J Sci Food Agric 1997, 75, 12È18

Comparison of Two Treatment Methods on thePurification of Shrimp Polyphenol OxidaseJ S Chen, D J Charest, M R Marshall* and C I Wei*

Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611-0370, USA

(Received 16 October 1996 ; accepted 3 March 1997)

Abstract : The e†ects of di†erent stirring time and two treatment methods (saltand organic solvent) on the recovery of shrimp polyphenol oxidase (PPO) wereinvestigated. Stirring for 30 min yielded maximal PPO recovery. With respect toPPO speciÐc activity, yield and puriÐcation fold enhancement, the use of butanoltreatment followed by Phenyl Sepharose CL-4B chromatography was shown tobe better than ammonium sulphate fractionation and then Phenyl Sepharosechromatography. White shrimp PPO was more susceptible than pink shrimpPPO to inactivation during puriÐcation.

J Sci Food Agric 75, 12È18 (1997)No. of Figures : 3. No. of Tables : 3. No. of References : 19

Key words : polyphenol oxidase, phenolase, enzymatic browning, shrimp

INTRODUCTION

Polyphenol oxidase (PPO; EC 1.14.18.1.), also knownas polyphenolase, phenolase, tyrosinase and catecholoxidase, has been implicated in the discolouration offruits, vegetables and crustaceans (Savagon and Sreeni-vasan 1978 ; Schwimmer 1981). Formation of melaninor black spot on the surface of crustaceans is of concernto the seafood industry, because the unappealing colourreduces the market value of the products (Simpson et al1987).

Affinity chromatography was shown by Simpson et al(1987, 1988) to yield a higher recovery and puriÐcationof shrimp PPO than ion-exchange chromatography.However, the former method is tedious and the use ofammonium sulphate to precipitate pro-[(NH4)2SO4]teins, if not performed cautiously, adversely a†ects PPOrecovery.

Stelzig et al (1972) used butanol to aid puriÐcation ofapple PPO; they obtained high enzyme yield with littlephenol oxidation. Since crustacean PPO employed inmost studies was semi-puriÐed, it is difficult to exclu-sively relate the Ðndings on crustacean browning toPPO (Simpson et al 1987). Therefore, it is important todevelop new procedures or modify established methods,

* To whom correspondence should be addressed.

like shortening the 3-h extraction time used by Simpsonet al (1987), to better extract crustacean PPO. Theobjectives of the present study were (1) to determine ifthe use of extended extraction period a†ects therecovery of shrimp PPO, and (2) to compare the e†ec-tiveness of ammonium sulphate precipitation andbutanol treatment on shrimp PPO yield, enzyme spe-ciÐc activity and puriÐcation fold.

MATERIALS AND METHODS

Cephalothorax of non-sulphited white shrimp (Penaeussetiferus) and pink shrimp (P duorarum) were frozen inliquid nitrogen and ground into a Ðne powder using aWaring blender at high speed. Shrimp powder wasstored at [20¡C until needed.

E†ect of di†erent extraction time on PPO recovery

Shrimp PPO was extracted according to the procedureof Simpson et al (1988) with slight modiÐcation. Shrimppowder (10 g) was added to three parts (30 ml) of0É05 M sodium phosphate bu†er (pH 7É2) containing 1 M

NaCl (extraction bu†er) and 0É2% Brij 35 (Fisher Scien-tiÐc Co, Orlando, FL, USA). The extract was stirred at4¡C for 30 min, or 1, 2 or 3 h, and the suspension was

121997 SCI. J Sci Food Agric 0022-5142/97/$17.50. Printed in Great Britain(

PuriÐcation of shrimp polyphenol oxidase 13

centrifuged at 8000g (4¡C) for 30 min. The supernatantwas checked for volume, protein content and PPOactivity. Triplicate samples were tested for each shrimpspecies.

PuriÐcation of PPO by ammonium sulphatefractionation

The above supernatant was fractionated withbetween 0 and 40% saturation, and protein(NH4)2SO4

precipitate was collected by centrifugation at 23 500g for30 min (4¡C). The pellet was resuspended in extractionbu†er containing 40% and the suspension(NH4)2SO4 ,was homogenised in a Dounce manual tissue grinder(Wheaton, Millville, NJ, USA) and centrifuged at23 500g (4¡C) for 20 min (Rolle et al 1991). The precipi-tate was again resuspended in extraction bu†er andhomogenised in the tissue grinder. The suspension wasthen subjected to hydrophobic interaction chromatog-raphy using a Phenyl Sepharose CL-4B (Sigma, StLouis, MO, USA) column (Flurkey and Jen 1978).

