Novel L-Asparaginases from the human gut microbiome: Genome mining, biochemical characterization, and in vitro anti-leukemic activity.

L-asparaginase is essential for acute lymphoblastic leukemia treatment; however, current Escherichia coli and Erwinia chrysanthemi formulations face significant limitations, including immunogenicity, glutaminase-associated toxicity, and short plasma half-life. The human gut microbiome represents an unexplored reservoir of therapeutic enzymes that may offer superior biocompatibility due to host-commensal co-evolution. We employed a systematic genome-mining approach to screen human gut metagenomic data for novel L-asparaginase candidates. Five candidate enzymes from the genera Bacteroides, Ruminococcus, Clostridium, and Prevotella were identified using virtual screening. These enzymes were subsequently codon-optimized and heterologously expressed in E. coli, thereby validating our computational selection strategy. Biochemical characterization revealed optimal activity at alkaline pH (8.0-9.0), robust performance at physiological temperature (37 °C), and excellent storage stability. Ruminococcus_seq7 exhibited exceptional kinetic properties (Km = 0.53 ± 0.19 mM; Vmax = 78.6 ± 5.59 U/mg), whereas Bacteroides_seq104 showed intermediate kinetics (Km = 2.04 ± 0.57 mM; Vmax = 75.4 ± 5.75 U/mg). These lead candidates demonstrated complementary anti-leukemic profiles: Ruminococcus_seq7 showed broad-spectrum activity against T-cell leukemias (IC₅₀: 5.1-8.6 U/mL for Jurkat, MOLT-4, and THP-1), while Bacteroides_seq104 exhibited remarkable potency against THP-1 cells (IC₅₀ = 0.9 U/mL) and successfully overcame resistance in REH cells (IC₅₀ = 36.9 U/mL). Both enzymes maintained >95% viability in healthy HUVEC cells. This study provides proof-of-concept for the discovery of therapeutic enzyme from the human gut microbiome. The identified L-asparaginases exhibited favorable biochemical properties, potent and selective anti-leukemic activity, and enhanced safety profiles. The absence of glutaminase activity and high biocompatibility position these gut microbiome-derived enzymes as promising biotherapeutic scaffolds for next-generation leukemia treatment, pending further optimization of substrate affinity to meet clinical standards.