Production, extraction, and purification of L-asparaginase from Penicillium sizovae - integrated downstream processing with aqueous biphasic systems

Oct 15, 2026

L-asparaginase (L-ASNase) is clinically and industrially important for treating acute lymphoblastic leukemia and for reducing acrylamide formation in baked foods, but current bacterial-derived formulations often trigger hypersensitivity reactions. A novel integrated process is presented to produce, extract, and purify L-ASNase from the fungus Penicillium sizovae using upstream cultivation plus DSP steps: solid–liquid extraction, liquid–liquid extraction in aqueous biphasic systems, and ion exchange chromatography. The ABS approach enables preconcentration directly from fungal lysates, achieving improved purification factors and an overall recovery of 42.3% while avoiding precipitation.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages13

Reading Time33 min

AccessFree to download

Introduction

Results and Discussion

Conclusions

Separation and Puriϧcation Technology 378 (2025) 134547 Contents lists available at ScienceDirect Separation and Purification Technology journal [homepage:](homepage: www.elsevier.com/locate/seppur)[ www.elsevier.com/locate/seppur](homepage: www.elsevier.com/locate/seppur) | Production, extraction, and purification of L-asparaginase from Penicillium sizovae: aqueous biphasic systems as an effective pre-concentration strategy
Marina Borges Guimar˜aes a,b , Carolina Castello Branco Rangel Helbourna,
Jorge Fernando Brand˜ao Pereira b,*, P´erola Oliveira Magalh˜aes a,*
a University of Brasilia, Health Sciences School, Brasilia, DF, Brazil
b University of Coimbra, CERES, Chemical Engineering Department, Faculty of Sciences and Technology, Coimbra, Portugal | | | | --- | --- | --- | | A R T I C L E I N F O | | A B S T R A C T | | Editor: Raquel Aires Barros | | L-asparaginase (L-ASNase) is a clinically and industrially important enzyme, widely used in the treatment of acute lymphoblastic leukemia and in reducing acrylamide formation in baked foods. However, its clinical application is often limited by hypersensitivity reactions, as current formulations are exclusively derived from bacterial sources. To address this limitation, eukaryotic sources such as fungi are being explored to reduce immunogenic effects. This study presents a novel, integrated process for the production, extraction, and purification of L-ASNase from Penicillium sizovae, combining upstream cultivation with downstream processing (DSP) strategies, including solid–liquid extraction (SLE), liquid–liquid extraction (LLE) using aqueous biphasic systems (ABS), and ion exchange chromatography. To the best or our knowledge, this is the first report of L-ASNase purification from a fungal source using ABS. Polymer-salt-based ABS served as a biocompatible and efficient preconcentration platform, enabling simultaneous extraction and concentration of L-ASNase from fungal cell lysates. Among three culture media tested, CM2 yielded the highest L-ASNase activity (2.47 IU/mg protein), attributed to its optimal nitrogen composition. The integrated DSP achieved a purification factor of 1.7 with ABS, followed by a final purification factor of 4.12 and an overall recovery yield of 42.3 % after ion exchange chromatography. This integrated approach eliminates the need for precipitation steps, allows high concentration with effective volume reduction using ABS, supports larger processing scales than fully multi-step chromatographic purification approaches, and shortens processing times compared to conventional DSP methods. The results highlight the potential of ABS as a sustainable and cost-effective alternative for the pre-purification of therapeutic enzymes. | | Keywords:
Enzymes Biopharmaceuticals Downstream processing Protein recovery Liquid–liquid extraction Ion exchange chromatography | | | 1. Introduction L-asparaginase (L-ASNase) is a therapeutic enzyme first discovered by Clementi in 1922 [1,2]. Today, it is widely used in the treatment of acute lymphoblastic leukemia (ALL), a cancer that predominantly affects children and accounts for approximately 25 % of all pediatric cancers and 80 % of all leukemia cases [3]. In pediatric patients, LASNase has been associated with remission rates approaching 90 %, although outcomes in adults remain significantly lower, with remission rates ranging from 20 to 40 %[4,5]. Beyond ALL, L-ASNase has shown therapeutic potential in treating acute monocytic leukemia, glioblastoma, colorectal cancer, pancreatic cancer, and other solid tumors [6]. Currently, all commercial L-ASNase formulations for ALL treatment are derived from bacterial sources, primarily Escherichia coli (native and PEGylated), and, as a second-line treatment, from Erwinia chrysanthemi. However, both formulations are associated with high rates of hypersensitivity reactions, affecting up to 60 % of patients [7]. These adverse effects are attrib

FAQs

Why is L-asparaginase purification a challenge for current clinical formulations?

Commercial L-asparaginase for ALL is mainly derived from bacterial sources, which have high immunogenicity and are associated with frequent hypersensitivity reactions.

What downstream processing steps are used to purify L-ASNase from Penicillium sizovae?

The integrated workflow combines solid–liquid extraction, liquid–liquid extraction using aqueous biphasic systems (ABS), and ion exchange chromatography.

How does aqueous biphasic systems (ABS) contribute to the process?

ABS based on polymer-salt enables biocompatible preconcentration, allowing simultaneous extraction and concentration of L-ASNase from fungal cell lysates and reducing reliance on precipitation and fully multi-step chromatographic purification.

Oct 15, 2026

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