PuriÐcation of PPO by butanol treatment

The method of Stelzig et al (1972) was followed withslight modiÐcations. Shrimp powder was added to threeparts of 0É1 M sodium phosphate bu†er (pH 6É0) con-taining 0É3 M sucrose. The suspension was stirred at 4¡Cfor 30 min, then centrifuged at 20 000g (4¡C) for 20 min.After the pellet was washed and resuspended in thesame bu†er, the suspension was made up to 2% TritonX-100 (v/v). Following incubation at 25¡C for 15 min,the suspension was centrifuged at 40 000g (4¡C) for30 min, and the supernatant was treated with ice-coldn-butanol ([20¡C). The aqueous phase was dialysedovernight (4¡C) against three changes of 0É05 M sodiumphosphate bu†er (pH 6É5) using dialysis tubings with amolecular weight exclusion limit of 6000È8000. Thedialysate was concentrated utilising an Omega stirredcell Ðtted with a YM 10 Ðlter (Amicon Co, Danvers,MA, USA). This preparation was then diluted in extrac-tion bu†er and loaded onto a Phenyl Sepharose CL-4Bcolumn.

Phenyl Sepharose CL-4B chromatography

High-performance hydrophobic interaction chromatog-raphy was performed on a Phenyl Sepharose CL-4Bcolumn (1É6 cm id ] 40 cm) Ðtted with a circulatingwater jacket and attached to a Dionex gradient pumpequipped with an eluent degas module (Dionex Corp,Sunnyvale, CA, USA). The column was pre-equilibratedwith extraction bu†er. PPO was eluted from the columnwith a stepwise gradient of elution bu†er (100% extrac-tion bu†er (9 ml), 50% extraction bu†er in water(24 ml), and 10% extraction bu†er in water (24 ml)), fol-

lowed by 50% ethylene glycol (12 ml) and Ðnally dis-tilled water (150 ml) at a Ñow rate of 0É2 ml min~1(4¡C) (Rolle et al 1991). A protein proÐle of the 3-mlfractions was determined spectrophotometrically at280 nm. Fractions showing PPO activity were pooledand concentrated.

Protein quantitation and PPO activity determination

Protein content of all preparations was determinedusing the Bio-Rad protein assay kit with bovine serumalbumin (Sigma) as standard. PPO activity was deter-mined at 25¡C for 5 min by mixing 80 kl enzyme prep-aration with 1É12 ml 10 mM L-DOPA in 0É05 M sodiumphosphate bu†er (pH 6É5). PPO activity was determinedby monitoring the rate of dopachrome formation at475 nm using a DU-7 spectrophotometer (BeckmanInstruments, Irving, CA, USA). One unit of PPO activ-ity was deÐned as an increase in one absorbance unitper minute at 25¡C. Unless otherwise speciÐed, experi-ments were conducted three times in duplicate.

Electrophoresis and molecular weight determination

Mini sodium dodecyl sulfate (SDS) polyacrylamide gel(SDS-PAGE; Laemmli 1970) (7] 8 cm, 1 mmthickness) containing 75 g acrylamide per 2 g bis(acrylamide) per 100 ml was prepared according to theMini-Protea II Dual Slab Cell Instruction Manual (Bio-Rad Laboratories 1985). PPO (10 kg protein) wasloaded onto the sample well and electrophoresis wascarried out at a constant voltage of 200 V for 35 minusing a Protea II Slab Cell System equipped with anEPS 500/400 power supply (Pharmacia Fine Chemicals,Piscataway, New Jersey, USA). The purity of enzymepreparations was determined by checking the proteinpatterns following staining of the gels with 10 mM

DL-DOPA in 0É05 M sodium phosphate bu†er (pH 6É5)(Constantinides and Bedford 1967) and then with 1 glitre~1 Commassie Blue R-250 (Eastman Kodak,Rochester, NY, USA) in a Ðxative solution (400 mlmethanol, 100 ml acetic acid per litre~1) for 30 min andthen destained with the Ðxative solution. The molecularweight of PPO was determined (Weber and Osborn1969 ; Weber et al 1972) by comparing the values ofRfprotein with those of non-denatured protein molecularweight standards (SDS-7, Sigma) containing a-lactalbumin (14É2 kDa), trypsin inhibitor (20É1 kDa),trypsinogen (24 kDa), carbonic anhydrase (29 kDa),glyceraldehyde-3-phosphate dehydrogenase (36 kDa),egg albumin (45 kDa) and bovine albumin (66 kDa).

