Development of a rapid assay for ß-etherase activity using a novel chromogenic substrate.

Biocatalytic processes play a crucial role in the valorization of lignin; therefore, methods enabling the monitoring of enzymes such as ß-etherases, capable of breaking ß-O-4 aryl-ether bonds, are of significant biotechnological interest. A novel method for quantifying ß-etherase activity was developed based on the ß-ester bond formation between a chromophore and acetovainillone. The chromogenic substrate ß-(ρ-nitrophenoxy)-α-acetovanillone (PNPAV), was chemically synthesized. Kintetic monitoring of ρ-nitrophenolate release at 410 nm over 10 min, using recombinant LigF from Sphingobium sp SYK-6, LigF-AB and LigE-AB from Althererytrobacter sp B11, yielded enzimatic activities of 404. 3 mU/mg, 72 mU/mg, and 50 mU/mg, respectively. This method is applicable in a pH range of 7.0-9.0, with a sensitivity of up to 50 ng of enzyme, exhibiting no interference with lipolytic, glycolytic, proteolytic, and oxidoreductase enzymes.

Inulin from halophilic archaeon Haloarcula: Production, chemical characterization, biological, and technological properties.

Halophilic archaea are capable of producing fructans, which are fructose-based polysaccharides. However, their biochemical characterization and biological and technological properties have been scarcely studied. The aim of this study was to evaluate the production, chemical characterization, biological and technological properties of a fructan inulin-type biosynthesized by a halophilic archaeon. Fructan extraction was performed through ethanol precipitation and purification by diafiltration. The chemical structure was elucidated using Fourier Transform-Infrared Spectroscopy and Nuclear Magnetic Resonance (NMR). Haloarcula sp. M1 biosynthesizes inulin with an average molecular weight of 8.37 × 106 Da. The maximal production reached 3.9 g of inulin per liter of culture within seven days. The glass transition temperature of inulin was measured at 138.85 °C, and it exhibited an emulsifying index of 36.47 %, which is higher than that of inulin derived from chicory. Inulin from Haloarcula sp. M1 (InuH) demonstrates prebiotic capacity. This study represents the first report on the biological and technological properties of inulin derived from halophilic archaea.

Identification, Antioxidant Capacity, and Matrix Metallopeptidase 9 (MMP-9) In Silico Inhibition of Haloarchaeal Carotenoids from Natronococcus sp. and Halorubrum tebenquichense.

Natural pigments from haloarchaea are of great interest; bacterioruberin is the major pigment, it shows higher antioxidant power when compared with ß-carotene. However, characterization of bacterioruberin and its isomers along with its antioxidant and the matrix metallopeptidase 9 (MMP-9) inhibition activities in extracts from Natronoccoccus sp. TC6 and Halorubrum tebenquichense SU10 was not previously described, being the aim of this work. The carotenoids profile was performed by UV-Vis spectrophotometry, thin-layer chromatography, nuclear magnetic resonance spectroscopy, and high-resolution mass spectrometry (UPLC-ESI-MS/MS). Antioxidant capacity was determined for DPPH, ABTS, and FRAP. In addition, MMP-9 inhibition was studied using docking simulations. The carotenoid profile of studied strains was composed of bacterioruberin, some derivatives like mono, bis, and tris anhydrobacterioruberin, and also some bacterioruberin cis isomers. The carotenoid pools showed antioxidant capacity for DPPH > ABTS > FRAP; Natronococcus sp. TC6 carotenoid pool was better for ABTS and DPPH, while Halorubrum tebenquichense SU10 carotenoid pool was better for FRAP. Additionally, docking and molecular dynamics suggest that bacterioruberin inhibits MMP-9 through hydrophobic interactions near the catalytic site. Bacterioruberin shows the higher binding energy of -8.3 (kcal/mol). The carotenoids profile of both strains was elucidated, their antioxidant activity and singular participation of each carotenoid on MMP-9 in silico inhibition were evaluated.

Physical Properties and Prebiotic Activities (Lactobacillus spp.) of Gelatine-Based Gels Formulated with Agave Fructans and Agave Syrups as Sucrose and Glucose Substitutes.

