Characterization of the amylolytic properties of the rumen ciliate protozoan Eudiplodinium maggii.

摘要

The rumen ciliate Eudiplodinium maggii is thought to be strongly cellulolytic. However, it preferentially ingested starch when the sheep were fed hay and ground barley. The study was conducted to characterize the amylolytic activity of E. maggii. The crude enzyme preparation obtained from the bacteria-free ciliates degraded starch and dextrin at the rate of 29.5 and 19.4 micro mol released glucose/mg protein/h, respectively, while the degradation rate of maltose and isomaltose was only 0.45 and 0.14 micro mol released glucose/mg protein/h. The pH and temperature optimum for starch, dextrin, maltose and isomaltose hydrolysis varied in the range of 4.5-7.5 and 45-55 degrees C relative to the 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 alpha -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 alpha -amylase 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 amyl1 consisted of 1625 bp and the amyla2-1593 bp and encoded enzymes of 505 and 431 amino acids, respectively.