Statistical analysis

Statistical analysis was performed using a PC SASpackage (SAS 1985). DuncanÏs multiple range test was

14 J S Chen et al

performed to determine any signiÐcant (P\ 0É05) di†er-ence among treatments.

RESULTS AND DISCUSSION

E†ect of extraction time on PPO recovery

A 3-h extraction time was used by Simpson et al (1987),Chen et al (1991) and Rolle et al (1991) to extract PPOfrom powder preparations of white shrimp, Floridaspiny lobster, Western Australian lobster and Taiwa-nese black tiger shrimp suspended in 0É05 M sodiumphosphate bu†er (pH 7É2) containing 1 M NaCl and0É2% Brij 35. While Chen et al (1992), in another study,used 30 min to extract PPO from brown shrimp andFlorida spiny lobster. Stirring of pink and white shrimppowder in extraction bu†er from 30 min to 3 h signiÐ-cantly (P\ 0É05) enhanced protein extraction (Table 1).However, the extractants showed signiÐcant decreasesin total PPO activity and speciÐc activity with increasedextraction time. Therefore, a 30-min stirring time waschosen to extract PPO.

Shrimp PPO is generally accompanied, though spa-tially separated, with phenolic substrates. When shrimptissue is disintegrated during enzyme extraction, brown-ing reaction could take place, and the pigments produc-ed following substrate polymerisation might lead toenzyme precipitation and render it insoluble. This irre-versible reaction might also lead to enzyme inactivation(Vamos-Vigyazo 1981). The release of proteolyticenzymes from shrimp preparations during extractioncould also damage PPO. The extended time interval

([30 min) for extracting shrimp PPO may cause sig-niÐcant reduction in enzyme activity, because protectiveagents (phenol scavengers) or inhibitors of PPO or pro-teases were not added during extraction.

Phenyl Sepharose chromatography

Chromatographic proÐles of the pink and white shrimpPPO following butanol treatment and ammonium sul-phate fractionation were similar, although the proteinelution proÐles for both shrimp were di†erent (Figs 1and 2). Apparently, butanol and treatments(NH4)2SO4were functioning di†erently classes of proteins ; pinkshrimp PPO eluted at fraction 39, while white shrimpPPO eluted at fraction 46. In general, shrimp PPO wasbetter separated from non-PPO proteins using butanoltreatment, as evidenced from the large protein peaks forammonium sulphate fractionation eluted before thePPO peaks.

An excessive amount of non-PPO proteins was elutedfrom Phenyl Sepharose column for white shrimp sub-jected to ammonium sulphate fractionation (Fig 2). Theerratic nature of the elution proÐle could be due to thepresence of pigments such as astaxanthine (3,3@-dihydroxy-4,4@-diketo-b-carotene) and its esters(Goodwin 1984), which were not thoroughly removedduring fractionation using These pigments(NH4)2SO4 .were eluted concomitantly with proteins and theytended to impede PPO elution. When butanol was used,pigments and lipid micelle were retained in the butanolfraction, while proteins were solubilised in the aqueousfraction. Removal of pigments was further veriÐed byscanning the butanol extracts of white and pink shrimp

TABLE 1E†ect of di†erent stirring times on the extraction of pink and white

shrimp PPOa

Extraction T otal protein T otal enzyme SpeciÐc activitytime (h) content activity (units mg~1)

(mg) (units)b

Pink shrimp0É5 166a 231a 1É39a1É0 179b 175b 0É98b2É0 209c 165b 0É79c3É0 241d 129c 0É54c

W hite shrimp0É5 171a 856a 5É01a1É0 174a 716b 4É11b2É0 190b 708b 3É73c3É0 213c 680c 3É19d

a Data within the same column and shrimp species with the samesuperscript letter are not signiÐcantly di†erent (P[ 0É05).b One unit \*A475 nm min~1.

PuriÐcation of shrimp polyphenol oxidase 15

Fig 1. Phenyl Sepharose CL-4B chromatographic proÐle of PPO from pink shrimp, employing ammonium sulphate fractionation(*, or butanol extraction min~1).A280 nm ; >, *A475 nm min~1) (K, A280 nm ; =, *A475 nm

PPO preparations ; maximal absorption occurred at470 nm (Chen and Meyers 1984).