This research developed model foods of gelatine-based gels, where carbohydrates from Agave tequilana Weber var. Azul (agave syrups or/and agave fructans) were incorporated into gel formulations as healthy sucrose and glucose substitutes. The sugars (sucrose and glucose) were substituted by agave carbohydrates (agave syrups and agave fructans), obtaining the subsequent gel formulations: 100% agave syrup (F2 gel), 100% agave fructan (F3 gel), and 50% agave syrup−50% agave fructan (F4 gel). The unsubstituted gel formulation was used as a control (F1 gel). The prebiotic activities, physical properties, thermal stability (HP-TLC), and texture of gelatine-based gels were evaluated. The gel formulations showed translucent appearances with approximately 36 g/100 g of water and water activities values between 0.823 and 0.929. The HP-TLC analysis validated that agave fructans did not hydrolyse during the thermal process of gels production. Gels produced with agave syrup and agave fructan (F2-F4 gels) provided higher hardness, gumminess, and springiness values (p < 0.05) than those produced with glucose and sucrose (F1 gel). Gelatine-based gel formulations displayed prebiotic activities correlated to the ability of Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus rhamnosus to use agave carbohydrates as carbon sources. Based on the prebiotic effect and physical and textural properties, the F2 and F4 gel formulations displayed the best techno-functional properties to produce gel soft candies.

Effect of Operating Conditions and Fructans Size Distribution on Tight Ultrafiltration Process for Agave Fructans Fractionation: Optimization and Modeling.

The objective of this work was to evaluate the effect of operating conditions and fructans size distribution on the tight Ultrafiltration process for agave fructans fractionation. A mathematical model of limiting mass flux transfer was used to represent the profile of concentrations over time at the outlet of a pilot scale ultrafiltration system. First, a Box-Behnken experimental design was performed for the optimization of the parameters that determine the operating conditions in their respective ranges: temperature, 30−60 °C; transmembrane pressure (TMP), 1−5 bar and feed concentration, 50−150 kg∙m−3, on the separation factor (SF) and permeate flux. Then, the validation of the model for different fructans size distribution was carried out. The results showed that for SF, the quadratic terms of temperature, TMP and feed concentration were the most significant factors. Statistical analysis revealed that the temperature-concentration interaction has a significant effect (p < 0.005) and that the optimal conditions were: 46.81 °C, 3.27 bar and 85.70 kg∙m−3. The optimized parameters were used to validate the hydrodynamic model; the adjustments conclude that the model, although simplified, is capable of correctly reproducing the experimental data of agave fructans fractionation by a tight ultrafiltration pilot unit. The fractionation process is favored at higher proportions of FOS:Fc in native agave fructans.

Optimization of Lipopeptide Biosurfactant Production by Salibacterium sp. 4CTb in Batch Stirred-Tank Bioreactors.

Halophilic microorganisms are potentially capable as platforms to produce low-cost biosurfactants. However, the robustness of bioprocesses is still a challenge and, therefore, it is essential to understand the effects of microbiological culture conditions through bioreactor engineering. Based on a design of experiments (DOE) and a response surface methodology (RSM) tailored and taken from the literature, the present work focuses on the evaluation of a composite central design (CCD) under batch cultures in stirred-tank bioreactors with the halophilic bacteria Salibacterium sp. 4CTb in order to determine the operative conditions that favor mass transfer and optimize the production of a lipopeptide. The results obtained showed profiles highlighting the most favorable culture conditions, which lead to an emulsification index (E24%) higher than 70%. Moreover, through the behavior of dissolved oxygen (DO), it was possible to experimentally evaluate the higher volumetric coefficient of mass transfer in the presence of lipopeptide (kLa = 31 1/h) as a key criterion for the synthesis of the biosurfactant on further cell expansion.

Validation of an Analytical Method for the Simultaneous Determination of Hyaluronic Acid Concentration and Molecular Weight by Size-Exclusion Chromatography.

The hyaluronic acid (HA) global market growth can be attributed to its use in medical, cosmetic, and pharmaceutical applications; thus, it is important to have validated, analytical methods to ensure confidence and security of its use (and to save time and resources). In this work, a size-exclusion chromatography method (HPLC-SEC) was validated to determine the concentration and molecular distribution of HA simultaneously. Analytical curves were developed for concentration and molecular weight in the ranges of 100-1000 mg/L and 0.011-2.200 MDa, respectively. The HPLC-SEC method showed repeatability and reproducibility greater than 98% and limits of detection and quantification of 12 and 42 mg/L, respectively, and was successfully applied to the analysis of HA from a bacterial culture, as well as cosmetic, and pharmaceutical products.

Effect of Carbon Sources in Carotenoid Production from Haloarcula sp. M1, Halolamina sp. M3 and Halorubrum sp. M5, Halophilic Archaea Isolated from Sonora Saltern, Mexico.

