Purification, characterization and kinetic properties of extracellular L-asparaginase produced by Cladosporium sp.

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 SDSPAGE
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 lmol/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, b-mercaptomethanol, exerted
inhibitory effect, suggesting a critical role of disulphide
linkages in maintaining a suitable conformation of the
enzyme. Metal ions such as Ca2?, Co2?, Cu2?, Mg2?, Na?,
K? and Zn2? significantly affected enzyme activity
whereas presence of Fe3?, Pb2
? 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.