Reactivity of a Recombinant Esterase from Thermus thermophilus HB27 in Aqueous and Organic Media.

The thermoalkalophilic membrane-associated esterase E34Tt from Thermus thermophilus HB27 was cloned and expressed in Kluyveromyces lactis (KLEST-3S esterase). The recombinant enzyme was tested as a biocatalyst in aqueous and organic media. It displayed a high thermal stability and was active in the presence of 10% (v/v) organic solvents and 1% (w/v) detergents. KLEST-3S hydrolysed triglycerides of various acyl chains, which is a rare characteristic among carboxylic ester hydrolases from extreme thermophiles, with maximum activity on tributyrin. It also displayed interfacial activation towards triacetin. KLEST-3S was also tested as a biocatalyst in organic media. The esterase provided high yields for the acetylation of alcohols. In addition, KLEST-3S catalyzed the stereoselective hydrolysis of (R,S)-ibuprofen methyl ester (87% ee). Our results indicate that KLEST-3S may be a robust and efficient biocatalyst for application in industrial bioconversions.

Optimisation of bovine ß-lactoglobulin hydrolysis using cardosins from dried flowers of Cynara cardunculus.

Bovine whey protein was hydrolysed using cardosins A and B purified from dried flowers of Cynara cardunculus by combining diafiltration, anion-exchange chromatography and ultrafiltration. The proteolysis experiments were performed using different whey protein concentrations and enzyme/substrate (E/S) ratios. Complete hydrolysis of the main whey proteins, ß-Lactoglobulin (ß-Lg) and α-lactalbumin (α-La), was achieved after 4 h, at E/S ratios of 1/150 U/mg, regardless the initial protein concentration. In previous reports, the authors suggested that cardosins could not hydrolyse ß-lactoblogulin. However, our promising results open up new possibilities to further explore the action of cardosins on whey proteins for the production of bioactive peptides.

Microencapsulation of Lactobacillus plantarum in W/O emulsions of okara oil and block-copolymers of poly(acrylic acid) and pluronic using microfluidic devices.

Okara oil is a by-product remaining from defatting okara, the solid residue generated after extracting the aqueous fraction of grounded soybeans in the elaboration of soy beverages. The goal of this work was to encapsulate the probiotic Lactobacillus plantarum CIDCA 83114 into W/O emulsions composed of a block-copolymer constituted of pluronic® and acrylic acid (PPP12) and okara oil, prepared in microfluidic devices. For comparative purposes, alginate was also included as a second dispersed phase. Lactobacillus plantarum CIDCA 83114 was suspended in PPP12 or alginate giving rise to dispersed phases with different compositions, named I, II, III and IV. Controls were prepared by suspending microorganisms in water as dispersed phase. 6-carboxyfluorescein was added as bacterial marker in all the emulsions. The presence of green dyed bacteria in the dispersed phases, inside the droplets of the emulsions and the absence of fluorescence outside them, confirmed the complete encapsulation of bacteria in the dispersed phases. After being prepared, emulsions were freeze-dried. The exposure to gastric conditions did not lead to significant differences among the emulsions containing polymers. However, in all cases bacterial counts were significantly lower than those of the control. After exposing emulsions to the simulated intestinal environment, bacterial counts in assays I, II and III (emulsions composed of only one dispersed phase or of two dispersed phases with bacteria resuspended in the PPP12 one) were significantly greater than those of the control (p < 0.05) and no detectable microorganisms were observed for assay IV (emulsions composed of two dispersed phases with bacteria resuspended in the alginate one). In particular, bacterial cultivability in emulsions corresponding to assay I (only PPP12 as dispersed phase) exposed to the intestinal environment was 8.22 ± 0.02 log CFU/mL (2 log CFU higher than the values obtained after gastric digestion). These results support the role of PPP12 as an adequate co-polymer to protect probiotics from the gastric environment, enabling their release in the gut, with great potential for food or nutraceutical applications.

One-step chromatographic method to purify α-lactalbumin from whey for nanotube synthesis purposes.

A one-step anion-exchange chromatography method (NaCl gradient elution on a DEAE Sepharose™ Fast Flow gel column) was developed to purify α-lactalbumin (α-LA) from whey protein isolate. α-LA nearly 100% pure (based on the total protein content) was obtained with a yield of about 39%. Besides pure α-LA, which was the main objective of this work, highly pure ß-lactoglobulin was also obtained with a yield of about 59%. The high purity of the obtained α-LA samples allowed its use to synthesise protein nanotubes with excellent gelation properties for their use as food thickeners and bioactive carriers. The samples' purity degree obtained (based on the total protein content) was critical in the formation of proper nanotubes instead of random aggregates, which produced opaque and weak gels, less useful for food applications.

Contribution of the Oligomeric State to the Thermostability of Isoenzyme 3 from Candida rugosa.

Thermophilic proteins have evolved different strategies to maintain structure and function at high temperatures; they have large, hydrophobic cores, and feature increased electrostatic interactions, with disulfide bonds, salt-bridging, and surface charges. Oligomerization is also recognized as a mechanism for protein stabilization to confer a thermophilic adaptation. Mesophilic proteins are less thermostable than their thermophilic homologs, but oligomerization plays an important role in biological processes on a wide variety of mesophilic enzymes, including thermostabilization. The mesophilic yeast Candida rugosa contains a complex family of highly related lipase isoenzymes. Lip3 has been purified and characterized in two oligomeric states, monomer (mLip3) and dimer (dLip3), and crystallized in a dimeric conformation, providing a perfect model for studying the effects of homodimerization on mesophilic enzymes. We studied kinetics and stability at different pHs and temperatures, using the response surface methodology to compare both forms. At the kinetic level, homodimerization expanded Lip3 specificity (serving as a better catalyst on soluble substrates). Indeed, dimerization increased its thermostability by more than 15 °C (maximum temperature for dLip3 was out of the experimental range; >50 °C), and increased the pH stability by nearly one pH unit, demonstrating that oligomerization is a viable strategy for the stabilization of mesophilic enzymes.

Lipases and esterases from extremophiles: overview and case example of the production and purification of an esterase from Thermus thermophilus HB27.

Extremophiles are organisms that have evolved to exist in a variety of extreme environments. They fall into a number of different classes that include thermophiles, halophiles, acidophiles, alkalophiles, psychrophiles, and barophiles (piezophiles). Extremophiles have the potential to produce uniquely valuable biocatalysts that function under conditions in which usually the enzymes of their nonextremophilic counterparts could not. Among novel enzymes isolated from extremophilic microorganisms, hydrolases, and particularly lipases and esterases are experiencing a growing demand. Lipases (EC 3.1.1.3) and esterases (EC 3.1.1.1) catalyze the cleavage of ester bounds in aqueous media and the reverse reaction in organic solvents. Both lipolytic enzymes have relevant applications in food, dairy, detergent, biofuel, and pharmaceutical industries. Here, we summarize the properties of lipases and esterases from the main extremophile groups: thermophiles and hyperthermophiles, psychrophiles, halophiles, alkalophiles/acidophiles, and solvent-resistant microorganisms.We report the biomass and lipolytic activity production by Thermus thermophilus HB27 in 5-L stirred-tank bioreactor at 70°C. Suitability of thermal spring water for culture media formulation is shown. In addition, a protocol to isolate and purify a cell-bound esterase from this microorganism is described.