PuriÐcation of PPO

The use of fractionation to purify PPO(NH4)2SO4from the crude extracts of pink and white shrimp prep-

arations produced samples with greater total proteincontents (8É1 vs 4É3 mg for pink shrimp, and 27É1 vs5É0 mg for white shrimp) than the use of butanol extrac-tion (Tables 2 and 3). Also enzyme activities (79 vs 60units for pink shrimp, and 80 vs 45 units for whiteshrimp) were higher for fractionation than(NH4)2SO4for butanol treatment. However, lower speciÐc activities(9É7 vs 14 units mg~1 protein for pink shrimp, and 2É95

Fig 2. Phenyl Sepharose CL-4B chromatographic proÐle of PPO from white shrimp, employing ammonium sulphate fractionation(*, or butanol extraction min~1).A280 nm ; >, *A475 nm min~1) (K, A280 nm ; =, *A475 nm

16 J S Chen et al

TABLE 2PuriÐcation scheme of PPO from pink shrimp

Step T otal volume T otal protein T otal activity SpeciÐc activity Y ield PuriÐcation(ml) (mg) (units)a (units mg~1 protein) (%) fold

Ammonium sulphate fractionationCrude supernatant 34É5 170 367 2É16 100 1Before HIC elutionb 10 8É1 79 9É7 21É5 4É4HIC eluant 9 0É3 38 126É7 10É4 64É0

Butanol treatmentCrude supernatant 16 58É7 83 1É41 100 1Before HIC elutionb 7 4É3 60 14 72É3 9É9HIC eluant 6 0É1 32 320 38É6 227

a One unit \ *A475 nm min~1.b Hydrophobic interaction chromatography using Phenyl Sepharose CL-4B column.

vs 9É0 units mg~1 protein for white shrimp), yields (21É5vs 72É3% for pink shrimp, and 33É6 vs 40É9% for whiteshrimp) and fold of puriÐcation (4É4 vs 9É9 for pinkshrimp, and 2É4 vs 5É6 for white shrimp) occurred for

fractionation compared to those with(NH4)2SO4butanol treatment. The reduction in protein recoveryduring butanol treatment could have resulted from theremoval of pigment-bound protein and/or lipoproteinby the solvent.

Samples subjected to butanol extraction and thenelution using a Phenyl Sepharose CL-4B column pro-duced greater PPO yield and puriÐcation fold as well ashigher speciÐc activity than those treated with

fractionation and then column chromatog-(NH4)2SO4raphy. For example, the respective percent yield andpuriÐcation fold of pink shrimp PPO following butanoltreatment and Phenyl Sepharose column chromatog-raphy were 3É7 and 3É5 folds higher than those byammonium sulphate fractionation (Table 2). The pinkand white shrimp PPO prepared by Simpson et al(1987, 1988) using fractionation and affinity(NH4)2SO4

chromatography also showed lower enzyme yield(21É4% for pink shrimp and 8É9% for white shrimp) andpuriÐcation fold (75É3 for pink shrimp and 65É6 forwhite shrimp) than the results of the present study usingbutanol treatment and Phenyl Sepharose chromatog-raphy. The e†ective removal of pigments or pigment-bound proteins from sample preparations duringbutanol treatment may have contributed to the higheryield and fold of puriÐcation of the Ðnal PPO prep-arations.

The speciÐc activity of PPO derived from pink (320units mg~1 protein) and white shrimp (3É5 units mg~1protein) employing butanol treatment and Phenyl Sep-harose chromatography are comparatively higher thanthose obtained by Simpson et al (1987, 1988) using

fractionation and affinity chromatography(NH4)2SO4(82É8 and 59É0 units mg~1 protein for pink and whiteshrimp, respectively). Di†erences in chromatographiesand conditions for PPO puriÐcation as well as di†er-ences of season and conditions of shrimp may beresponsible for the di†erences in PPO speciÐc activity.

TABLE 3PuriÐcation scheme of PPO from white shrimp

Step T otal volume T otal protein T otal activity SpeciÐc activity Y ield PuriÐcation(ml) (mg) (units)a (units mg~1 protein) (%) fold

Ammonium sulphate fractionationCrude supernatant 34 194 238 1É23 100 1Before HIC elutionb 16 27É1 80 2É95 33É6 2É4HIC eluant 7 0É56 31 55É4 13É0 45

Butanol treatmentCrude supernatant 16 68É3 110 1É161 100 1Before HIC elutionb 8 5É0 45 9É0 40É9 5É6HIC eluant 6 0É06 21 350 19É1 217

a One unit \ *A475 nm min~1.b Hydrophobic interaction chromatography using Phenyl Sepharose CL-4B column.

PuriÐcation of shrimp polyphenol oxidase 17

Fig 3. SDS-PAGE proÐles of pink shrimp (PSPPO) and white shrimp (WSPPO) polyphenol oxidase employing ammoniumsulphate fractionation (I) or butanol extraction (II) followed by Phenyl Sepharose chromatography. Protein standards (SDS-7) are

also included. The numerical designations indicate molecular weights of the protein bands.

