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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.

Evaluation of non-linear equations to model different animal growths with mono and bisigmoid profiles

Experimental data of different animals (e.g. cocks, pigs, cats, dogs, cattles, etc.) from recent bibliography were selected to evaluate the capability of five classical sigmoidal equations (i.e. Bertalanffy, Weibull, logistic, Gompertz, and modified Hill) to model growth. These functions were used in different reparameterized forms in order to define all growth phases and to characterize significant kinetic parameters. The results indicated that logistic and Weibull equations were the best options to simulate the data with mono-sigmoid profiles. A subsequent formulation of logistic and Gompertz equations was constructed to describe accurately the biphasic trends for cock and foal growths.

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.

Inhibition kinetics of lipid oxidation of model foods by using antioxidant extract of fermented soybeans

Fermentation by using Aspergillus oryzae has been reported to increase antioxidant activity of soybeans significantly. The effectiveness of the extract from fermented soybeans was studied in 3 model foods with different complexities, i.e., linoleic acid emulsion, sunflower oil emulsions and bulk sunflower oil. For the emulsion systems, oxidation at two different pH values (4.5 and 7) was also compared. A reparameterised logistic equation was used to describe and to predict the experimental data. In general, a good agreement between experimental trends and simulated data from the model was found. A crude antioxidant extract (5 mg/g) showed a comparable antioxidant activity to 0.26 mg/g of butylated hydroxytoluene (BHT) in the linoleic acid emulsions. The extract exhibited a better capability to retard primary products in the linoleic acid systems than the secondary products. The opposite effect was observed in the bulk sunflower oil and its emulsion systems.

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 and characterization of two N-terminal truncated esterases from Thermus thermophilus HB27 in a mesophilic yeast: effect of N-terminus in thermal activity and stability.

Two N-terminally truncated variants of the esterase E34Tt from Thermus thermophilus HB27 (YP_004875.1) were expressed in Kluyveromyces lactis. Production and biochemical properties of both recombinant proteins were investigated. The esterase activity was greatly increased compared to the wild-type strain. In particular, the extracellular production of the ΔN16 variant (KLEST-3S) was 50-fold higher than that obtained with T. thermophilus HB27. Response surface methodology was applied to describe the pH and temperature dependence of both activity and stability. When compared with the wild type esterase, the optimal temperature of reaction decreased 35 and 15 °C for ΔN16 and ΔN26, respectively. KLEST-3S showed a maximum of activity at pH 7.5 and 47.5 °C, and maximal stability at pH 8.1 and 65 °C. KLEST-5A (ΔN26) did not show an absolute maximum of activity. However, best results were obtained at 40 °C and pH 8.5. KLEST-5A showed also a lower stability. In the presence of a surfactant, both proteins showed lower stability at 85 °C (t(½)< 5 min) than the wild-type enzyme (t(½)=135 min). However, in the absence of detergent, the stability of KLEST-3S was higher (t(½)=230 min, at 85 °C) than that of the mutant KLEST-5A (12 min) or the wild type enzyme (19 min). Minor differences were observed in the substrate specificity. Our results suggest that the N-terminal segment is critical for maintaining the hyperthermophilic function and stability.

Effects of spill-treating agents on growth kinetics of marine microalgae.

The effects of four spill-treating agents (STAs) (CytoSol, Finasol(®) OSR 51, Agma OSD 569 and OD4000) on the growth kinetics of three marine microalgae (Isochrysis galbana, Chaetoceros gracilis, Phaeodactylum tricornutum) were studied. Chlorophyll a concentration and optical density at 700 nm were assessed to describe the logistic growth of algae in batch cultures. The optical density data were initially analyzed as described for standard algal growth inhibition tests and subsequently modelled by a bivariate model, as a function of time and dose, to assess the toxic effects on growth parameters. Increasing trends in EC50 and EC10 values with time were found with the standard approach. In 8 of the 11 tests, the lag phase (λ) or the time required to achieve half the maximum biomass (τ) was significantly dependent on the STA concentration. A global parameter (EC50,τ) was calculated to summarize the effects of STAs on growth parameters in the bivariate model. The ranking of sensitivity as EC50,τ values was I. galbana>C. gracilis>P. tricornutum. For all species tested, the least toxic agent was Agma OSD 569, followed by CytoSol. The mathematical model allowed successful ecotoxicological evaluation of chemicals on microalgal growth.

