Expression of Bacillus amyloliquefaciens transglutaminase in recombinant E. coli under the control of a bicistronic plasmid system in DO-stat fed-batch bioreactor cultivations.

We studied the expression of Bacillus amyloliquefaciens transglutaminase cloned in Escherichia coli BL21(DE3)pLysS harboring the plasmid pBAD/3C/bTGase, a bicistronic expression system, in bioreactor cultivation. Batch and fed-batch controlled as DO-stat strategies were employed for the production of the recombinant enzyme. In 30 h-batch cultivations using Terrific broth (TB), 6 g/L of biomass and 3.12 U/mgprotein of transglutaminase activity were obtained. DO-stat fed-batch cultivations under the control of oxygen concentration (DO-stat) using TB as medium but fed with glucose allowed the increment in biomass formation (17.5 g/L) and enzyme activity (6.43 U/mgprotein). DO-stat fed-batch using mineral medium (M9) and fed with glucose under the same conditions produced even higher enzymatic activity (9.14 U/mgprotein). The pH effect was investigated, and the best enzymatic activity could be observed at pH 8. In all cultivations, the bicistronic system remained stable, with 100% of plasmid-bearing cells. These results show that E. coli bearing bicistronic plasmid constructs to express recombinant TGase could be cultivated in bioreactors under DO-stat fed-batch using mineral medium and it is a promising strategy in future optimizations to produce this important enzyme.

Cloning and expression of the Bacillus amyloliquefaciens transglutaminase gene in E. coli using a bicistronic vector construction.

Transglutaminases (TGases) are a class of transferases widely used in the food and biotechnology industries. In this work, we describe the production of recombinant Bacillus amyloliquefaciens TGase in Escherichia coli, obtaining the protein in its soluble and active form. In order to reduce TGase activity inside host cells and consequently its toxicity, we constructed a bicistronic plasmid containing the B. amyloliquefaciens TGase gene fused to the inhibitory Streptomyces caniferus prodomain. To make the enzyme active and avoid the need of prodomain removal in vitro, we also cloned the 3C protease gene into the same plasmid. After a fast single-step purification protocol, we obtained a partially purified recombinant TGase with 37 mU/mg protein activity, that crosslinked bovine serum albumin (BSA). This is the first report on the expression of B. amyloliquefaciens TGase in E. coli in its mature and active form.

Biocatalytic action of proteases in ionic liquids: Improvements on their enzymatic activity, thermal stability and kinetic parameters.

This study evaluated the effect of the addition of the following ionic liquids (IL): choline chloride (CC), tetramethylammonium bromide (TB) and 1­ethyl­3­methylimidazolium bromide (EM), on some biochemical properties including enzymatic activity and different kinetic parameters of commercial proteases. The enzyme-IL combinations that showed the highest increases in enzyme activities were as follows: CC (0.5mM) and Neutrase® 0.8L; CC (5mM) and Flavourzyme® 500L; TB (2000mM) and Alcalase® 2.4L, with relative increases of 20, 15 and 150% in protease activities, respectively, compared to the control assays. The combination TB and Alcalase® 2.4L showed a reduction of 50% of the activation energy (Ea), an increase of the relation Vmax/Km of 35% and a 16-fold rise in the values of t1/2, and D. Neutrase® 0.8L combined with CC showed an increase of 20% in the relation Vmax/Km. The combination Flavourzyme® 500L and CC presented a 20% higher value of the relation Vmax/Km and a 2-fold increase in the values of t1/2 and D compared to the control assay. In summary, the most positive effects observed in this study included proteases with improved activity and stability properties and a greater affinity for the substrate.