Optimization, purification, and characterization of L-asparaginase from Actinomycetales bacterium BkSoiiA

ABSTRACT

Actinobacteria are promising source of a wide range of important enzymes, some of which are produced in industrial scale, with others yet to be harnessed. L-Asparaginase is used as an antineoplastic agent. The present work deals with the production and optimization of L-asparaginase from Actinomycetales bacterium BkSoiiA using submerged fermentation in M9 medium. Production optimization resulted in a modified M9 medium with yeast extract and fructose as carbon and nitrogen sources, respectively, at pH 8.0, incubated for 120 hr at 30 ± 2°C. The crude enzyme was purified to near homogeneity by ammonium sulfate precipitation following dialysis, ion-exchange column chromatography, and finally gel filtration. The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed an apparent molecular weight of 57 kD. The enzyme was purified 95.06-fold and showed a final specific activity of 204.37 U/mg with 3.49% yield. The purified enzyme showed maximum activity at a pH 10.0 and was stable at pH 7.0 to 9.0. The enzyme was activated by Mn²⁺ and strongly inhibited by Ba²⁺. All these preliminary characterization suggests that the L-asparaginase from the source may be a tool useful to pharmaceutical industries after further research.

KEYWORDS:

• Actinomycetales bacterium BkSoiiA
• enzyme
• L-asparaginase
• optimization
• purification
• submerged fermentation

Acknowledgments

We are thankful to the principal CCF (wildlife) and chief wildlife warden, Odisha, for their permission for collection of samples from Bhitarkanika National Park, and to HOD, Department of Microbiology O.U.A.T, Bhubaneswar, for carrying out the work in the Department of Microbiology.