1/2 1/2 ification, for ification, for ification, for ification, for ification, for ) of2.1(±0.16)dat5 °C decreasingto20minat30 ) of2.1(±0.16)dat5 °C. °C decreasingto20minat30 °C. ) of2.1(±0.16)dat5 °C decreasingto20minat30 °C. ) of2.1(±0.16)dat5 °C decreasingto20minat30 ) of2.1(±0.16)dat5 °C. °C decreasingto20minat30 °C. P P Preparation ofgingerproteaseextract Preparation ofgingerproteaseextract Chemicals andmaterials Chemicals andmaterials (DTT; 0.3m pared similarlywiththefollowing bufferadditives;dithiothreitol °C. OtherGPextractswerepre- tant, designatedGPwasstoredat5 the filtratecentrifuged(12000× P Preparation ofgingerproteaseextract Chemicals andmaterials with 5 parts (v/w)coldacetone followedwith 5parts GP by airdrying. Whatman nr1paper. Suction washes wascontinued withfurther precipitate wascollectedby formed vacuumfiltrationthrough °C) for90sandallowedtostand1520min.The acetone (–30 Tris-HCl buffer(0.05 fuge (Eppendorfmodel5415C,Eppendorf,Germany). (Beckman, Fullerton,Calif.,U.S.A.)oranEppendorfmicrocentri- trifugation waswitharefrigeratedBeckmanJS2-HScentrifuge standard configuration(Pharmacia-LKB,(Uppsala,Sweden).Cen- Chromatography studiesusedtheGradiFrac-100systemwitha a Bio-Rad3000Xipowersupply(Bio-Rad,Hercules,Calif.,U.S.A.). Scientific Instruments,SanFrancisco,Calif.,U.S.A.)connectedto phoresis (SDS-PAGE) involved aHoefer unit(Hoefer SE600vertical (Cecil, U.K.).Sodiumdodecylsulphate-polyacrylamidegelelectro- tometric measurementswereusingaCecilmodel2002Instrument U.K.). Singledistilledwaterwasusedinallexperiments.Spectropho- other chemicalwerepurchasedfromSigma-Aldrich,Ltd.(Poole, support wasfromPharmacia-Biotechnology(Uppsala,Sweden).All thane (DEAE) Toyopeal-650 ion-exchange chromatography (IEC) crushed, sievedcrushed, toremove andstored coarseparticles, zyme extracts were prepared usingGP P P Preparation ofgingerproteaseextract Preparation ofgingerproteaseextract Chemicals andmaterials Chemicals andmaterials est asameattender est asameattender est asameattender forGPfrom2to20dat5 forGPfrom2to20dat5 °C. Dithiothreitolor °C. Dithiothreitolor est asameattender est asameattender forGPfrom2to20dat5 °C. Dithiothreitolor forGPfrom2to20dat5 forGPfrom2to20dat5 °C. Dithiothreitolor °C. Dithiothreitolor r r r r r epar epar epar epar epar Diced ginger was frozen at –30 °C, homogenizedwith4partscold Diced gingerwasfrozenat–30 Freshly dicedgingerwashomogenizedwith4parts(w/v)cold Ginger wasobtainedfromalocalmarket.Diethylaminoethyle- ation ofgingeracetonepo ation ofgingeracetonepo ation ofgingeracetonepo ation ofgingeracetonepo ation ofgingeracetonepo M mulations mulations mulations mulations mulations ), EDTA (1m . A . A . A . A . A cetone po cetone po cetone po cetone po cetone po Materials andMethods xic orexpensiv xic orexpensiv xic orexpensiv xic orexpensiv xic orexpensiv M , pH8)filtered through4layersofmuslin and izing agent. izing agent. izing agent. izing agent. izing agent. , ascorbate , ascorbate , ascorbate , ascorbate , ascorbate M w w w w w ), orsodiumascorbate(0%to1%w/w). der pr der pr der pr der pr der pr g ) at 5 °C for20min.Thesuperna- ) at5 e additiv e additiv e additiv e additiv e additiv epar epar epar epar epar AC w w w w w The objectiv The objectiv The objectiv The objectiv The objectiv asneeded. der der der der der ations fr ations fr ations fr ations fr ations fr es es es es es . GPwaspar . GPwaspar . GPwaspar . GPwaspar . GPwaspar om ginger om ginger om ginger om ginger om ginger e ofthis e ofthis e ofthis e ofthis e ofthis at 5°C.En- AC was - - - - -

