Indicator dye based screening of glutaminase free L-asparaginase producer and kinetic evaluation of enzyme production process

Abstract

Several soil isolates from 1 g of soil sample were isolated and screened for the production of L-asparaginase. Primary screening was performed using rapid plate assay; dye indicator studies were conducted, and phenol red with 0.005% concentration was found to be optimum. The secondary screening was carried out using the Nesslerization method. The bacteria screened for L-asparaginase production with no glutaminase activity was identified as Bacillus subtilis. Crude L-asparaginase enzyme was partially purified 1.57 folds of purity and 110 U/mg of specific activity. The glutaminase-free L-asparaginase activity was also confirmed using LC-MS analysis. The presence of mass peaks at 147.0 in the reaction mixture suggested an absence of glutaminase activity. An optimized medium obtained comprised of Dextrose 1.5 g/L, K₂HPO₄ 1.2 g/L, L-asparagine 15 g/L, and Tryptone 5 g/L. The highest L-asparaginase activity was observed at 6.0 pH and 30 °C. Kinetic parameters associated with biomass and L-asparaginase production were also studied. The computed values were µ_m 0.104 h⁻¹, X_m 6g/L P₀ 1.7U/mL P_m 8.2 U/mL Y_X/S 4 g-cell/g-glucose µP_m 0.35 h⁻¹ q_p 5.46 U/g/h Y_P/x 13.6667 U/g-cell. The novel bacterial isolates showed promise as a potential glutaminase-free L-asparaginase producer, which can prove to be of industrial applications.

Keywords:

• Bacillus subtilis
• glutaminase
• L-asparaginase
• phenol red

Acknowledgments

The authors acknowledge Sophisticated Analytical Instrument Facility, Council of Scientific and Industrial Research—Central Drug Research Institute (SAIF-CSIR-CDRI), Lucknow, Uttar Pradesh, India to provide instrumentation facilities for LC-MS analysis.

Disclosure statement

No potential conflict of interest was reported by the author(s).