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.

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.

Asparaginase is an integral component in the treatment of acute lymphoblastic leukemia. The asparaginase products available in our country are Escherichia Coli (E. Coli)-derived asparaginase (Leunase), pegaspargase (Oncaspar), and, to a lesser extent, Erwinia chrysanthemi-derived asparaginase. Hypersensitivity reactions (HSR) are reported less frequently in those receiving pegaspargase, ranging from 3% to 15%; however, these reactions can limit its use in this population.

L-Asparaginase (E.C.3.5.1.1; L-ASNase) hydrolyzes L-asparagine (an essential amino acid for the growth of leukemic cells) to aspartic acid and ammonia. It is obtained from various sources, including: bacteria, yeast, fungi, plants, and animals. It is used as a chemotherapeutic agent to treat acute lymphoblastic leukemia (ALL) and reduce acrylamide formation in baked and fried foods. Globally, various recombinant and pegylated L-ASNases formulations, including Escherichia coli and Erwinia-derived variants, are in Phase II or active clinical trials for ALL and related conditions across the USA, Brazil, and Canada. Some drugs are recruiting, and other variants of E. coli and E. chrysanthemi L-ASNases are already approved for marketing. Although L-ASNase is used as a therapeutic agent, immunogenic reactions and other adverse effects continue to limit its use. To enhance yield, L-ASNase isoforms are cloned and expressed in host cells, generating recombinants with distinct physicochemical properties and kinetic parameters. The enzymes' temperature and pH ranges vary from 25 to 100 °C and 6 to 10, respectively. Nowadays, enzymatic modifications, such as immobilization (chemical, physical, and PEGylation), mutagenesis, and PASylation, are used to overcome the limitations of commercialized L-ASNase-based drugs. Therefore, this review is a timely effort to compile and analyze the properties of recombinant L-ASNases and the contemporary techniques used to improve L-ASNase. A comprehensive study would help us better understand the kinetic parameters, biochemical properties, and modification trends of L-ASNase, enabling the development of robust, reliable therapeutics in the future.

L-Asparaginase (LA; EC 3.5.1.1) is a clinically and industrially relevant enzyme that hydrolyzes L-asparagine to L-aspartate and ammonia. LA has demonstrated efficacy as a chemotherapeutic agent by depleting L-asparagine, an amino acid essential for leukemic cell survival. FDA-approved formulations derived from Escherichia coli and Erwinia chrysanthemi face challenges, including a short half-life and adverse side effects, necessitating continuous efforts to develop more stable and specific variants. Concurrently, LA's role in acrylamide mitigation during food processing has garnered attention, supported by thermostable and high-yield fungal and bacterial strains. This review provides a comprehensive overview of LA's multifaceted roles, highlighting its therapeutic significance in treating acute lymphoblastic leukemia (ALL) and its role in acrylamide mitigation during food processing. It highlights exciting developments in the field, from discovering novel microbial sources to engineering the enzyme for improved performance. It also examines scalable production methodologies like submerged and solid-state fermentation, which are key to meeting growing demand. The review also highlights innovative strategies such as structural modifications and immobilization techniques aimed at enhancing enzyme stability, substrate specificity and therapeutic efficacy. This review demonstrates the transformative potential of LA in both healthcare and industrial contexts through integrating recent breakthroughs and innovative approaches. The insights provided aim to guide future research and innovation, paving the way for the development of optimized and sustainable applications of this crucial enzyme.

Asparaginase is a standard treatment for acute lymphoblastic leukemia (ALL) of childhood. As a bacteria-derived enzyme, asparaginase is highly immunogenic, and hypersensitivity reactions (HSRs) routinely lead to drug discontinuation. HSRs remain common even with the introduction of pegaspargase, a PEGylated version of Escherichia coli-derived asparaginase. Asparaginase Erwinia chrysanthemi (recombinant)-rywn (recombinant Erwinia) is an alternative for those with HSRs to pegaspargase. Here, we describe an 11-year-old boy with relapsed ALL who developed HSRs to both pegaspargase and recombinant Erwinia. This is the report of a novel desensitization protocol for calaspargase pegol-mknl (calaspargase) with no adverse events and adequate serum asparaginase activity.