The isolation and molecular and chemo-taxonomic identification of seventeen halophilic archaea from the Santa Bárbara saltern, Sonora, México, were performed. Eight strains were selected based on pigmentation. Molecular identification revealed that the strains belonged to the Haloarcula, Halolamina and Halorubrum genera. Neutral lipids (quinones) were identified in all strains. Glycolipid S-DGD was found only in Halolamina sp. strain M3; polar phospholipids 2,3-O-phytanyl-sn-glycerol-1-phosphoryl-3-sn-glycerol (PG), 2,3-di-O-phytanyl-sn-glycero-1-phospho-3'-sn-glycerol-1'-methyl phosphate (PGP-Me) and sodium salt 1-(3-sn-phosphatidyl)-rac-glycerol were found in all the strains; and one unidentified glyco-phospholipid in strains M1, M3 and M4. Strains M1, M3 and M5 were selected for further studies based on carotenoid production. The effect of glucose and succinic and glutamic acid on carotenoid production was assessed. In particular, carotenoid production and growth significantly improved in the presence of glucose in strains Haloarcula sp. M1 and Halorubrum sp. M5 but not in Halolamina sp. M3. Glutamic and succinic acid had no effect on carotenoid production, and even was negative for Halorubrum sp. M5. Growth was increased by glutamic and succinic acid on Haloarcula sp. M1 but not in the other strains. This work describes for first time the presence of halophilic archaea in the Santa Bárbara saltern and highlights the differences in the effect of carbon sources on the growth and carotenoid production of haloarchaea.

Combinations of Legume Protein Hydrolysates Synergistically Inhibit Biological Markers Associated with Adipogenesis.

The objective was to investigate the anti-adipogenesis potential of selected legume protein hydrolysates (LPH) and combinations using biochemical assays and in silico predictions. Black bean, green pea, chickpea, lentil and fava bean protein isolates were hydrolyzed using alcalase (A) or pepsin/pancreatin (PP). The degree of hydrolysis ranged from 15.5% to 35.5% for A-LPH and PP-LPH, respectively. Antioxidant capacities ranged for ABTS•+ IC50 from 0.3 to 0.9 Trolox equivalents (TE) mg/mL, DPPH• IC50 from 0.7 to 13.5 TE mg/mL and nitric oxide (NO) inhibition IC50 from 0.3 to 1.3 mg/mL. LPH from PP-green pea, A-green pea and A-black bean inhibited pancreatic lipase (PL) (IC50 = 0.9 mg/mL, 2.2 mg/mL and 1.2 mg/mL, respectively) (p < 0.05). For HMG-CoA reductase (HMGR) inhibition, the LPH from A-chickpea (0.15 mg/mL), PP-lentil (1.2 mg/mL), A-green pea (1.4 mg/mL) and PP-green pea (1.5 mg/mL) were potent inhibitors. Combinations of PP-green pea + A-black bean (IC50 = 0.4 mg/mL), A-green pea + PP-green pea (IC50 = 0.9 mg/mL) and A-black bean + A-green pea (IC50 = 0.6 mg/mL) presented synergistic effects to inhibit PL. A-chickpea + PP-lentil (IC50 = 0.8 mg/mL) and PP-lentil + A-green pea (IC50 = 1.3 mg/mL) interacted additively to inhibit HMGR and synergistically in the combination of A-chickpea + PP-black bean (IC50 = 1.3 mg/mL) to block HMGR. Peptides FEDGLV and PYGVPVGVR inhibited PL and HMGR in silico, showing predicted binding energy interactions of -7.6 and -8.8 kcal/mol, respectively. Combinations of LPH from different legume protein sources could increase synergistically their anti-adipogenic potential.

Performance Evaluation of Tight Ultrafiltration Membrane Systems at Pilot Scale for Agave Fructans Fractionation and Purification.

Ceramic and polymeric membrane systems were compared at the pilot scale for separating agave fructans into different molecular weight fractions that help to diversify them into more specific industrial applications. The effect of the transmembrane pressure of ultrafiltration performance was evaluated through hydraulic permeability, permeate flux and rejection coefficients, using the same operating conditions such as temperature, feed concentration and the molecular weight cut-off (MWCO) of membranes. The fouling phenomenon and the global yield of the process were evaluated in concentration mode. A size distribution analysis of agave fructans is presented and grouped by molecular weight in different fractions. Great differences were found between both systems, since rejection coefficients of 68.6% and 100% for fructans with degrees of polymerization (DP) > 10, 36.3% and 99.3% for fructooligosaccharides (FOS) and 21.4% and 34.2% for mono-disaccharides were obtained for ceramic and polymeric membrane systems, respectively. Thus, ceramic membranes are better for use in the fractionation process since they reached a purity of 42.2% of FOS with a yield of 40.1% in the permeate and 78.23% for fructans with DP > 10 and a yield of 70% in the retentate. Polymeric membranes make for an efficient fructan purification process, eliminating only mono-disaccharides, and reaching a 97.7% purity (considering both fructan fractions) with a yield of 64.3% in the retentate.

