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

Abstract

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.

Keywords: Culture medium; Dodecane; Glycine; Luria–Bertani; Permeabilisation.

Fig. 1

Pareto chart showing effects of the analysed factors and their combined impact on evaluated responses. Evaluated responses: extracellular ASNase activity (A); intracellular ASNase activity (B); biomass (C); cell viability (D). The vertical line indicates the p value ≤ 0.05. Factor in linear form (L) and quadratic form (Q). Software used: Statistica software 10.0 (StatSoft Inc.)

Fig. 2

RS from a CCD, with α = $\sqrt{2}$ and 5 replicates of the central point, to ASNase secretion by using glycine and n-dodecane. Responses: EC ASNase from culture supernatant (A); IC ASNase after osmotic shock (B); biomass, X (C); viability (D)

Fig. 3

Profile of ASNase activity expression and EC ASNase secretion. Induced cultures at 25 °C (A and B) or 37 °C (C and D). Modified LB in the absence (A and C) or presence (B and D) of 0.8% w/v glycine and 6.0% v/v n-dodecane. EC activity (□); IC activity (■);% ASNase secreted (○); total volumetric productivity (◊)

Fig. 4

EC volumetric productivity (●; ○) and specific activity (■;□) at different times post-induction. Induced cultures at 37 °C. Modified LB (●;■); modified LB with 0.8% (w/v) glycine and 6.0% (v/v) n-dodecane (○;□)

Fig. 5

Native-PAGE stained with Coomassie R-250 (A) and zymography (B). EC production, supernatants of cultures with 0.8% w/v glycine and 6.0% v/v n-dodecane (1–2); IC production, cells growth without glycine/n-dodecane and disrupted by glass beads (3–4). Induced cultures for 24 h (1)(3); non-induced cultures (2)(4). Commercial ASNase (5). NativeMark™ Unstained Protein (MW). Arrows: positive for ASNase activity

Fig. 6

SDS-PAGE of culture supernatants induced at 37 °C. Modified LB in the presence (A) or absence (B) of 0.8% w/v glycine and 6.0% v/v n-dodecane. Samples were applied to a 12% gel and stained with Coomassie R-250. Precision Plus Protein marker (MW); post-induction time (h): 0 (1); 4 (2); 8 (3); 12 (4); 16 (5); 20 (6); 24 (7). Commercial ASNase (8)