Isolation and optimization of L-Asparaginase production from native halotolerant Bacillus subtilis bacteria isolated from Central Iran.

L-Asparaginase is an important enzyme with therapeutic applications. Current commercial enzymes from E. coli and Erwinia chrysanthemi have limitations, such as high glutaminase activity, creating a need for more stable alternatives. This study aimed to isolate and optimize the production of L-Asparaginase from native, halotolerant Bacillus subtilis strains (DAR and D6A) isolated from central Iran, to find an alternative source for this therapeutically important enzyme. The enzyme was initially isolated using polyethylene glycol (PEG) precipitation. Key factors affecting enzyme production were screened using the Plackett-Burman design and subsequently optimized via the Box-Behnken response surface methodology. PEG precipitation at concentrations of 15-20% for PEG 4000/6000 and 10-15% for PEG 8000/10000 was most effective for initial enzyme separation, yielding specific activities of 4.8 U/mg and 5.2 U/mg for strains DAR and D6A, respectively. Optimization revealed that the most influential factors for maximum L-Asparaginase production were 17.6 g/L NaCl, 4.99 g/L KH2PO4, and 10 g/L asparagine for strain DAR, and 1.49 g/L glucose, 10 g/L NaCl, and 9.98 g/L asparagine for strain D6A. The isolated halotolerant B. subtilis strains are capable of producing L-Asparaginase. PEG precipitation is a suitable method for initial purification, and statistical optimization successfully identified the critical culture conditions to enhance enzyme yield significantly. These strains represent promising sources for the production of this clinically valuable enzyme.