L-asparaginases (EC 3.5.1.1) are amidohydrolase enzymes that predominantly catalyze conversion of L-asparagine to L-aspartic acid and ammonia. In addition, some exhibit secondary L-glutaminase activity. Escherichia coli and Erwinia chrysanthemi L-asparaginases are widely used in the pharmaceutical industry to produce therapeutically important compounds. In the therapeutic use of enzymes, bacterial L-asparaginases can trigger immune responses, leading to a high rate of adverse effects that diminish the effectiveness of the treatment. This situation has forced scientists to search for promising L-asparaginases from new sources. Yeast L-asparaginases could be useful in reducing toxicity and enhancing efficacy but they have been poorly studied to date. Here, we characterized the yeast Lachancea thermotolerans L-asparaginase (LtASNase) purified by affinity chromatography. It has a specific activity of 313.8 U/mg and a high kcat value (312.4 s). We demonstrated through a semi-rational design that the mutations of Lys99 show varying effects on catalytic activity, with the Lys99Ala mutant increasing specific activity 3.3-fold. Furthermore, the in vitro antileukemic activity of the non-formulated form of Lys99Ala LtASNase was evaluated against SUP-B15 and REH cell lines. The results demonstrated that LtASNase exhibits significant antileukemic potential, comparable to commercial type II bacterial enzymes. The understanding of the mutant L-asparaginases examined in this study will significantly contribute to the development of new and more effective yeast-derived asparaginases.

Polyethylene-glycolated Escherichia coli-derived l-asparaginase (pegaspargase, pASP) is an essential component of paediatric-inspired regimens for the treatment of acute lymphoblastic leukaemia/lymphoma; nonetheless, is characterised by severe and potentially life-threatening toxicities, such as hypertriglyceridemia. Grades 3-4 events have been reported in ~1%-18% of paediatric patients and in sparse reports in adults. There is limited evidence on the safety of asparaginase rechallenge in patients experiencing severe pASP-related hypertriglyceridemia. Herein we present the case of a young adult patient diagnosed with T-LBL who experienced an asymptomatic severe pASP-related hypertriglyceridemia and was safely re-exposed to ASP using Erwinia chrysanthemi asparaginase (crisantapase), with only mild transient hypertriglyceridemia recurrence.

L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with L-asparaginase have been observed, mainly caused by immunogenicity and allergenicity. Some strategies have been adopted, such as searching for new microorganisms that produce the enzyme and applying protein engineering. Therefore, this work aimed to elucidate the molecular structure and predict the immunogenic profile of L-asparaginase from Penicillium cerradense, recently revealed as a new fungus of the genus Penicillium and producer of the enzyme, as a motivation to search for alternatives to bacterial L-asparaginase. In the evolutionary relationship, L-asparaginase from P. cerradense closely matches Aspergillus species. Using in silico tools, we characterized the enzyme as a protein fragment of 378 amino acids (39 kDa), including a signal peptide containing 17 amino acids, and the isoelectric point at 5.13. The oligomeric state was predicted to be a homotetramer. Also, this L-asparaginase presented a similar immunogenicity response (T- and B-cell epitopes) compared to Escherichia coli and Dickeya chrysanthemi enzymes. These results suggest a potentially useful L-asparaginase, with insights that can drive strategies to improve enzyme production.

Amino acid depletion therapy is a promising approach for cancer treatment. It exploits the differences in the metabolic processes between healthy and cancerous cells. Certain microbial enzymes induce cancer cell apoptosis by removing essential amino acids. L-asparaginase is an enzyme approved by the FDA for the treatment of acute lymphoblastic leukemia. The enzymes currently employed in clinics come from two different sources: Escherichia coli and Erwinia chrysanthemi. Nevertheless, the search for improved enzymes and other sources continues because of several factors, including immunogenicity, in vivo instability, and protease degradation. Before determining whether L-asparaginase is clinically useful, research should consider the Michaelis constant, turnover number, and maximal velocity. The identification of L-asparaginase from microbial sources has been the subject of various studies. The primary goals of this review are to explore the most current approaches used in the search for therapeutically useful L-asparaginases and to establish whether these investigations identified the crucial characteristics of L-asparaginases before declaring their therapeutic potential.