C: Food Chemistry & Toxicology 1/ ) with or ), DTT (0.3 AC M /min/mg) to 440 A ⌬ are shown in Fig- ( 0.16) d at 5 °C to 17 0.16) d at 5 AC –3 was 18 mo due to the Ϯ AC for GP 1/2 ). The t M Results and Discussion and Results (100 m ) is adequate for domestic applications (Thomp- for domestic applications ) is adequate /min/mg). Higher ascorbate concentrations (0.2% /min/mg). Higher ascorbate 2 1/2 440 A ⌬ ( –3 ), and Ca Cl Sodium ascorbate (0.2% w/v) stabilized GP and no activity loss Sodium ascorbate (0.2% Crude GP extract from ginger was unstable (Figure 1), although unstable (Figure 1), from ginger was Crude GP extract Results for thermal stability of GP and GP M value for GP, determined by fitting data to a semi-log plot of ln fitting data to a semi-log plot determined by value for GP, Figure 2—Accelerated testing of the shelf-life stability for extracts. (a) Stability curves for gin- crude ginger protease (GP) with or without 0.2% (w/v) ascorbate; (b) ger protease ginger acetone powder (GP enzyme extracts from heated at the without 0.2% (w/v) ascorbate. Enzymes were tempera- for 10 min, cooled to room indicated temperatures and assayed (see text for details). ture, Stabilization of zingibain Stabilization of zingibain Stabilization son and others 1973; Lee and others 1986). In this study, there was there study, this 1986). In 1973; Lee and others son and others at 5 was stored for 4 d activity after crude GP 80% loss of °C. The t (activity) versus time, decreased from 2.1 ( (activity) versus time, min at 30 is undesirable in a com- °C. Because low storage stability this strategies were examined in mercial enzyme, 2 stabilization additives, and (2) acetone powder prep- study: (1) use of enzyme aration. GP 1). Ascorbate also increased after 14 d at 5°C (Figure occurred 5-fold from 1.0 × 10 specific activity by about 5.5 × 10 the half-life (t the half-life Stabilization of zingibain Stabilization 2 15% loss of activity after 15 weeks of storage at 5 15% loss of activity after 15 weeks of storage °C. to 1.0% w/v) led to no further benefits, and 0.2% ascorbate was further benefits, and 0.2% ascorbate to 1.0% w/v) led to no The following compounds had negligi- used in all further studies. (1 m ble effect on GP stability or specific activity: EDTA m Stabilization of zingibain Stabilization of zingibain Stabilization , , g M , pH M = GP ᭺ , 50 m M , pH 7; ± 0.2% w/w sodium M of NaCl dissolved in Na phosphate sample was precipitated with solid sample was precipitated M AC for 10 min) and absorbance measurements at and absorbance measurements for 10 min) g = GP with 0.2% ascorbate, and , and 300 m ᭿ M Tris-HCl buffer, pH 8.0) at 38 °C as described Sarath 38 pH 8.0) at by buffer, Tris-HCl , 200 m was prepared as described previously and extracted with 4 was prepared as described = GP; M M ٗ AC Dialized GP was fractionated by ion exchange chromatography Dialized GP was fractionated Enzyme molecular weight (MW)Enzyme determination was using discon- GP Enzyme extracts were assayed using azocasein (0.5 mL; 2 mg/mL azocasein (0.5 mL; were assayed using Enzyme extracts Partial purification of GP from acetone powder Partial purification of GP from acetone powder Partial purification Determination of ginger protease specific activity specific protease of ginger Determination activity specific protease of ginger Determination 440 nm. An assay time of 10 to 50 min led to initial rate measure- 50 min led to initial assay time of 10 to 440 nm. An by of ginger extracts were determined ments. The protein content (Peterson 1977). the modified Lowry assay ammonium sulphate (70% saturation), centrifuged (12000 × (70% saturation), centrifuged (12000 ammonium sulphate 5 °C), resuspended in a minimum volume of buffer, and exhaustive- of buffer, in a minimum volume 5 °C), resuspended phosphate buffer. Na against ly dialyzed 650 column (16 × (DEAE)-Toyopeal (IEC) using diethylaminoethyl buffer (20 m 450 mm) pre-equilibrated with Na phosphate 7.0) at 5 °C. First, 20 mL of enzyme (0.6 to 0.8 mg [total protein]/mL) buffer (1 mL/min) and was applied followed by 2 bed volumes of each of 25 m successive applications of 2 bed volumes buffer. Chromatographic fractions (5 mL) were collected for enzyme Chromatographic