Purification and partial characterization of beta-galactosidase from Tritrichomonas foetus.
The work presented in this paper describes the purification and properties of a beta-galactosidase from the protozoan Tritrichomonas foetus. An inexpensive and straightforward method for extraction of the enzyme involving ammonium sulphate precipitation, ion exchange and affinity chromatography resulted in a high level of purification. After purification beta-N-acetylglucosaminidase was the only enzyme present as a contaminant at a significant level. The beta-galactosidase isolated had a pH optimum of 5.8. The Km determined at pH 5.8 was found to be 2.2 mM. Interesting results were obtained when studies were carried out to determine the effect of various metal ions on enzyme activity. Of the metal ions used in this study only manganese ions were found to activate the enzyme. This seems to be a characteristic of trichomonad enzymes, as N-acetyl-beta-glucosaminidase, alpha-galactosidase and N-acetyl-alpha-galactosaminidase are also activated by manganese ions. The strongest inhibition was recorded with lead and to a lesser extent by zinc. The result with lead is not unexpected as the heavy metal is known to cause irreversible inhibition by binding to the amino-acid backbone of the enzyme. The result with zinc is interesting as high levels of zinc are present and trichomonads are known to be apathogenic in semen. The purified beta-galactosidase was found to have the capacity to hydrolyse lactose (Gal beta1-4 Glc), lacto-N-biose 1 (Gal beta1-3 GlcNAc) and N-acetyllactosamine (Gal beta1-4 GlcNAc). When the enzyme was applied to a non-denaturing polyacrylamide gel a single band was observed when stained with Coomassie brilliant blue. This band coincided with that obtained when the gel was stained with p-nitrophenyl beta-galactopyranoside. When the same gel was incubated with p-nitrophenyl N-acetyl beta-glucopyranoside a band was detected which did not coincide with that of beta-galactosidase. Since the beta-N-acetylglucosaminidase enzyme does not move to the same position on a non-denaturing gel as the beta-galactosidase, we will use this technique to isolate the latter enzyme and determine the N-terminal sequence as a prelude to cloning and further study of the gene.
Vella
M
M
School of Biological and Health Sciences, University of Westminster, London, UK.
Greenwell
P
P
D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D013379:Substrate Specificity; D016835:Tritrichomonas foetus / Q000201:enzymology*; D001616:beta-Galactosidase / Q000302:isolation & purification* / Q000378:metabolism
D001616:beta-Galactosidase