Electrophoresis and molecular weight determination

The pink and white shrimp PPO prepared by butanoltreatment had two isoforms on the SDS-PAGE gel,while only one isoform was found for those subjected to

fractionation (Fig 3). The molecular weights(NH4)2SO4for the two pink shrimp isoforms were determined as 30and 35 kD, and 20 and 25 kD for white shrimp PPOisoforms. The molecular weights for pink and whiteshrimp PPO with one isoform were calculated as 30and 20 kD, respectively. In this study, the molecularweight of pink shrimp PPO with 30 kD and whiteshrimp PPO with 20 kD were identical to those report-ed by Simpson et al (1988). The multiplicity of enzymeisoforms have been reported in many plants and crusta-ceans PPO, eg mushroom, apple, peach, potato andlobster (Patil and Evans 1963 ; Constantinides andBedford 1967 ; Flurkey and Jen 1978 ; Chen et al 1991).Di†erent preparation procedures might contribute tovariation in the multiplicity forms of pink and whiteshrimp PPO between this study and the work ofSimpson et al (1988).

CONCLUSIONS

This study indicates that a stirring for 30 min of shrimpcephalothorax powder in 0É05 M sodium phosphatebu†er (pH 7É2) containing 1 M NaCl and 0É2% Brij 35 isadequate for higher PPO recovery. Butanol treatmentwas better than ammonium sulphate fractionation for

white and pink shrimp PPO with respect to speciÐcactivity, yield and puriÐcation enhancement.

REFERENCES

Bio-Rad Laboratories 1985 ProteanTM II Mini Slab CellInstruction Manual. Bio-Rad Laboratories, Richmond, CA,USA.

Chen H M, Meyers S P 1984 A rapid quantitative method fordetermination of astaxanthine pigment concentration in oilextracts. J Am Oil Chem Soc 61 1045È1047.

Chen J S, Rolle R S, Marshall M R, Wei C I 1991 Compari-son of phenoloxidase activity from Florida spiny lobsterand Western Australian lobster. J Food Sci 56 154È160.

Chen J S, Balaban M O, Wei C I, Marshall M R, Hsu W Y1992 Inactivation of polyphenol oxidase by high-pressurecarbon dioxide. J Agric Food Chem 40 2345È2349.

Constantinides S M, Bedford C L 1967 Multiple forms of phe-noloxidase. J Food Sci 32 446È450.

Flurkey W H, Jen J 1978 Peroxidase and polyphenol oxidaseactivities in developing peaches. J Food Sci 43 1826È1831.

Goodwin T W 1984 T he Biochemistry of the Carotenoids, Vol.II Animals. Chapman & Hall, New York, USA, p 80.

Laemmli U K 1970 Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227 680È685.

Patil S, Evans H J 1963 Electrophoretic separation of the phe-nolases from potato tubers. Nature 200 1322È1323.

Rolle R S, Guizani N, Chen J S, Marshall M R, Yang J S, WeiC I 1991 PuriÐcation and characterization of phenoloxidaseisoforms from Taiwanese black tiger shrimp (Penaeusmonodon). J Food Biochem 15 17È32.

SAS 1985 UserÏs Guide For Personal Computers, 6th edn. Sta-tistical Analysis System Institute, Cary, NC, USA.

Savagaon K A, Sreenivasan A 1978 Activation mechanism ofpre-phenoloxidase in lobster and shrimp. Fishery T echnol15 49È55.

18 J S Chen et al

Schwimmer S 1981 Source Book of Food Enzymology. AVIPublishing Co, Westport, CT, USA, p 89.

Simpson B K, Marshall M R, Otwell W S 1987 Phenoloxidasefrom shrimp (Penaeus setiferus) : puriÐcation and someproperties. J Agric Food Chem 35 918È921.

Simpson B K, Marshall M R, Otwell W S 1988 Phenoloxidasefrom pink and white shrimp: kinetic and other properties. JFood Biochem 12 205È217.

Stelzig D A, Akhtar S, Ribeiro S 1972 Catechol oxidase of reddelicious apple peel. Phytochem 11 535È539.

Vamos-Vigyazo L 1981 Polyphenoloxidase and peroxidase infruits and vegetables. CRC Crit Rev Food Sci Nutr 9 49È127.

Weber K, Osborn M 1969 The reliability of molecular weightdeterminations by dodecyl sulfate-polyacrylamide gel elec-trophoresis. J Biol Chem 244 4406È4412.

Weber K, Pringle J R, Osborn M 1972 Measurement ofmolecular weights by electrophoresis on SDS-acrylamidegel. Meth Enzymol 26 3È27.