fractions (5 mL) were buffer. activity determination as described previously. gel resolving analysis of Laemmli (1970) with 15% tinuous SDS-PAGE Brilliant Blue R- and a 4.0% stacking gel and stained with Coomasie was performed as described SDS-PAGE addition, substrate 250. In with 0.01% (w/ previously using polyacrylamide gel copolymerized and others 1993). v) bovine serum albumin (Garcia-Carreno 100 m Partial purification of GP from acetone powder Partial purification and others (1989). The enzyme reaction was stopped by addition of was stopped (1989). The enzyme reaction and others micro-cen- by acid (TCA) followed (30% w/w), Trichloroacetic 0.5 mL (12000 × trifugation in 0.05 Determination of ginger protease specific activity specific protease of ginger Determination ascorbate). The GP resulting volumes of Na phosphate buffer (20 m volumes of Na phosphate Partial purification of GP from acetone powder Partial purification of GP from acetone powder Partial purification Determination of ginger protease specific activity specific protease of ginger Determination activity specific protease of ginger Determination Figure 1—The storage stability of ginger protease (GP) at 5°C. acetone powder. Stabilization and purification, ginger protease . . . . . protease ginger purification, and Stabilization

C: Food Chemistry & Toxicology Possible mechanismofzingibain Possible mechanismofzingibain inactivation andstabilization inactivation andstabilization tion. BothDTTandascorbicacidareinhibitorsforbrowning nucleophilic residues GP, from thereby causingenzymedeactiva- The quinonescouldthenreactwithamine,sulfhydryl,andother quinones catalyzedbyanendogenouspolyphenoloxidase(PPO). The processprobablyinvolved theoxidation ofpolyphenols toform interactions.electrostatic autolysis. GPcouldalsobindtotanninsby hydrogen bondingor ofanamino-quinoneadduct,and with heavymetalions, formation exchange, formationofaquinone-thioladducts, SH-groupbinding deactivation are oxidation oftheactivesiteSH-group, SH/disulfide require afree SH-groupforactivity. Some possibleroutesforGP thiolproteases better techniquesforenzymestabilization.Plant In general,GP °C, GPweretotallystabilized byascorbate. temperatures of<50 sence orpresence of0.2%(w/v)ascorbicacid,respectively. At ascorbate (Figure2b). ure 2.At60 °C, thet Stabilization andpurification,gingerprotease. Possible mechanismofzingibain inactivation andstabilization Possible mechanismofzingibain Possible mechanismofzingibain inactivation andstabilization inactivation andstabilization purification fold; nostabilizerswere used. total gingerprotease (GP)activity recovered; (b)enzyme- 77 m chromatography withstepwiseelutionusing25m Figure 3—Fractionation ofgingerprotease byion-exchange Crude GPextractsfromfreshgingerunderwentrapidbrowning. Improved understandingofGPdeactivationmayhelptodesign M , 100m AC M , 200m solutionswereonlymodestlystabilizedby 1/2 forGPwas2.3minor24intheab- M , 300m M , and400m M NaCl.(a)% M , 50m M , Stabilization andpurificationofgingerprotease Stabilization andpurificationofgingerprotease Protease stabilizationasacetonepowder Protease stabilizationasacetonepowder Degree ofpurificationandyieldforgingerprotease Degree ofpurificationandyieldforgingerprotease bate isthussurprisingandsomewhatcounterintuitive. tolytic deactivationprocess forGP. The stabilizationofGPby ascor- bilizing effectofascorbateisnoteasytoexplaininrelationanau- of GPwithcystineorp-chloromercuribenzoicacid(PCMB).Thesta- Autolysis canusually beavoidedby reacting theactive siteSH-group does notexplainthestabilizationofGPbyascorbate. GP. didnotstabilize the former Apparently, inhibitionofbrowning (Matheis and Whitaker 1983;Jervis andPierpoint1989).However, others 1995). viously reported as29kDa(Ichikawaandothers 1973;Ohtsukiand PAGE isduetoenzymebindingwith(BSA). The MWforGPwaspre- for papain.TheslightlyhigherMW