Endopolygalacturonase from tomato fruit

Phytochemlstry 1974 Vol I?, pp 2725lo 2727 Pergamon Pren

Prmtrd m England

ENDOPOLYGALACTURONASE TOMATO FRUIT* WILLIAMJ. HUNTERand Department

of Mlcroblology,

North

Carohna (Recwd

GERALD

State Umverslty, Retrwd

FROM

H. ELKAN Raleigh,

North

Carolma

27607, U S A

3 Aprrl 1974)

Solanaceae, endopolygalacturonase. Key Word Index-Lycoperslcon esculentum, enzymes, pectlc acid, tomato pectlc enzymes, tomato fruit ripening

tomato

enzymes,

pectlc

Abstract-A polygalacturonase was extracted from rlpenmg tomato fruit A four step procedure was developed producing a 44-fold increase m specific activity with 9”/;, recovery The enzyme was found to rapIdly degrade pectlc acid but not pectin N-0 transehmmase actlvlty was detected Vlscoslty and per cent hydroIysls studies formed a basis for suggesting that this enzyme cleaves Its substrate m a random manner and 1s likely to be an endopolygalacturonase

INTRODUCTION

ripening, the mtercellular pectin material of the tomato fruit 1sweakened by pectlc enzymes. Pectinesterase de-esterlfies the pectin material, converting it to pectlc acid.’ Pectic acid IS then depolymerized by the action of hydrolytic polygalacturonases.2,3 Two polygalacturonases appear to be involved m the process. One produces a more rapid reduction in viscosity compared with percentage hydrolysis of the substrate than the other; the former is characteristic of an endopolygalacturonase and the latter of an exopolygalacturonase.4 The present investigation concerns the purification of tomato endopolygalacturonase. Some time ago Pate1 and Phaffj obtained a 55 x increase m specific activity of tomato polygalacturonase. DURING

RESULTS

AND

DISCUSSION

The current work obtained a higher degree of purification. A 44-fold increase in specific activity was achieved, with a 9% recovery. The procedure involved four maJor steps which are summarized in Table 1. Column chromatography, the last step in the purification procedure, yielded three protein peaks The first peak eluted with or near the void volume, a second peak came off shortly thereafter, and a third peak trailed. Polygalacturonase activity corresponded with the second peak. Characterization studies suggest that this enzyme 1s a hydrolytic endopolygalacturonase. The substrate specificity indicates that this enzyme rapidly degrades pectlc acid but * Paper

number

427X of the Journal

Series of the North

’ H~BSON,G E (1963) B~ochrm J 86, 358 2 3 4 s

Carolma

Agricultural

DEMAIN,A L and PHAFF, H J (1957) J Ayr Food Chem $60 BESFORD,R T and HOBSON,G E (1972) Phytochemstry 11, 2201 PRESSEY.R and AVANTS, J K (1973) &othm Bzophyc Acta 309,363 PATEL, D S and PHAFF,H J (1960) Food Rer 25, 37 2725

Experiment

Statlon,

Raleigh

W J Huvr~~andG

H

Era~w

not pectm. Pectic aad degradation proceeded with no detectable Increase m the ahsorbance at 235 nm This suggests that the enzyme IS hydrolytic ’ Activltj was such that an mcrcase m A235 nm of 1 5 units would have been expected if the enzyme cleaved by a transelunmase mechamsm Studies comparing per cent decrease m v~scoslt> suggest that this enzyme cleaves Its substrate m ;I random, rather than sequential, manner Thzsc results are presented m Table 3 Random cleavmg enzymes product a loo rate of hydrol>sls, about 2Z3”;. at 50”,, dccrcase m vlscoslty 7 ‘)With a sequentlal attack the rate of hqdrolysls 15 higher. about 2@ 25”(1 at 501,,“’ decrease m viscosity.

Endopolygalacturonase

from tomato

fruit

__ ‘777

at 4”, then centrifuged (150009 for 30mm) to remove msoluble material, and filtered (Whatman No 1) (Step 2) The flmd from Step 1 was cooled to 0” made 50% saturated by the slow addnlon of granular (NH&SO,, and centrifuged (ISo q for 30 mm) The supernatant was made 80% saturated with (NH&SO, and receqtrifuged The resultmg pellet was solubdlzed m a mmlmum vol of buffer (0 05 M Na citrate, pH 5 5) (Step 3) The fluid from Step 2 was dialyzed agamst 4 changes of H,O and centrifuged (15000y for 30 mm) The pellet was suspended m 25 ml cold buffer and placed on a stnrer at 4” overmght. Insoluble material was removed by centrlPM-30 filter (Step 4) This concentrate was placed on a Sephadex G-100 column and eluted with buffer at 4” or Enzyme actlvlty was measured by a vlscoslty method, I’ by followmg the mcrease m reducmg umts,” A235 nm ’ Size 300 Cannon-Fenske vlscometers were used to follow viscosity changes Substrates were 1% soln of pectlc acid (Na Polypectate No 6024) and pectm (Pectin, L M No 3446) obtamed from Sunkist Growers, Ontario, Cahforma Buffer was used to suspend the substrates Merthlolate was added to a final concentration of 0 002% to prevent bacterlal growth Acknowledgements-This GB-8383, Pubhc Health Diseases, and by USDA

mvestlgatlon was supported by Natlonal Science Foundation grants GB-4738X and Service Research Grant AI-07247 from the Natlonal Institute of Allergy and Infectious Cooperative State Research Service grant 916-15-04

” LILLICH.T T and ELKAN, G H (1968) Ca,l J M~rob~ol I2 NELSON, N (1944) J B~ol Chem 153, 375

14, 617