Purification and characterisation of a glutaminase from Debaryomyces spp.

A glutaminase was purified from the cell-free extract of Debaryomyces spp. CECT 11815 by protamine sulphate treatment and several chromatographic procedures including anion exchange chromatography and gel filtration. The purified enzyme consisted of two subunits, with molecular masses of 65 and 50 kDa, respectively. Activity was optimal at 40 °C and pH 8.5, and the Km value for l-glutamine was 4.5 mM. The glutaminase exhibited activity against l-γ-Glu-methyl ester, l-γ-Glu-hydrazide, and l-albiziin, while l-asparagine, CBZ-l-Gln, CBZ-l-Gln-Gly, glutathione, l-γ-Glu-pNA and l-γ-Glu-AMC were not hydrolysed. The enzyme was not affected by PMSF, DTT and EDTA. However, the enzyme was inhibited by sulfhydryl group reagents, DON, l-albizziin, l-asparagine and high concentrations of l-glutamine and ammonium, while l-aspartate did not affect the activity. Phosphate and acetate did not produce any significant effect on the glutaminase activity, but it was slightly stimulated by lactate and borate.