Partial Separation and Characterization of Papaya Endo- and Exo-Polygalacturonase

Partial Separation and Characterizat on of Papaya Endo- and Exo-Pblygalactu -0nase HARVEY

T. CHAN, JR. and STEVEN

ABSTRACT Exo- and endo-polygalacturonases (EC 3.2.1.40 and EC 3.2.1.15) in papayas were extracted, purified 20- and go-fold, respectively, and characterized. Both enzymes functioned optimally at pH 4.6 and 45°C. Heat inactivation of the papaya PGaseswas biphasic and both phases followed first order kinetics. Decreasing the pH from 4.6 to 3.6 decreasedthe time required for their heat inactivation. The activation energies for the thermal inactivation at pH 3.6 and 4.6 were 85 and 92 kcal/mol, respectively, for endo-PGase(PC I) and 140 and 102 kcal/mol, respectively, for exe-PGase (PC II). The apparent molecular weight was determined by gel filtration to. be 164,000 daltons for PC I and 34,000 daltons for PG II.

INTRODUCTION PAPAYA PGase activity

was reported to increase with fruit ripeness and to be highest in the placenta, decreasing towards the exocarp (Chan et al., 198 1). The effect of excessive heat treatments on the ripening process and on the polygalacturonase activity of papaya fruit was also reported. Extended hot water treatments (46’C for 65 and 90 mm) delayed softening of the fruit tissue which was correlated with a decrease in PGase activity. The decrease in PGase activity after the heat treatments was more severe in the riper fruits than the less ripe fruits. In this study, we report the isolation and partial characterization of an endo-PGase (EC 3.2.1.15) and an exo-PGase (EC 3.2.1.40) from papaya fruit.

MATERIALS&METHODS Extraction of papaya PGase Fresh ripe papayas (Cuticu papaya) Solo cultivar, were chilled, sliced, the seeds discarded and the placenta tissue placed in a chilled beaker. Tissue (1OOg) was macerated with 400 ml of cold acetone (-18°C) in a Waring Blendor for 1 min and the homogenate filtered (Schleicher and Schuell #598) under vacuum. The residue was extracted twice by the above procedure and filtered to near dryness on the last extraction. The residue was then dried under nitrogen and stored at -18’C. The yield was 5.3g acetone powder per 1OOg fresh placenta. Extraction and separation of endo- and exo-PGase Preliminary experiments separated endo-PGase(PC I) from exoPGase (PG II) on the basis of their solubilities in buffers containing high or low NaCl concentrations. PG I is soluble in 0.03M acetate at pH 4.6 containing 0.96M NaCl (high-salt buffer, HSB) and is insoluble in the same buffer containing 0.06M NaCl (low-salt buffer, LSB). PG II is soluble in both HSB and LSB. All enzymes were purified in a room at 7°C. PG I was extracted from acetone powder (5.Og) with 100 ml of HSB. The slurry was stirred 5 min, blended in an ultra Turrax (Tekmar, Inc.) for 1 mm, and squeezed through a Nitex cloth with 30 micron orifices (Tobler, Ernest, Troble, Inc.). The expressedjuices were centrifuged (30 mm, 12,000 x g). The enzymes were precipitated from the supernatant by adding (NH&SO4 to 0.80 saturation. The final mixture was centrifuged (30 min, 12,000 x g). The precipitate was resolubilized with 7 ml LSB and dialysed overnight Authors Research,

Chan and Tam Western Region,

7478-JOURNAL

OF

are with the USDA-SEA, Agricultural P. 0. Box 9 17, Hilo, HI 96720.

FOOD

SCIENCE-Volume

47

(1982)