Carrier-bound and carrier-free immobilization of type A feruloyl esterase from Aspergillus niger: Searching for an operationally stable heterogeneous biocatalyst for the synthesis of butyl hydroxycinnamates.

Feruloyl esterases synthesize butyl hydroxycinnamates, molecules possessing interesting biological properties, nonetheless, they exhibit a low stability under synthesis conditions in organic solvents, restricting its use. To enhance its operational stability in synthesis, we immobilized type A feruloyl esterase from Aspergillus niger (AnFAEA) using several carrier-bound and carrier-free strategies. The most active biocatalysts were: 1) AnFAEA immobilized on epoxy-activated carriers (protein load of 0.6 mgenzyme x mg-1carrier) that recovered 91 % of the initial hydrolytic activity, and 2) AnFAEA aggregated and cross-linked in the presence of 5 mg of BSA and 15 mM of glutaraldehyde (AnFAEA-amino-CLEAs), which exhibited 385 % of its initial hydrolytic activity; both using 4-nitrophenyl butyrate as substrate. The AnFAEA-amino-CLEAs were 12.7 times more thermostable at 60 °C than the AnFAEA immobilized on epoxy-activated carrier, thus AnFAEA-amino-CLEAs were selected for further characterization. Interestingly, during methyl sinapate hydrolysis (pH 7.2 and 30 °C), AnFAEA-amino-CLEAs KM was 15 % higher, while during butyl sinapate synthesis the KM was reduced in 63 %, both compared with the soluble enzyme. The direct esterification of butyl sinapate at solvent free conditions using sinapic acid 50 mM, reached 95 % conversion after 24 h employing AnFAEA-amino-CLEAs, which could be used for 10 cycles without significant activity losses, demonstrating their outstanding operational stability.

Comparative data of molecular weight distribution of agave fructans fractions using MALDI-ToF and HPLC-SEC.

A comparative data of two generally accepted mass techniques for estimate the molecular weight distribution of polysaccharides are presented. The data were obtained from agave fructans samples of different molecular weight, which were analyzed authored independently. The two analytical techniques used were Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-ToF) and High Pressure Size Exclusion Chromatography (HP-SEC). The data set here are related to the research paper entitled "Size-exclusion chromatography (HPLC-SEC) technique optimization by simplex method to estimate molecular weight distribution of agave fructans" by Moreno-Vilet et al. [1]. This article present the comparative figures as histograms obtained from mathematically processed MALDI-ToF spectra and HPLC-SEC chromatograms. And also, the calculated polymer parameters as number and mass molecular weight, number and mass average degree of polymerization and dispersity index.

Isolation of halophilic bacteria associated with saline and alkaline-sodic soils by culture dependent approach.

Cultivable halophilic microorganisms were isolated and identified from saline and alkaline-sodic soils: Cuatro Cienegas, Sayula and San Marcos lakes. Physicochemical characteristics of soils were determined to understand the relationship between those and the microorganisms isolated. The Cuatro Cienegas soils had a neutral pH, EC of 2.3-8 dS cm-1, classified as moderately saline. Whereas, the soils from Sayula and San Marcos lakes, had an alkaline pH, EC 15 to 65 dS m-1, typical of saline-sodic. We identified 23 cultivable halophilic bacteria using 16s rDNA, being Halobacillus sp., Marinococcus sp., and Alkalibacillus sp. the predominant genus by culture dependent approach. We found a correlation between the soils anion and cation content with the occurrence of different genus of halophilic bacteria in each studied site. Alkalibacillus sp. was predominant in Sayula and San Marcos lakes and was related to the high Na+ content; while Bacillus sp. and Halobacillus sp. were predominant in Cuatro Cienegas, their occurrence was related to a high content of Ca2+, Mg2+, and SO4 2-.

Carbohydrate Esterases: An Overview.

Carbohydrate esterases are a group of enzymes which release acyl or alkyl groups attached by ester linkage to carbohydrates. The CAZy database, which classifies enzymes that assemble, modify, and break down carbohydrates and glycoconjugates, classifies all carbohydrate esterases into 16 families. This chapter is an overview of the research for nearly 50 years around the main groups of carbohydrate esterases dealing with the degradation of polysaccharides, their main biochemical and molecular traits, as well as its application for the synthesis of high added value esters.

Size-exclusion chromatography (HPLC-SEC) technique optimization by simplex method to estimate molecular weight distribution of agave fructans.

