Effectiveness of proteolytic enzymes to remove gluten residues and feasibility of incorporating them into cleaning products for industrial purposes.

The development of protocols for efficient gluten elimination is one of the most critical aspects of any allergen management strategy in the industry. The suitability of different proteolytic enzymes to be included in a cleaning formulation that allows the effective elimination of gluten residues was studied. Alcalase (ALC), neutrase (NEUT) and flavourzyme (FLAV) were selected from in silico analysis. The presence of 1% (v/v) of linear alkylbenzene sulphonate (LAS), a common anionic detergent, improved the gluten solubility, which may favour its elimination. Chromatographic analysis showed that the three enzymes studied were able to hydrolyse gluten in the presence of LAS. The highest percentage of short peptides (< 5 kDa) was achieved with ALC, what increases the probability of reducing the gluten antigenicity. Besides, in the presence of ALC and detergent LAS have detected the lowest levels of gluten with ELISA kits. So, effective amounts of ALC and LAS were added to a cleaning formulation, where its proteolytic activity was maintained above 90% after 37 days at 4 °C and 25 °C (under dark). Preliminary validation of the effectiveness enzymatic cleaning formulation to hydrolyse gluten was performed in a ready-to-eat/frozen food company, in which previous episodes of cross-contamination with gluten have been detected. The gluten content decreased to values below 0.125 µg/100 cm2 when the cleaning formulation was tested on different surfaces with different cleaning protocols, demonstrating the high suitability of the enzymatic cleaning formulation developed.

Influence of pH on viscoelastic properties of heat-induced gels obtained with a ß-Lactoglobulin fraction isolated from bovine milk whey hydrolysates.

A ß-Lactoglobulin fraction (r-ßLg) was isolated from whey hydrolysates produced with cardosins from Cynara cardunculus. The impact of the hydrolysis process on the r-ßLg structure and the rheological properties of heat-induced gels obtained thereafter were studied at different pH values. Differences were observed between r-ßLg and commercial ß-Lg used as control. Higher values for the fluorescence emission intensity and red shifts of the emission wavelength of r-ßLg suggested changes in its tertiary structure and more solvent-exposed tryptophan residues. Circular dichroism spectra also supported these evidences indicating that hydrolysis yielded an intermediate (non-native) ß-Lg state. The thermal history of r-ßLg through the new adopted conformation improved the microstructure of the gels at acidic pH. So, a new microstructure with better rheological characteristics (higher conformational flexibility and lower rigidity) and greater water holding ability was founded for r-ßLg gel. These results were reflected in the microstructural analysis by scanning electron microscopy.

Heterologous expression of an esterase from Thermus thermophilus HB27 in Saccharomyces cerevisiae.

In this work, a system for high-level secretion by Saccharomyces cerevisiae of the Thermus thermophilus HB27 putative esterase YP_004875.1 was constructed. The recombinant protein was purified and partially characterised. Its lipolytic activity dropped abruptly when the acyl chain length of the substrate increased from 12 to 18 carbon atoms, and variation of the reaction rate as function of substrate concentration followed Michaelis-Menten kinetics. These results suggested that the enzyme was an esterase. The recombinant enzyme was N-glycosylated and both the glycosylated and non-glycosylated forms showed activity. Compared to the native enzyme, thermal stability (half-life of 4.3h at 85 degrees C) was higher, optimum temperature (40 degrees C) was lower and optimum pH (7.5-8.5) was similar. These characteristics support potential biotechnological applications of the recombinant esterase.

Production of thermostable lipolytic activity by thermus species.

A quantitative screening for intra- and extracellular lipolytic activity was performed in submerged cultures of four Thermus strains using two different media (named T or D medium). Major differences in the extracellular lipolytic activity were observed in T medium, the highest values being for Thermus thermophilus HB27 and Thermus aquaticus YT1 strains (18 and 33 U/L, respectively). Two enzymes with lipase/esterase activity were identified in the four Thermus strains by zymogram analysis, with molecular weights of 34 and 62 kDa. No kinetic typification of the enzymes as primary metabolites was possible for any of the Thermus strains, because of the lack of a good fitting of the experimental lipolytic activity production rates to the Luedecking and Piret model. However, a linear relationship was found between the absolute values of biomass and total lipase/esterase activity (sum of intracellular and extracellular). For T. thermophilus HB27, an increase in the aeration rate caused the increase in the production of biomass and, particularly, intracellular lipolytic activity but the extracellular lipolytic activity was not affected except for the series with the strongest oxygen limitation. Transmission electronic microscopy revealed that T. thermophilus HB27 formed rotund bodies surrounded by a common membrane in cultures in the early stationary phase. The results suggest the occurrence of a specific mechanism of lipase/esterase secretion that might be due to the different composition and permeability of the cell membranes and those surrounding the rotund bodies.

Structural insights into the lipase/esterase behavior in the Candida rugosa lipases family: crystal structure of the lipase 2 isoenzyme at 1.97A resolution.

The yeast Candida rugosa produces several closely related extracellular lipases that differ in their substrate specificity. Here, we report the crystal structure of the isoenzyme lipase 2 at 1.97A resolution in its closed conformation. Lipase 2 shows a 79.4% amino acid sequence identity with lipase 1 and 82.2% with lipase 3, which makes it relevant to compare these three isoenzymes. Despite this high level of sequence identity, structural comparisons reveal several amino acid changes affecting the flap (residue 69), the substrate-binding pocket (residues 127, 132 and 450) and the mouth of the hydrophobic tunnel (residues 296 and 344), which may be responsible for the different substrate specificity and catalytic properties of this group of enzymes. Also, these comparisons reveal two distinct regions in the hydrophobic tunnel: a phenylalanyl-rich region and an aliphatic-rich region. Whereas this last region is essentially identical in the three isoenzymes, the phenylalanyl content in the first one is specific for each lipase, resulting in a different environment of the catalytic triad residues, which probably tunes finely their lipase/esterase character. The greater structural similarity observed between the monomeric form of lipase 3 and lipase 2 concerning the above-mentioned key residues led us to propose a significant esterase activity for this last protein. This enzymatic activity has been confirmed with biochemical experiments using cholesteryl [1-14C]oleate as substrate. Surprisingly, lipase 2 is a more efficient esterase than lipase 3, showing a twofold specific activity against cholesteryl [1-14C]oleate in our experimental conditions. These results show that subtle amino acid changes within a highly conserved protein fold may produce protein variants endowed with new enzymatic properties.