The Degradation of Polygalacturonic Acid by Rumen Ciliate Protozoa

Abstract

295The depolymerase activity of cell-free extracts of nine species of rumen ciliate protozoa and two mixed protozoal preparations, grown in vivo and in vitro, towards polygalacturonic acid was examined. The highest activity was found with Eremoplastron bovis and Ostracodinium obtusum bilobum while there was none in the spined or spineless forms of Entodinium caudatum and little in Polyplastron multivesiculatum. On the basis of the rapid drop in viscosity, inhibition by EDTA and the production of u.v.-absorbing material, the enzymes from all active species were designated as endopectate lyases (EC 4 . 2 . 2 . 2 ) although some polygalacturonase may be present. Neither pectin nor polygalacturonic acid supported the survival or growth of any of the protozoal species tested. I N T R O D U C T I O NThere are several reports (e.g. Wright, 1960Wright, , 1961 ; Abou Akkada & Howard, 1961) that rumen ciliate protozoa contain pectin esterase and enzymes that depolymerize polygalacturonic acid. There is little information on the activity possessed by individual protozoal species although Mah & Hungate (1965) reported that Ophryoscolex purkynei contained polygalacturonate transeliminase (now called pectate lyase). This produced dehydrogenogalacturonyl digalacturonic acid (altered trimer) and dehydrogenogalacturonyl galacturonic acid (altered dimer) as final products and was similar to the bacterial enzyme studied by Hasegawa & Nagel (1962). The present paper records the activities against polygalacturonic acid of cell-free supernatant fluids obtained after the disruption of pure and mixed suspensions of rumen ciliates, grown in vivo and in vitro, and provides evidence that the enzymes are lyases. METHODSProtozoa. Sheep (Clun Forest wethers) containing single protozoal species were obtained by defaunation of the rumen by the method of Orpin (1977) followed by the introduction of a suspension of a single protozoal species isolated from the rumen and grown in vitro as described by Coleman (1978). Mixed rumen protozoa were taken from animals with a normal A-type population (Eadie, 1962(Eadie, , 1967. Entodinium bursa and the spined form of Entodinium caudatum were grown together in the rumen as the former would only grow in the presence of the latter and the latter only formed spines in the presence of the former. The sheep were fed once a day on hay chaff (800 g) and cats (100 g) and rumen contents were removed via a cannula 17 h after feeding. The preparation of washed suspensions of protozoa and the separation of a mixed population into fractions containing small or large protozoa were carried out as described by Coleman & Sandford (1979).Protozoa were grown in vitro as described by Coleman (1978) and harvested as described by Coleman (1969) except that mineral salt solution D [containing (g 1-I): KzHP04, 6.3; KH,P04, 5.0; NaCI, 0.65; CaCI, (dried), 0.045; MgSOl. 7H@, 0.091 was used throughout. The protozoa were finally suspended in water (for disruption) or in salt solution D (for experiments with living protozoa) when N2/...

Effects of microbial synergism on fibre digestion in the rumen

“…Orpin (1983-4) has summarized the information available on the occurrence of the pectin-degrading enzymes found in cell-free extracts of different genera and species of rumen ciliates. Pectinesterase (EC 3.1.1.11) and polygalacturonase (EC 3.2.1.15) activity occurs in the lsotrichidae and P. multivesiculatum, while endopectate lyases occur only in the higher ophryoscolecids (Coleman et al 1980;Orpin, 1983-4;Williams, 1986). Entodinia appear to be devoid of any enzymes active against pectin (Coleman et al 1980).…”

Microbial pectic transeliminases

“…Very few reports are available on PGL from protozoa. Endo-PGL activity was observed in cell free extracts of rumen ciliate protozoa, viz., Ostracodinium obtusum, Entodinium caudatum (Coleman et al 1980). A human anaerobic intestinal bacterium, Clostridium butyricum-beijerinckii, produced endo PGL (0.21 U/ml) and exo PGL (0.11 U/ml) when pectic acid was the carbon source (Nakajima et al 1999).…”

The metabolism of rumen ciliate protozoa

“…Many of the rumen ciliates contain a soluble endopectate lyase which depolymerises polygalacturonic acid [38]. In general, the cellulolytic species contain enzyme with the highest specific activity and Entodinium spp.…”