Agave fructans are increasingly important in food industry and nutrition sciences as a potential ingredient of functional food, thus practical analysis tools to characterize them are needed. In view of the importance of the molecular weight on the functional properties of agave fructans, this study has the purpose to optimize a method to determine their molecular weight distribution by HPLC-SEC for industrial application. The optimization was carried out using a simplex method. The optimum conditions obtained were at column temperature of 61.7°C using tri-distilled water without salt, adjusted pH of 5.4 and a flow rate of 0.36mL/min. The exclusion range is from 1 to 49 of polymerization degree (180-7966Da). This proposed method represents an accurate and fast alternative to standard methods involving multiple-detection or hydrolysis of fructans. The industrial applications of this technique might be for quality control, study of fractionation processes and determination of purity.

Correction: Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation.

[This corrects the article DOI: 10.7717/peerj.3524.].

Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation.

Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging to Streptomyces (73%) and Micromonospora (10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity. Then, a colorimetric HTS assay for general PLA activity detection (cHTS-PLA) using enriched PC (≈60%) as substrate and cresol red as indicator was developed and applied; this cHTS-PLA assay was validated with known PLAs. For the first time, actinomycete strains were cultivated by solid-state fermentation (SSF) using PC as inductor and sugar-cane bagasse as support to produce high PLA activity (from 207 to 2,591 mU/g of support). Phospholipase activity of the enzymatic extracts from SSF was determined using the implemented cHTS-PLA assay and the PC hydrolysis products obtained, were analyzed by TLC showing the presence of lyso-PC. Three actinomycete strains of the Streptomyces genus that stood out for high accumulation of lyso-PC, were selected and analyzed with the specific substrate 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine (EEPC) in order to confirm the presence of PLA activity in their enzymatic extracts. Overall, the results obtained pave the way toward the HTS of PLA activity in crude microbial enzymatic extracts at a larger scale. The cHTS-PLA assay developed here can be also proposed as a routine assay for PLA activity determination during enzyme purification,directed evolution or mutagenesis approaches. In addition, the production of PLA activity by actinomycetes using SSF allow find and produce novel PLAs with potential applications in biotechnology.

Estimation of bacterioruberin by Raman spectroscopy during the growth of halophilic archaeon Haloarcula marismortui.

Halophilic archaea are interesting microorganisms that produce low biomass and metabolites, complicating their quantification. Raman spectroscopy (RS) is a powerful technique, which requires small samples, attractive for using in archaeal research. The objective of this work was the estimation of bacterioruberin content along with Haloarcula marismortui growth and their correlation with biomass concentration. RS was used to detect characteristic bands of bacterioruberin (vibrational modes C═CH, C─C, and C═C) in H. marismortui culture samples. The intensity of Raman spectra in bacterioruberin and the biomass concentration were adequately correlated. The highest production of bacterioruberin occurred at 60 h. RS is revealed as a reliable technique for the estimation of bacterioruberin in the biomass of H. marismortui, which could be considered as a promising qualitative and quantitative technique to assay metabolites in cell cultures.

Effects of Agave tequilana fructans with different degree of polymerization profiles on the body weight, blood lipids and count of fecal Lactobacilli/Bifidobacteria in obese mice.

Fructans are dietary fibers with beneficial effects on the gastrointestinal physiology and offer a promising approach for the treatment of some metabolic disorders associated with obesity. In vitro and in vivo studies were developed to test the safety of fructans obtained from Agave tequilana Weber var. azul. Additionally, an in vivo experiment using a diet-induced obesity model was performed to compare the effect of agave fructans with different degree of polymerization (DP) profiles: agave fructans with DP > 10 (LcF), agave FOS with DP < 10 (ScF), and agave fructans with and without demineralization (dTF, TF) versus commercial chicory fructans (OraftiSynergy1™) on the body weight change, fat, total cholesterol, triglycerides and count of fecal Lactobacillus spp. and Bifidobacterium spp. Results showed that A. tequilana fructans were not mutagenic and were safe even at a dose of 5 g per kg b.w. Obese mice that received ScF showed a significant decrease in body weight gain, fat tissue and total cholesterol without increasing the count of fecal Bifidobacteria. Whereas, obese mice that received LcF and TF showed decreased triglycerides and an increased count of fecal Bifidobacteria. Interestingly, although obese mice that received dTF did not show changes in body weight gain, fat tissue, total cholesterol or triglycerides, they showed an increase in the count of Bifidobacteria. These results demonstrate that both the degree of polymerization and the demineralization process can influence the biological activity of agave fructans.