Improvement in extracellular secretion of recombinant L-asparaginase II by Escherichia coli BL21 (DE3) using glycine and n-dodecane.

L-asparaginase II (ASNase) is the biopharmaceutical of choice for the treatment of acute lymphoblastic leukaemia. In this study, E. coli BL21 (DE3) transformed with the pET15b + asnB vector which expresses recombinant ASNase was used as a source to obtain this enzyme. The ideal conditions to produce ASNase would be a high level of secretion into the extracellular medium, which depends not only on the application of molecular biology techniques but also on the development of a strategy to modify cell permeability such as the addition of substances to the culture medium that stimulate destabilisation of structural components of the cell. Thus, the growth of E. coli BL21 (DE3) in modified Luria-Bertani broth, supplemented with 0.8% (w/v) glycine and 6% (v/v) n-dodecane, increased the total yield of ASNase by about 50% (15,108 IU L-1) and resulted in a 16-fold increase in extracellular enzymatic productivity (484 IU L-1 h-1), compared to production using the same medium without addition of these substances. Most of the enzyme (89%) was secreted into the culture medium 24 h after the induction step. This proposed approach presents a simple strategy to increase extracellular production of ASNase in E. coli.

L-asparaginase Production by Leucosporidium scottii in a Bench-Scale Bioreactor With Co-production of Lipids.

L-asparaginase (ASNase) is a therapeutical enzyme used for treatment of acute lymphoblastic leukemia. ASNase products available in the market are produced by bacteria and usually present allergic response and important toxicity effects to the patients. Production of ASNase by yeasts could be an alternative to overcome these problems since yeasts have better compatibility with the human system. Recently, it was found that Leucosporidium scottii, a psychrotolerant yeast, produces ASNase. In order to advance the production of ASNase by this yeast, the present study aimed to select suitable process conditions able to maximize the production of this enzyme in a bench-scale bioreactor. Additionally, the accumulation of lipids during the enzyme production process was also determined and quantified. Experiments were carried out with the aim of selecting the most appropriate conditions of initial cell concentration (1.0, 3.5, and 5.6 g L-1), carbon source (sucrose and glycerol, individually or in mixture) and oxygen transfer rate (k L a in the range of 1.42-123 h-1) to be used on the production of ASNase by this yeast. Results revealed that the enzyme production increased when using an initial cell concentration of 5.6 g L-1, mixture of sucrose and glycerol as carbon source, and k L a of 91.72 h-1. Under these conditions, the enzyme productivity was maximized, reaching 35.11 U L-1 h-1, which is already suitable for the development of scale-up studies. Additionally, accumulation of lipids was observed in all the cultivations, corresponding to 2-7 g L-1 (32-40% of the cell dry mass), with oleic acid (C18 : 1) being the predominant compound (50.15%). Since the L-asparaginase biopharmaceuticals on the market are highly priced, the co-production of lipids as a secondary high-value product during the ASNase production, as observed in the present study, is an interesting finding that opens up perspectives to increase the economic feasibility of the enzyme production process.