Effect of Agave Fructan Bioconjugates on Metabolic Syndrome Parameters in a Murine Model.

Metabolic syndrome is a complex disorder that combines abdominal obesity, dyslipidemia, hypertension, and insulin resistance. Metabolic syndrome affects 25% of the world's population. Agave fructans have shown positive effects on alterations related to metabolic syndrome, so some investigations have focused on their bioconjugation with fatty acids to increase their biological activity. The objective of this work was to evaluate the effect of agave fructan bioconjugates in a rat model with metabolic syndrome. Agave fructans enzymatically bioconjugated (acylated via food-grade lipase catalysis) with propionate or laurate were administered orally for 8 weeks in rats fed a hypercaloric diet. Animals without treatment were used as the control group, as well as animals fed with a standard diet. The data indicate that the group of animals treated with laurate bioconjugates showed a significant decrease in glucose levels, systolic pressure, weight gain, and visceral adipose tissue, as well as a positive effect of pancreatic lipase inhibition. These results allow us to demonstrate the potential of agave bioconjugates, particularly laurate bioconjugates, for the prevention of diseases associated with metabolic syndrome.

Yeasts Inhabiting Extreme Environments and Their Biotechnological Applications.

Yeasts are microscopic fungi inhabiting all Earth environments, including those inhospitable for most life forms, considered extreme environments. According to their habitats, yeasts could be extremotolerant or extremophiles. Some are polyextremophiles, depending on their growth capacity, tolerance, and survival in the face of their habitat's physical and chemical constitution. The extreme yeasts are relevant for the industrial production of value-added compounds, such as biofuels, lipids, carotenoids, recombinant proteins, enzymes, among others. This review calls attention to the importance of yeasts inhabiting extreme environments, including metabolic and adaptive aspects to tolerate conditions of cold, heat, water availability, pH, salinity, osmolarity, UV radiation, and metal toxicity, which are relevant for biotechnological applications. We explore the habitats of extreme yeasts, highlighting key species, physiology, adaptations, and molecular identification. Finally, we summarize several findings related to the industrially-important extremophilic yeasts and describe current trends in biotechnological applications that will impact the bioeconomy.

Beneficial Effects of Fructooligosaccharides Esterified with Lauric Acid in a Metabolic Syndrome Model Induced by a High-Fat and High-Carbohydrate Diet in Wistar Rats.

Metabolic syndrome (MS) is a group of abnormalities in which obesity, insulin resistance (IR), oxidative stress, and dyslipidemia stand out. This pathology predisposes to the development of cardiovascular diseases and diabetes. The ingestion of linear fructooligosaccharides (FOS) such as inulin reduces conditions such as hyperinsulinemia, increased body fat, and triglyceridemia. When FOS are esterified with fatty acids, they present emulsifying and surfactant properties; however, there are no reports of their function at the biological level. The purpose of this investigation was to evaluate the effect of Agave tequilana Weber's FOS (AtW-FOS) and FOS esterified with lauric acid (FOS-LA) in MS markers in a rat model induced by a HFHC diet. Supplementation with AtW-FOS and FOS-LA decreased IR, improved glucose tolerance, reduced liver weight (19%), plasma triglycerides (24%), and blood pressure (16%) when compared with the untreated MS group. In conclusion, the ingestion of AtW-FOS and FOS-LA has beneficial effects in the prevention of MS alterations, showing a high potential for their application in functional foods.

Enzymatic synthesis of phlorizin fructosides.