Modeling the angiotensin-converting enzyme inhibitory activity of peptide mixtures obtained from cheese whey hydrolysates using concentration–response curves

Three mathematical models, two logistic models (previously published in previous works) and one mechanistic, developed in this work and based on Michaelis–Menten kinetics, were compared to select the most adequate model in describing the angiotensin‐converting enzyme (ACE)‐inhibitory activity of bioactive peptide mixtures obtained from cheese whey protein. The significance of both the model and its parameters as well as the value of the regression coefficient was used as criteria to select the most adequate model for obtaining the IC50 values corresponding to each bioactive peptides mixture. The best results were obtained with the Michaelis–Menten‐based model because it provided the best fits and in addition the values for its parameters were always significant. As parameters of this model have a physical meaning, it could be used for inhibition‐testing experiments in the development of novel bioactive peptides. The results obtained indicated that the peptide mixture derived from the neutrase hydrolysis exhibited strong ACE inhibition activity. The main active peptides were short, with molecular masses below 1 kDa (IC50 = 40.37 ± 2.66 μg/mL) and represent 38% of the initial protein content in the hydrolysate.

Optimization of Antimicrobial Combined Effect of Organic Acids and Temperature on Foodborne Salmonella and Escherichia coli in Cattle Feed by Response Surface Methodology

Despite the importance of feedstuffs and feed ingredients in the food chain, few studies have analyzed the effectiveness of usual methods for the microbial decontamination of feeds. This work aimed to study the combined effect of temperature and organic acids (formic or lactic) on the inactivation of 10 isolates of Salmonella enterica and Escherichia coli obtained from vegetable feed ingredients, including cereals and other processed materials, in cattle feed. A central composite design was used with acid concentrations ranging from 0 to 0.2% (vol/wt) and temperatures from 50°C to 70°C. Second-order models were obtained to predict the survival inhibitions. The results reported in the present study indicate that temperature affects the survivability more significantly than acid concentration, whose effect was isolate dependent. However, after 2 min of treatment, optimal microbial inhibitions were generally achieved at temperatures above 65°C, using 0.1% formic acid or 0.2% lactic acid for Salmonella isolates and around 0.1% lactic acid or 0.2% formic acid for E. coli isolates, respectively. This work provides a set of guidelines helpful to reduce microbial contamination of feeds and highlights the importance of feed-ingredient monitoring to reduce pathogen contamination risk during feed processing.

Recombinant Candida rugosa lipase 2 from Pichia pastoris: Immobilization and use as biocatalyst in a stereoselective reaction

The characterization of the recombinant Candida rugosa Lip2 (r‐Lip2) isoenzyme obtained from fed‐batch cultures of Pichia pastoris under PAOX promoter was carried out, determining the optimal pH and temperature as well as their catalytic performance in both hydrolysis and synthesis reactions comparing with purified native Lip2 (n‐Lip2) previously determined. The substrate specificity of r‐Lip2 in hydrolysis reactions was determined with a series of triacylglycerols and p‐nitrophenyl esters of variable acyl chain length. r‐Lip2 showed the maximum specificity for both substrates towards medium‐chain esters (C‐8), similar behavior was observed with n‐Lip2. However, significant differences were observed towards unsaturated substrates (triolein) or short‐chain esters. A statistical design applied to study the effect of pH and temperature on lipase stability shown that r‐Lip2, like n‐Lip2, was more sensitive to pH than temperature changes. Nevertheless, the overall stability of soluble r‐Lip2 was lower than soluble n‐Lip2. The stability of r‐lip2 was significantly improved by immobilization onto EP100, an excellent support for lipases with yields around 95% for offered lipolytic activity lower than 600 AU/mL. Finally, immobilized r‐Lip2 was tested in the resolution of ibuprofen in isooctane by means of enantioselective esterification using 1‐butanol as esterifying agent. r‐Lip2 showed a better performance in terms of enantiomeric excess (74%) and enatiomeric factor (96%) than n‐Lip2 (56 and 80%, respectively) for the same conversion (40%). Thus, r‐Lip2 should be considered a good and pure biocatalyst, easy to produce and with a remaining activity of ca. 90% after one reaction cycle when immobilized on EP100.