estimateswithsubstrate-SDS- PAGE theMWforGPwas35.1to36.4kDacomparedwith29.2 kDa SDS-PAGEgle visiblebandduring analysis. With substrate-SDS- (control). Allenzymefractionshad thesameMWandproduceasin- activity increaseforGP 21.1%, respectively, ofthetotalGPappliedtocolumn. The peak recoveriesprised 14.3%,19.1%,and of61.3%(225-foldpurification), Stabilization andpurificationofgingerprotease Protease stabilizationasacetonepowder Degree ofpurificationandyieldforgingerprotease GP tive or2-MEbefore (MB-GP)wasreactivated assay. usingDTT modified crudeGPwithPCMB.Themercuribenzoate-GPderiva- cysteine. To autolysis, avoid Ohtsuki andothers(1995)chemically tion ofGP. Ichikawa andothers(1973)stabilized GPwith2m others 2000). role ofwaterasaplasticizer(SladeandLevine1991;Champion rationalized byreferencetoglasstransitiontemperatureandthe (Lui andothers1991;Apenten1998).Sucheffectscanbereadily Enzyme 3-Dstructureismorerigidwithinlowwateractivitysystems of plantpigmentsand(2)reducedwateractivityinGPpowders. from freshgingercanbeexplainedbythe(1)lowerconcentration 1979). ThehighstabilityofGPacetonepowdercomparedwith becomes dehydratedbyacetoneatlowtemperatures(Caygill polyphenols were removed from theginger. also The pulpmaterial ing 25m of ascorbicacid.In summary, GP sults showedthatGPwasactivatedwhenassayedinthepresence thepurificationprocess; moreover,inhibitor during previous re- recovery isperhapsexplainedbytheremovalofanendogenousGP covery rangedbetween54%(pH8)and118%7).The>100% stabilizer (Figure 3a). With additionof0.2%ascorbate, enzymere- ranged between36%(pH8)and98.5%6)withoutascorbateas elution usingNaCl.ThetotalactivityrecoveredbyIECfractionation sulphate, dialyzed,andfractionatedbyIECwithstepwisegradient ascorbate) wasthenprecipitated with70%saturated ammonium ated atpH6to7withoutaddedstabilizer. shown). The peaksdesignatedGP into 4fractions by IECatpH7.0 occurred withGP 17.6%, respectively. The highestdegree ofpurification(940-fold) 210-fold, 127-fold,and102-foldwitharecovery of37%,14.5%, and enzyme fractions designatedGP Stabilization andpurificationofgingerprotease Stabilization andpurificationofgingerprotease Protease stabilizationasacetonepowder Protease stabilizationasacetonepowder Degree ofpurificationandyieldforgingerprotease Degree ofpurificationandyieldforgingerprotease Autolysis isanotherwellknowmodeforproteasedeactivation. The MWofGPwas34.8kDacompared with25.1kDaforpapain The GPstabilizationstrategyadoptedinthisstudywastouse Some formofenzymestabilizationisessentialduringtheisola- During acetonepowderpreparation,hydrophobicplant Four GPvarietieswereelutedfromtheIECcolumnatpH7us- AC asastableenzymesource. Abuffered GP M , 50m 25 M (6.6%recovery) atpH8.0.GPwasalsoseparated , and75m 25 was662-fold(10.8%yield). M without addedascorbate NaCl andaddedascorbate. The AC 25 25 couldbesuccessfullyfraction- , GP , GP 50 50 , andGP , GP 75 AC , andGP solution( 75 werepurified (datanot 100 Ϯ com- 0.2% M

C: Food Chemistry & Toxicology 1986. Tenderization of meat with ginger rhi- of Tenderization 1986. obial proteinases and biotechnology. In: Forgarty and biotechnology. obial proteinases teinase inhibitors. Anal Biochem 214:65–9. Anal Biochem inhibitors. teinase Nutria 26:377–83. technol 11:161–98. T4. Nature 227:680–5. bacteriophage J Food Sci 51:1558–9. zome protease. 37:177–84. Boeing Biotechnol in the solid state. 8:137–62. Biochem J Food and their products. peroxidase Jpn 38:923–6. Econ muscle collagen. J Home 1243:181–4. Acta Biophys Biochim and isoelectric focusing. DEAE-sepharose WM, Kelly CT, editors. Microbial enzyme biotechnology. 2nd ed. New York: Elsevi- York: 2nd ed. New enzyme biotechnology. Microbial editors. CT, WM, Kelly er Science p 227–54. 