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.

Valorization of Opuntia monacantha (Willd.) Haw. cladodes to obtain a mucilage with hydrocolloid features: Physicochemical and functional performance.

Opuntia monacantha mucilage was investigated for its physicochemical characteristics and functional properties. The mucilage extraction yield was 12% (DW) and it consisted of 80.12% carbohydrates, 15.14% ashes, 3.55% proteins, and 1.19% lipids. Monosaccharide profiling demonstrated a predominantly presence of galactose, glucuronic acid, and arabinose. Viscosimetric measurements gave an intrinsic viscosity of 9.02 dL/g and a molar mass of 1.12 × 106 g/mol. Reconstituted mucilage solution (1% w/v) had a mean particle diameter (D4,3) of 648 nm and solubility above 85%. Its emulsifying capacity improved with the increment of mucilage solution in the emulsion; likewise, it provided high emulsion stability through different ratios of oil to polysaccharide solution. It displayed good foaming capacity, although its foam stability progressively reduced over time. In addition, its blending with ovalbumin resulted in a foaming capacity enhancement and in a markedly greater foam stability compared to ovalbumin alone. The rheological studies indicated the mucilage solutions exhibited shear-thinning behavior at concentrations between 1 and 10% and fairly stable viscous properties in the temperature range of 5-80 °C. These outcomes support that O. monacantha mucilage may find potentially useful applications in food systems, particularly as an emulsifying, foaming and thickening agent, or as a stabilizer.

Kinetics and Thermodynamics of Thermal Inactivation of β-Galactosidase from Aspergillus oryzae

ABSTRACT For optimization of biochemical processes in food and pharmaceutical industries, the evaluation of enzyme inactivation kinetic models is necessary to allow their adequate use. Kinetic studies of thermal inactivation of β-galactosidase from Aspergillus oryzae were conducted in order to critically evaluate mathematical equations presented in the literature. Statistical analysis showed that Weibull model presented the best adequacy to residual enzymatic activity data through the processing time and its kinetic parameters as a function of the temperature, in the range of 58-66 ºC. The investigation suggests the existence of a non-sensitive heat fraction on the enzyme structure, which is relatively stable up to temperatures close to 59 ºC. Thermodynamic parameters were evaluated and showed that such β-galactosidase presents activation energy of 277 kJ mol-1 and that the enzyme inactivation is due to molecular structural changes. Results shown that the enzyme is quite stable for biotechnological applications.