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.

Production and Characterization of Extremophilic Proteinases From a New Enzyme Source, Barrientosiimonas sp. V9.

Microbial proteases are widely used as commercial enzymes, which have an active role in several industrial processes. The aim of this study was to investigate the production and properties of extracellular proteases from Barrientosiimonas sp. strain V9. The cultivation conditions for protease production were studied using different carbon and nitrogen sources. Maximum protease production was obtained in medium containing 25 g L-1 sucrose, 7 g L-1 KNO3, and initial pH 7.0 at 35 °C and 150 rpm during 72 h. Under these conditions, maximum proteolytic activity reached 1200 U mL-1. The enzyme extract showed optimum activity at 60 °C, pH 9.0, and was stable from 30 to 50 °C within a pH range from 4.0 to 10.0 and NaCl concentration up to 2.5 M. The enzyme was stable in the presence of EDTA, urea, Triton X-100 and laundry detergent (sodium lauryl sulfate as main component). The addition of 1% sodium dodecyl sulfate, Tween-80, or Tween-20 increased the activity by 183% and 119% respectively, while 2-mercaptoethanol reduced the activity to 71%. Casein zymogram analysis revealed three hydrolysis zones suggesting that Barrientosiimonas sp. V9 expresses proteases with molecular weights about 60, 45, and 35 kDa, which were inhibited in the presence of phenylmethylsulfonyl fluoride. Barrientosiimonas sp. V9 produces halotolerant serine proteases with great biotechnological potential.

Microbial cell disruption methods for efficient release of enzyme L-asparaginase.

The efficacy of a simple laboratory method for cell disruption based on the glass bead stirring, sonication, osmotic shock, freezing and grinding, or use of solvents and detergents was assessed in this study, via measurements of the release of total protein and L-asparaginase activity. Three different microbial sources of L-asparaginase were used: Escherichia coli BL21 (DE3), Leucosporidium muscorum, and Aspergillus terreus (CCT 7693). This study adjusted and identified the best procedure for each kind of microorganism. Sonication and glass bead stirring led to obtaining filamentous fungus cell-free extracts containing high concentrations of soluble proteins and specific activity; however, sonication was the best since it obtained 4.61 ± 0.12 IU mg-1 after 3 min of operation time. Mechanical methods were also the most effective for yeast cell disruption, but sonication was the technique which yielded a higher efficiency releasing 7.3 IUtotal compared to glass bead stirring releasing 2.7 IUtotal at the same operation time. For bacterium, sonication proved to be the best procedure due to getting the highest specific activity (9.01 IU mg-1) and total enzyme activity (61.7 IU). The data presented lead to conclude that the mechanical methods appeared to be the most effective for the disintegration of the all microbial cells studies. This is the first report related to the experimental comparison of L-ASNase extraction procedures from different microorganisms, which can also be used for extracting periplasm located enzymes from other organisms.