Bioprocess optimization of L-glutaminase production by a novel <i>Klebsiella pneumoniae</i> strain SCPRC using response surface methodology

L-Glutaminase (EC 3.5.1.2) is an industrially and therapeutically relevant enzyme that hydrolyzes L-glutamine to L-glutamic acid and ammonia. In this study, a potent L-glutaminase-producing bacterium, <i>Klebsiella pneumoniae</i> strain SCPRC, was isolated from soil samples in Kerala, India, using Minimal Glutamine Agar medium with phenol red indicator. The strain was identified by biochemical characterization and 16S rRNA sequencing (GenBank Accession No. OQ338363). Under unoptimized conditions, enzyme activity was 67.18 U/mL. Optimization of culture parameters using Response Surface Methodology with Central Composite Design (RSM-CCD) identified ammonium sulfate, K₂HPO₄, and incubation time as critical factors. The optimized conditions yielded 131.77 U/mL, representing a nearly two-fold improvement compared to baseline production. ANOVA confirmed the model's significance (p < 0.01) with a non-significant lack of fit. These findings establish <i>K. pneumoniae</i> SCPRC as a novel source of L-glutaminase and demonstrate the utility of statistical optimization for enhancing enzyme yields, supporting its potential application in therapeutic and food biotechnology.