rumen ciliates

starch

amylolytic enzymes

amylase genes

The rumen ciliates Eudiplodinium maggii are thought to be strongly celluloloytic. We observed, however, that they preferentially ingested starch when the sheep were fed hay and ground barley. The studies reported in this paper were undertaken in order to characterize the amylolytic activity of these protozoa. The crude enzyme preparation obtained from the bacteria-free ciliates degraded starch and dextrin at the rate of 29.5 and 19.4 μmol released glucose/mg protein/h, respectively, while the degradation rate of maltose and isomaltose was only 0.45 and 0.14 μmol released glucose/mg protein/h. The pH and temperature optimum of starch, dextrin, maltose and isomaltose hydrolysis varied in the range of 4.5-7.5 and 45-55ºC relative to substrate. Pullulan was not degraded. Four protein bands with the ability to degrade starch were identified by a zymogram technique following the electrophoretic separation of protozoal protein. The enzymes were α-amylase in nature, as they degraded starch mainly to maltose and maltotriose. Ion-exchange chromatography of a crude enzyme preparation resulted in the separation of numerous fractions which were able to degrade starch. The most amylolytic fractions were very rich in protein and also exhibited a strong ability to digest carboxymethylcellulose. Partial sequences from two genes coding for synthesis of α-mylase enzymes were identified in a cDNA library of Eudiplodinium maggii. The rest of the sequences were reconstructed using GeneRacer and both complete genes were sequenced and cloned. Gene amyl 1 consisted of 1625 bp and the amyla 2 - 1593 bp. They encoded enzymes of 505 and 431 amino acids, respectively.