Abstract
<title>Abstract</title> <p> L-asparaginase (EC 3.5.1.1) remains an essential therapeutic enzyme for acute lymphoblastic leukemia. Current formulations from <italic>Escherichia coli</italic> and <italic>Erwinia chrysanthemi</italic> , however, suffer from short half-lives and immunogenicity rates of 30–70%. This study reports the first systematic screening of L-asparaginase production by <italic>Pseudomonas reptilivora</italic> ATCC 13626 (NRRL B-6bs). A 2³⁻¹ fractional factorial design (Resolution III; four factorial runs and two center points) was used to evaluate the effects of agitation (150–230 rpm), urea (0–10 g/L), and asparagine (1–5 g/L) on volumetric activity (U/mL), specific activity (U/mg protein·min), and specific production rate (qP, U/g·h). Enzyme activity was measured by aspartic acid hydroxyaminolysis. Urease activity was assessed through three independent methods: Christensen’s agar, growth in peptone-free media, and urease-coupled Berthelot quantification. The maximum volumetric activity (113.76 ± 7.60 U/mL) and specific activity (0.30 ± 0.02 U/mg protein·min) occurred after 48 h at low agitation (150 rpm) with 5 g/L asparagine and no urea. Agitation was the dominant significant factor for specific activity (F = 1688.58; p = 0.015) and qP (F = 2629.35; p = 0.012). Significant curvature across all the responses (p = 0.011–0.037) revealed nonlinear behavior, warranting a response surface methodology (RSM) follow-up. Multiple independent lines of evidence also support an inducible ureolytic capacity—the first such report for this species—challenging its classical urease-negative classification, although molecular confirmation via <italic>ureABC</italic> sequencing remains pending. Under unoptimized conditions, <italic>P. reptilivora</italic> ATCC 13626 is a highly productive L-asparaginase source. Low agitation and asparagine supplementation without urea act as key process levers; a Box–Behnken RSM is recommended for further optimization. </p>