Optimization, Purification and Antitumor Activity of Kodamaea ohmeri ANOMY L-Asparaginase Isolated from Banana Peel

Abstract

Objective: L-Asparaginase is an important enzyme that converts L-asparagine to L-aspartate and ammonia. Microbial L-asparaginase has important applications as anticancer and food processing agents.

Methods: This study reported the isolation, screening of a local yeast isolate from banana peel for L-asparaginase production using submerged fermentation, optimization of the production, purification, and anticancer assay of L-asparaginase. The yeast isolate was identified as Kodamaea ohmeri ANOMY based on the analysis of nuclear large subunit (26S) rDNA partial sequences. It was a promising L-asparaginase producer with a specific activity of 3059±193 U/mg in a non-optimized medium. The classical one-variable-at-a-time method was used to optimize the production medium components, and it was found that the elimination of K2HPO4 from the medium increased L-asparaginase specific activity (3100.90±180 U/mg).

Results: Statistical optimization of L-asparaginase production was done using Plackett-Burman and Box-Behnken designs. The production medium for the maximum L-asparaginase specific activity (8500±578U/mg) was as follows (g/L): L-asparagine (7.50), NaNO3 (0.50), MgSO₄.7H₂O (0.80), KCl (0.80) associated with an incubation period of 5 days, inoculum size of 5.60 %, and pH (7.0). The optimization process increased L-asparaginase production by 2.78-fold compared to the non-optimized medium. L-Asparaginase was purified using ammonium sulphate precipitation followed by gel filtration on a Sephadex G-100 column. Its molecular weight was 66 KDa by SDS-PAGE analysis.

Conclusion: The cell morphology technique was used to evaluate the anticancer activity of L-asparaginase against three different cell lines. L-Asparaginase inhibited the growth of HepG-2, MCF-7, and HCT-116 cells at a concentration of 20, 50, and 60 μL, respectively.

Keywords: L-Asparaginase; anticancer activity.; banana peel; purification; statistical optimization; yeast.