title: Purification, characterization and kinetic properties of extracellular L-asparaginase produced by Cladosporium sp.
abstract: L-asparaginase from Cladosporium sp. grown on wheat bran by SSF was purified. Enzyme appeared to be a trimer with homodimer of 37 kDa and another 47 kDa amounting to total mass of 121 kDa as estimated by SDS-PAGE and 120 kDa on gel filtration column. The optimum temperature and pH of the enzyme were 30 °C and 6.3, respectively with Vmax of 4.44 μmol/mL/min and Km of 0.1 M. Substrate specificity studies indicated that, L-asparaginase has greater affinity towards L-asparagine with substrate hydrolysis efficiency (Vmax/Km ratio) eightfold higher than that of L-glutamine. L-asparaginase activity in presence of thiols studied showed decrease in Vmax and increase in Km, indicating nonessential mode of inactivation. Among the thiols tested, β-mercaptomethanol, exerted inhibitory effect, suggesting a critical role of disulphide linkages in maintaining a suitable conformation of the enzyme. Metal ions such as Ca(2+), Co(2+), Cu(2+), Mg(2+), Na(+), K(+) and Zn(2+) significantly affected enzyme activity whereas presence of Fe(3+), Pb(2+) and KI stimulated the activity. Detergents studied also enhanced L-asparaginase activity. In-vitro half-life of purified L-asparaginase in mammalian blood serum was 93.69 h. The enzyme inhibited acrylamide formation in potato chips by 96 % making it a potential candidate for food industry to reduce acrylamide content in starchy fried food commodities.
authors.affiliation: Fermentation Technology and Bioengineering Department, Central Food Technological Research Institute (CFTRI), Mysore 570 020, India.
authors.forename: N S
authors.initials: NS
authors.lastname: Mohan Kumar
authors.forename: H K
authors.initials: HK
authors.lastname: Manonmani
mesh_terms: D001215:Asparaginase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D001216:Asparagine / Q000378:metabolism; D002850:Chromatography, Gel; D002963:Cladosporium / Q000201:enzymology*; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D005973:Glutamine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D055503:Protein Multimerization; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D003470:Culture Media; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D005973:Glutamine; D001216:Asparagine; D001215:Asparaginase