Y.T. TAM

in Spectrapor membrane tubing No. 1 (Spectrum Medical Ind.) against the same buffer. The dialysate was centrifuged (30 min, 12,000 x g) to remove the PG I precipitate. The supernatant, a semipure extract of PG II with minor amounts of PG I, was labeled supernatant 1. The precipitate containing PG I was rinsed twice with LSB, resolublized in 10 ml HSB and centrifuged (30 min, 12,000, x g). The supernatant containing PC I (Supernatant 2) was loaded on a Sephadex G-200 column and- eluted with HSB. Only the eluted: enzyme peak fraction was kept for further characterization. In another purification scheme PG II was extracted and purified solely through the use of LSB. The purification steps were the same as the steps for PG I with exceptions of the exclusive use of LSB. Supernatant 3 (equivalent to supernatant 1) labeled low-salt extract and containing PG II was layered on a Sephadex G-200 column and eluted with LSB. The eluted enzyme peak fraction was stored at 0°C for further characterization. Enzyme assays PGase activity .was measured yiscometrically (Arakji, 1968). Polygalacturonic acid (PGA) (Sigma Chemical Co.) repurified according to Pressey.and Avants (1973a), was used as the substrate. The reaction mixture consisted of 1.0 ml enzyme solution in either HSB or LSB and 5.0 ml-of 1.2% PGA substrate in LSB. The viscometer (Canon 100) with PGA substrate was equilibrated to 37°C in a water bath. The enzyme at 37’C was added rapidly to the PGA substrate and mixed by bubbling air through the mixture. Differences in viscosity were determined from the flow times of the reaction mixture at initial time (t) and at various intervals. Initial rates were used to calculate PGase activity which then was expressed as the increase in fluidity (Pharr and Dickinson, 1973) per unit time. Fluidity is defined as the inverse of specific viscosity (l/Nsp). Specific viscosity was calculited as follows: flow time of sample -1 Nsp = flow time of water PGase activity was also determined through the spectrophotometric assay of reducing groups by the method of Liu and Luh (1978). The reaction mixture consisted of 2.0 ml 1% PGA substrate in LSB plus 0.2 ml of enzyme extract in either HSB or LSB. The mixture was incubated at 37°C for 2 hr. The reaction was stopped with 0.37 ml 2N HCl and made slightly alkaline with 5.0 ml 0.167N NaOH. One ml of the alkaline reaction mixture was mixed with 0.5 ml of copper reagent, heated in boiling water for 20 min, and cooled to room temperature. The cooled solution was mixed with 0.5 ml arsenomolybdate reagent for color development, diluted with 5 ml distilled water, and centrifuged (15 min, 12,000 xg). The absorbance of the colored solution was read at 730 nm and converted to pmole/ ml/2 hr reducing groups by use of a standard curve of o-D-galacturonic acid (Sigma Chemical Co.). The mode of attack, terminal or random, can be determined by measuring viscosity changes and percent cleavagesof the substrate simultaneously. One-ml samples were drawn from a duplicate viscometric reaction mixture and acidified with 0.17 ml 2N HCI. The samples were made alkaline with 2.3 ml 0.167N NaOH and the reducing group assay was continued as above. Molecular weight determination by gel filtration A column (2.6 x 67 cm) packed with rehydrated Sephadex G-206 (Pharmacia) was equilibrated with LSB and rigged for reverse flow. The column was calibrated for molecular weight determination with the standard proteins, aldolase, ovaibumin, chymotrypsinogen A, catalase, and bovine serium albumin (Pharmacia Calibration Kit). Two ml of dialyzed supernatant 3 were centrifuged (30 min, 12,000 x g). Ths supernatant, then 5 ml of 10% of sorbitol (w/w) were

I

tion scheme was based on their relative solubilities in salt solution (Table 1). The LSB extracts of acetone powder (supernatant 3) consisted mainly of PG II because PC I was insoluble at low salt concentrations. The LSB extracts (supernatant 3) did not reduce substrate viscosity appreciably in 8 hr which indicated the probable presence of exoPGase. HSB extracts of acetone powder consisted of a mixture of PG I and PG II. PG I was precipitated from the mixture by dialysis in LSB. The HSB extracts reduced the substrate’s viscosity appreciably within an hour, indicating the probable presence of an endo-PGase. Papaya endo- and exo-polygalacturonases were purified 1.08- and 4.5-fold, respectively, by ammonium sulfate orecioitation and dialysis (Table 1). This dialysate was stable at pH 4.6 for at least 31 days at 4’C, retaining 91% of the original activity. Gel filtration through Sephadex G-200 resulted in a 90-fold purification for PG I and a 20-fold purification for PC II (Table 1).

introduced into the column through a 4-way valve. The enzymes were eluted with LSB. For supernatant 1 and 2, the G-200 column was reequilibrated with HSB and recalibrated with the standard proteins. Supematant 1 in low salt buffer was first dialyzed against HSB before being introduced into the column. The enzyme sample, followed by 5 ml 10% sorbitol, was introduced into the column and eluted with HSB.

Thin-layerchromatography Thin-layer chromatography (TLC) was used to identify the mono-, di-, and oligogalacturonic acid end-products of PGase degradation. Reaction mixtures with acid inactivated enzymes were desalted through a Biorad AG-SOW-X8 cation exchange column H+ form. The desalted samples were concentrated by lyophilization, resolubilized in methanol and centrifuged 10 min at 12,000 x g. The method of Liu and Luh (1978) was followed for TLC. The supernatant was spotted on thin layer cellulose sheets (Eastman 13255). The spots were eluted twice in the ascending direction in a solvent mixture of ethyl acetate, acetic acid and water (4:2:3, v/v). The acids were detected by spraying with 10% NH40H followed by bromophenol blue (50 mg/lOO ml 95% ethanol at pH 7.0) and finally bromocresol green (0.4% in ethanol).

