Therapeuticl-asparaginase: upstream, downstream and beyond

Abstract

l-asparaginase (l-asparagine amino hydrolase, E.C.3.5.1.1) is an enzyme clinically accepted as an antitumor agent to treat acute lymphoblastic leukemia and lymphosarcoma. It catalyzes l-asparagine (Asn) hydrolysis to l-aspartate and ammonia, and Asn effective depletion results in cytotoxicity to leukemic cells. Microbial l-asparaginase (ASNase) production has attracted considerable attention owing to its cost effectiveness and eco-friendliness. The focus of this review is to provide a thorough review on microbial ASNase production, with special emphasis to microbial producers, conditions of enzyme production, protein engineering, downstream processes, biochemical characteristics, enzyme stability, bioavailability, toxicity and allergy potential. Some issues are also highlighted that will have to be addressed to achieve better therapeutic results and less side effects of ASNase use in cancer treatment: (a) search for new sources of this enzyme to increase its availability as a drug; (b) production of new ASNases with improved pharmacodynamics, pharmacokinetics and toxicological profiles, and (c) improvement of ASNase production by recombinant microorganisms. In this regard, rational protein engineering, directed mutagenesis, metabolic flux analysis and optimization of purification protocols are expected to play a paramount role in the near future.

Recombinant L-asparaginase 1 from Saccharomyces cerevisiae: an allosteric enzyme with antineoplastic activity

“…Despite structural differences, type I and type II L-ASNases present similarity in their amino acid sequences, and their functional active site residues are conserved throughout evolution11638. The mechanism of action proposed for L-ASNases relies on EcASNase2 observations, in which a first nucleophilic attack of T12 on the substrate is followed by the release of ammonia and formation of the acyl-enzyme complex.…”

Current applications and different approaches for microbial l-asparaginase production

“…Although l -asparaginase production is observed in animals, plants,12, 30 the microorganisms are considered mainly source for l -asparagine synthesis 30, 31…”

“…The production of this enzyme is mainly proceeded by submerged fermentation 30 . Several researchers have studied the isolation of microbial strains that produce this important enzyme, such as Pseudomonas fluorescens , 32 Serratia marcescens , 33 Escherichia coli , 34 Erwinia carotovora , 35 Proteus vulgaris , 36 Saccharomyces cerevisiae , Karnatakensis Streptomyces, Streptomyces venezuelae and several genres of fungi as Aspergillus, Penicillium and Fusarium 37 …”

“…Concerning to bacteria, the best producers of l -asparaginase are members of the Enterobacteriaceae family 30 . For example, in pharmaceutical industry, this enzyme is produced mainly from bacteria such as Escherichia coli and Erwinia carotova , (also known as Erwinia chrysanthemi ), generally used for leukemia and lymphoma treatment 4 .…”

“…Another interesting technique for asparaginase production is the solid-state fermentation, that allows the use of agroindustrial residues as substrate or support 30 . Within this context, recently, Dias et al (2015) 76 presented the use of different organic residues (wheat bran, soybean meal, cottonseed meal and orange peel), evaluating the production of l -asparaginase from Aspergillus niger .…”