Phlorizin is a low soluble dihydrochalcone with relevant pharmacological properties. In this study, enzymatic fructosylation was approached to enhance the water solubility of phlorizin, and consequently its bioavailability. Three enzymes were assayed for phlorizin fructosylation in aqueous reactions using sucrose as fructosyl donor. Levansucrase (EC 2.4.1.10) from Gluconacetobacter diazotrophicus (Gd_LsdA) was 6.5-fold more efficient than invertase (EC 3.2.1.26) from Rhodotorula mucilaginosa (Rh_Inv), while sucrose:sucrose 1-fructosyltransferase (EC 2.4.1.99) from Schedonorus arundinaceus (Sa_1-SST) failed to modify the non-sugar acceptor. Gd_LsdA synthesized series of phlorizin mono- di- and tri-fructosides with maximal conversion efficiency of 73 %. The three most abundant products were identified by ESI-MS and NMR analysis as ß-D-fructofuranosyl-(2→6)-phlorizin (P1a), phlorizin-4'-O-ß-D-fructofuranosyl-(2→6)-D-fructofuranoside (P2c) and phlorizin-4-O-monofructofuranoside (P1b), respectively. Purified P1a was 16 times (30.57 g L-1 at 25 °C) more soluble in water than natural phlorizin (1.93 g L-1 at 25 °C) and exhibited 44.56 % free radical scavenging activity. Gd_LsdA is an attractive candidate enzyme for the scaled synthesis of phlorizin fructosides in the absence of co-solvent.

Fructosylation of phenolic compounds by levansucrase from Gluconacetobacter diazotrophicus.

Fructosylation can significantly improve the solubility, stability and bioactivity of phenolic compounds, increasing their health benefits. Levansucrase from Gluconacetobacter diazotrophicus (LsdA, EC 2.4.1.10) was found to transfer the fructosyl unit of sucrose to different classes of phenolic compounds. Among the various acceptors tested, the isoflavone puerarin and the phenol coniferyl alcohol were the most efficiently fructosylated compounds, with conversion rates of 93% and 25.1%, respectively. In both cases, mono-, di-, and trifructosides were synthesized at a ratio of 37:14:1 and 32:8:1, respectively. Structural characterization of the puerarin mono-fructoside revealed that the enzyme transferred the fructosyl moiety of sucrose to the O6-position of the glucosyl unit of puerarin. The water solubility of fructosyl-ß-(2→6)-puerarin was increased 23-fold, up to 16.2 g L-1, while its antioxidant capacity was only decreased 1.25-fold compared with that of puerarin.

Evaluation of Yarrowia lipolytica Oil for Biodiesel Production: Land Use Oil Yield, Carbon, and Energy Balance.

Oils from yeasts have emerged as a suitable alternative raw material to produce biodiesel, due to their similar composition to common raw materials such as vegetable oils. Additionally, they have the advantage of not competing with human or animal feed, and, furthermore, they do not compete for arable land. In this work, a carbon and energy balance was evaluated for Yarrowia lipolytica as a model yeast, using crude glycerol from biodiesel as the only carbon source, which improves biodiesel overall yield by 6%. The process presented a positive energy balance. Feasibility of yeast oil as biodiesel substrate was also evaluated by determination of the lipid fatty acid profile and cetane number. Moreover, a comparison of oil yields, in terms of land use, between vegetable, microalgae, and yeast oils is also presented. The results showed that Y. lipolytica oil yield is considerably higher than vegetable oils (767 times) and microalgae (36 times).

Optimization of Regioselective α-Glucosylation of Hesperetin Catalyzed by Cyclodextrin Glucanotransferase.

The regioselective α-glucosylation of hesperetin was achieved by a transglycosylation reaction catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. using soluble starch as glucosyl donor. By combining mass spectrometry (ESI-TOF) and 2D-NMR analysis, the main monoglucosylated derivative was fully characterized (hesperetin 7-O-α-d-glucopyranoside). In order to increase the yield of monoglucoside, several reaction parameters were optimized: Nature and percentage of cosolvent, composition of the aqueous phase, glucosyl donor, temperature, and the concentrations of hesperetin and soluble starch. Under the optimal conditions, which included the presence of 30% of bis(2-methoxyethyl) ether as cosolvent, the maximum concentration of monoglucoside was approximately 2 mM, obtained after 24 h of reaction. To our knowledge, this is the first report of direct glucosylation of hesperetin employing free enzymes instead of whole cells.

Lipases: An Overview.

Lipases are ubiquitous enzymes, widespread in nature. They were first isolated from bacteria in the early nineteenth century, and the associated research continuously increased due to the characteristics of these enzymes. This chapter reviews the main sources, structural properties, and industrial applications of these highly studied enzymes.

Functional Expression of Plant Lipases: The Case of CpLip1 from Carica papaya.