83:346–56. Anal Biochem applicable. generally which is more Press. Academic York: 4. New Suppl. Res. Food Food J Jpn Soc containing protease. some additives by maboko (boiled fish-paste) 27:371–6. Sci Technol OIRL Press. p 25–56. Bond JS, editors. Proteolytic enzymes. Oxford, U.K.: Crit. Rev. to the assessment of food quality and safety. alternative approach Food Sci Nutria 30:115-360. istry 25:2701–9. enzyme. J Food Sci 38:652–5. J Food enzyme. Ichikawa Y, Sasa H, Michi K. 1973. Purification of ginger protease. J Jpn Soc Food Jpn Soc J protease. of ginger H, MichiK. 1973. Purification Sasa Y, Ichikawa for plant proteins. J Bio- technologies WS. 1989. Purification L, Pierpoint Jervis assembly of the heat of during the protein of structure 1970. Cleavage Laemmli UK. CR. DJ, Ashmore Sehnert YB, Lee proteins of lyophilized AM. 1991. Moisture-aggregation R, Klibanov WR, Langer Lui and polyphenol oxidase by of protein Whitaker JR. 1983. Modification G, Matheis of ginger proteases on M, Arakawa N. 1987. Effects Tajima T, A, Mitsuhashi Mega by M. 1995. Purification of ginger protease K, Kawabata K, Sato Taguchi K, Ohtsuki 1990. Micr COL. H, Boyce Outtrup Peterson GL. 1977. A simplification of the protein assay method of Lowry and others simplification of the protein assay method of Lowry GL. 1977. A Peterson Adv. technology. and condiments: chemistry microbiology JS. 1980. Spices Pruthi in elasticity of Ka- J. 1980. Reduction Yamamoto A, K, Nobuhara N, Hayashi Sakasai RJ, Beynon In: assay methods. 1989. Protease FW. Wagne RS, G, De La Motte Sarath advantages based on an Slade L, Levine H. 1991. Beyond water activity - recent Phytochem- for uniformity. need Plant proteases—a FW.1986. Wagner RD, Storey of proteolytic rhizome: a new source Allen CE. 1973. Ginger ID, Wolf Thompson EH, . Eur . Bio- officinale officinale

Zingiber Zingiber oteases. Enzyme Microbiol Technol 1:233–42. Technol Microbiol Enzyme oteases. aard NF. 1983. Substrate-gel electrophoresis 1983. Substrate-gel NF. aard References Conclusions Acknowledgments

age life for GP for storage and enzyme isolation. Sodium ascor- isolation. Sodium GP for storage and enzyme age life for wo stabilization methods are describedwo stabilization stor for extending the

protease with proline specificity from ginger rhizome protease with proline specificity from ginger rhizome J Biochem 267:1516–26. chemistry 38:11624–33. protease with proline specificity from ginger rhizome protease with proline specificity from ginger rhizome irreversible denaturation. Food Hydrocolloids 12:1-8. irreversible denaturation. mobility in the glass of glass transition and relaxations in foods: molecular Sci 11:41–55. Food Trends range. transition or proteinaceous pro- for composition and molecular weight of proteinases Choi H, Laursen RA, Allen KN. 1999. The 2.1-angstrom structure of a cysteine Choi H, Laursen RA, Allen KN. 1999. The 2.1-angstrom FL, Dimes LE, H Garcia-Carreno bate stabilized GP solutions by 20-fold at 5 GP solutions by bate stabilized Ginger protease ac- °C. charac- stability excellent to have also found were etone powders of GP Stabilization enzyme fractionation. or storage teristics for An ef- commodity. commercial attractive this a more could make GP was de- purifying the stabilized ficient method for partially scribed, to 940-fold maximum purification. leading Caygill JC. 1979. Sulfhydryl plant pr Caygill of cysteine structures glycan Choi KH, Laursen RA. 2000. Amino-acid sequence and Apenten RKO. 1988. The effect of protein unfolding stability on their rates of rates unfolding stability on their The effect of protein 1988. RKO. Apenten understanding an improved Toward 2000. D. M, Simatos Le Meste Champoin D,

The authors are grateful to The Royal Thai Government for a schol- Thai Government The Royal to grateful The authors are Prince of Agro-Industry of thank the Faculty We arship for PA. a study leave. PA for granting Univ. Sonkla T Stabilization and purification, ginger protease . . . . . protease ginger purification, and Stabilization

C: Food Chemistry & Toxicology