Mechanism of action

Comparisonratio

The mechanisms of PG I and PG II action were identified both by comparison of their viscometric and reductometric

A comparison ratio (CR) (Tam, 1982) was used to differentiate endo- from exo-PGaseby comparing their rates of loss in viscosity with their rates of bonds hydrolyzed. The CR was calculated by dividing the rate of increase in fluidity by the rate of hydrolysis, as measured by the increase in reducing groups in two identical reaction mixtures. fluidity (l/Nsp)/min CR = nmole reducing groups/min CR was calculated from the initial rates of increase in fluidity and in bonds hydrolyzed; the enzyme concentrations and the time units cancel out and become dimensionless. Endo-PGase cleaves PGA substrate randomly and thus reduces initial viscoisty faster than exe-PGasefor the same number of bonds hydrolyzed. Therefore, the CR for endo-PGasesis higher than the CR for exe-PGases.We have calculated CRs for our enzyme extracts. The CR of purified endo-PGases(gel filtration peak 1) was 11.0 or higher and that of purified exo-PGases(gel filtration peak 2) was 0.10 or lower. Intermediate values of CR constitute mixtures of endo- and exo-PGase. Thus, the value of the CR identifies pure endo- from exo-PGaseextracts and the magnitude of the value can be used as an indication of their purity of their separation.

assays and by TLC end-product identification. Supernatant 2 was made up mostly of PG I. It reduced initial viscosity much faster (Fig. 1) than supernatant 3 (low-salt extracts) (Fig. 2) or supernatant 1 extracts and had a CR of 1.89 (Table

2). Supernatant

2 was purified

by gel filtration

yielding peak 1 (PG I). Peak 1 had a CR of 11 .l and reduced the initial viscosity by 50% through the hydrolysis of only 0.19% of the total available bonds. The 0.19% of

bonds cleaved is between the 3-5% estimated Whitaker (1972)

to be needed to attain a 50% decrease in viscosity

for an endo-PGase, and the 0.03% obtained by Pressey and Avants (1973a) for peach endo-PGase. TLC data on supernatant 2 end-products confirmed the endo mechanism of degradation. The TLC end-products of

supernatant 2 acting on PGA yielded an alcohol solubleimmobile spot at the origin which at first formed a diffuse band of oligogalacturonides of greater than 4 subunits. As the degradation progressed, tetra-, tri-, di- and finally monogalacturonic acids appeared. This pattern is consistent with the mode of action for endo-PGase (Whitaker, 1972; Liu and Luh, 1978).

RESULTS & DISCUSSION

Supernatant I was a mixture consisting mainly of PG II

Separation and purification

and some PG I. It required

The separation and purification of endo-PGase (PG I) from exo-PGase (PG II) in the initial stages of the purifica-

initial viscosity and resulted in a CR of 0.92 (Table 2). Gel

Table 1 -Partial

Procedure

purification

Cone activityb ~mole/ml/2

Low-salt extract, exo-PGases Acetone powder extracta fNH4)$04 ppt dialysate Sephadex G-200 Chromatography Peak 2 (PG II) fexo-PGasest High-salt extract, endo- and exo-PGases Acetone powder extracta (NH4)+04 ppt dialysate Sephadex G-200 Chromatography Peak 1 (PG I) Peak 2 (PG II) fNH4)+04 ppt dialysate Sephadex G-200 Chromatography Peak 1 (PG I)

hr

filtration

of papaya

Total units

of supernatant

100 m m to reach 50% of the

1 yielded

two peaks. Peak 1 was

endo- and exo-PGeses

Protein Img/ml)

Specific activity units/m9

Yield (s/D)

0.86 6.43

191 97.0

1.72 2.83

0.50 2.27

100 50.7

3.72

12

0.075

9.93

6.2

0.86 0.76

192 11.46

4.96 4.24

0.174 0.19

0.015 0.19 11.3

0.075 0.56 113

0.002 0.017 2.18

2.94 2.18 5.17

0.80

2.35

0.010

16.0

100 6.0

Purification 1 4.5 20 1 1.1

0.04 0.29 58.8

17 I3 30

I .23

92

“, 6g Acetone Powder equivalent to 1209 papaya. Reaucing group assay.

Volume 47 (1982bJOURNAL

OF FOOD SCIENCE-1479

PAPA YA POL YGALACTURONASE.

0

’

20

’

’

40

8

’ 60

*

’ SO

8

’ 100

..

a

’

’

120

J

,400

TIME (MINUTES) Fig. I-Changes during degradation

in

TIME (HOURS)

viscosity and reducing by supematant 2 (PG II.

groups

of pectic

acid

Table 2-Comparison

Enzymes PG I (Endo-) & PG II (Exe-)

1 1 - gel filtration

PG I (Endo-

Supernatant

PG I (Endo-)

Supernatant 2 - gel filtration Peak 1 Supernatant 3 (low-salt extract) Supernatant 3 -gel filtration Peak 2

PG II (Exe-1 E CR = fluidity/pmole - Values calculated

viscosity and reducing by supernatant 3 (PG ill.

groups

of pectic

acid

fractions

Fluidity/min

CW

% Bonds hydrolyzed at 50% vise. loss

min

1.15 x 10-Z

1.25 x lo-*

0.92

2.23

7.05 min

3.7 9.0 x 10-5 10-g 1.78 x 10-l

100

2

purification

in

Reducing groups pmole/min

47

hr

-

x 10-4 10-5 x 10-2

0.119

(19.3vJ

1.89

1.07

1.8 x lo-* 5.47 x 10-4

9.8 x lo-* 5.82 x 10-3

11.08

3.5

6.2

x 10-5