# Recombinant L-asparaginase from Stenotrophomonas maltophilia: a promising low-immunogenic anticancer agent

## TL;DR

A new low-immunogenic L-asparaginase enzyme from Stenotrophomonas maltophilia is developed for better cancer treatment with fewer side effects.

## Contribution

A novel recombinant L-asparaginase with reduced immunogenicity is cloned and optimized for expression in E. coli.

## Key findings

- Recombinant L-asparaginase showed low IC50 values on HEP-G2 and K-562 cell lines.
- The enzyme exhibited significantly lower immunological response in mice compared to existing E. coli L-asparaginase.
- Optimized expression conditions yielded a 17 kDa protein with favorable kinetic parameters.

## Abstract

L-asparaginase is a crucial enzyme used in chemotherapy regimens for the treatment of acute lymphoblastic leukemia (ALL), its incorporation in the pediatric treatment protocols helped in achieving a high cure rate. However, immunogenic side-effects restrict its application and frequently result in stopping treatment. There is a current need for the identification of novel L-asparaginase with improved properties and lower adverse effects compared to those available in the market. L-asparaginase gene from Stenotrophomonas maltophilia (S. maltophilia), an isolated organism that was mentioned as a novel and excellent source for L- asparaginase, was cloned and expressed using E. coli DH5α and E. coli BL21(DE3). Investigations of different conditions of expression of recombinant L-asparaginase in E. coli BL21(DE3) using Box–Behnken design predicted maximum expression at 37 °C temperature, 250 rpm agitation, 0.83 mM isopropylthio-β-D-galactoside (IPTG) concentration after incubation for 17 h. The optimized expression conditions were validated using L-asparaginase activity assay. The obtained recombinant protein was purified using Ni-NTA spin column. SDS-PAGE demonstrated a single band of 17 KDa apparent molecular weight. The kinetic parameters were determined, and they exhibited a low Km value of 2.94 × 10− 2 M and Vmax of 14.73 IU/ml. Cytotoxicity on various cell lines was tested in relation to marketed E. coli L-asparaginase and exhibited low IC50 of 1.92 IU/ml and 2.03 IU/ml for HEP-G2 and K-562 cell lines, respectively. Additionally, mice treated with recombinant L-asparaginase displayed a significantly lower immunological response (IgG) compared to mice treated with marketed E. coli L-asparaginase (p-value < 0.0001). These findings demonstrate the potentiality of recombinant L-asparaginase for its development as a chemotherapeutic drug.