title: Purification and properties of an L-asparaginase from Cylindrocarpon obtusisporum MB-10.
abstract: An L-asparaginase producing mesophilic fungus Cylindrocarpon obtusisporum MB-10 was isolated from soil. The constitutive intracellular L-asparaginase from the organism was purified. The enzyme after 65-fold purification with an overall yield of 11% and specific activity of 100 unit.mg-1 seemed to be homogeneous in native, SDS-PAGE and thin layer isoelectric focusing gel. The apparent Mr of the enzyme was 216,000, and it constituted four identical subunits. The pI of the enzyme was 5.5. It was a conjugate protein with 37.3% (w/w) carbohydrate. The enzyme was stable to storage at -20 degrees C and to repeated freezing and thawing. The L-asparaginase from the organism was very much specific for L-asparagine and did not hydrolyze D-asparagine and L-glutamine. The pH and temperature optima for the enzyme activity were 7.4 and 37 degrees C, respectively. The Km of the L-asparaginase was found to be 1 x 10(-3)M. Metal ions, such as Zn2+, Fe2+, Cu2+, Hg2+ and Ni2+ potentially inhibited the enzyme activity, while metal chelators like EDTA, CN-, cysteine, etc., enhanced the activity indicating that the enzyme was not a metalloprotein. Its activity was also enhanced in the presence of reduced glutathione but not with dithiothreitol and 2-mercaptoethanol. Differential inhibition of the enzyme activity was observed with iodoacetamide and p-chloromercuribenzoate, thus indicating possible involvement of free-SH group in the enzyme catalysis.
authors.affiliation: Department of Microbiology, Bose Institute, Calcutta, India.
authors.forename: S K
authors.initials: SK
authors.lastname: Raha
authors.forename: S K
authors.initials: SK
authors.lastname: Roy
authors.forename: S K
authors.initials: SK
authors.lastname: Dey
authors.forename: S L
authors.initials: SL
authors.lastname: Chakrabarty
mesh_terms: D001215:Asparaginase / Q000737:chemistry* / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000502:physiology; D007525:Isoelectric Focusing; D003904:Mitosporic Fungi / Q000201:enzymology*; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001215:Asparaginase