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

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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 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.

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Acknowledgments

The authors gratefully acknowledge the Director, CFTRI, for providing necessary laboratory facilities. The first author expresses his gratitude and sincere thanks to Council of Scientific and Industrial Research (CSIR), New Delhi for providing Senior Research Fellowship.

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  1. Fermentation Technology and Bioengineering Department, Central Food Technological Research Institute (CFTRI), Mysore, 570 020, India

    N. S. Mohan Kumar & H. K. Manonmani

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  1. N. S. Mohan Kumar
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  2. H. K. Manonmani
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Correspondence to H. K. Manonmani.

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Mohan Kumar, N.S., Manonmani, H.K. Purification, characterization and kinetic properties of extracellular l-asparaginase produced by Cladosporium sp.. World J Microbiol Biotechnol 29, 577–587 (2013). https://doi.org/10.1007/s11274-012-1213-0

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