Carica papaya latex is one of the most studied sources of plant lipases. However, the complexity of the matrix composition makes it difficult to isolate and purify the lipolytic enzymes present in Carica papaya latex. Therefore, diverse strategies have been developed to study the catalytic properties of these enzymes.Recently the first lipase from Carica papaya latex (CpLip1) has been successfully cloned and expressed in order to study their catalytic properties. In order to improve the catalytic properties and increase the potential for its use at industrial scale.In this chapter, a practical protocol to recombinant CpLip1 lipase is given.

Solid-State Fermentation as an Economic Production Method of Lipases.

Solid-state fermentation (SSF) has been largely employed during the last three decades to produce different biomolecules of industrial interest, particularly enzymes. Through the use of agroindustrial wastes as SSF substrates, an economic process of lipases production can be achieved. In this chapter we describe a comprehensive SSF method for producing an economical preparation of Rhizomucor miehei lipase, employing sugarcane bagasse and used vegetal oil as substrates. To demonstrate the usefulness of the lipase produced by this method, we utilized directly the dried fermented solid, as a heterogeneous biocatalyst for the ethanolysis of different fats and oils. Final ethyl ester conversions (>90%, 24 h) were similar with those obtained using a commercial immobilized Rhizomucor miehei lipase at our best conditions. In this work we demonstrated that SSF is an easy and economical method for the production of lipases that can be used directly as heterogeneous biocatalysts for biodiesel production, employing low-cost feedstocks.

Lipase-Catalyzed Synthesis of Fatty Acid Esters of Trisaccharides.

Carbohydrate fatty acid esters have a broad spectrum of applications in the food, cosmetic, and pharmaceutical industries. The enzyme-catalyzed acylation is significantly more selective than the chemical process and is carried out at milder conditions. Compared with mono- and disaccharides, the acylation of trisaccharides has been less studied. However, trisaccharide esters display notable bioactive properties, probably due to the higher hydrophilicity of the sugar head group. In this chapter, we describe the acylation of two trisaccharides, maltotriose and 1-kestose, catalyzed by different immobilized lipases, using vinyl esters as acyl donors. To illustrate the potential of such compounds, the antitumor activity of 6″-O-palmitoyl-maltotriose is shown.

Lipases as Biocatalyst for Biodiesel Production.

Global shortages of fossil fuels, significant rise in the price of crude oil, and increased environmental concerns have stimulated the rapid growth of biodiesel production. Biodiesel is generally produced through transesterification reaction catalyzed either chemically or enzymatically. Enzymatic transesterification is of interest since it shows advantages over the chemical process and, in addition, is considered a "green" process. This chapter reviews the current status of biodiesel production with a lipase biocatalysis approach, including sources of lipases, kinetics, lipase immobilization techniques, and lipase reaction mechanism for biodiesel production. Factors affecting biodiesel production and the economic feasibility of lipase biodiesel production are also covered.

Lipase, Phospholipase, and Esterase Biosensors (Review).

A biosensor is a device composed by a biological recognition element and a transducer that delivers selective information about a specific analyte. Technological and scientific advances in the area of biology, bioengineering, catalysts, electrochemistry, nanomaterials, microelectronics, and microfluidics have improved the design and performance of better biosensors. Enzymatic biosensors based on lipases, esterases, and phospholipases are valuable analytical apparatus which have been applied in food industry, oleochemical industry, biodegradable polymers, environmental science, and overall the medical area as diagnostic tools to detect cholesterol and triglyceride levels in blood samples. This chapter reviews recent developments and applications of lipase-, esterase-, and phospholipase-based biosensors.

A Fast and Simple Qualitative Method for Screening Oleaginous Yeasts on Agar.

Finding new oleaginous yeasts is of great interest due to their many important applications. Currently available screening procedures are time-consuming, and most of these require liquid cultures. In this work, a new, fast, economical, and simple qualitative method for screening oleaginous yeasts was developed. The fluorescent dye, Rhodamine B, was selected because its fluorescence is directly correlated to lipid content, and no additional steps or special equipment are needed. This method only requires growing the yeasts on dyed agar plates. Under visible light, it is easy to observe that nonpigmented oleaginous yeasts become colored, whereas non-oleaginous yeasts remain uncolored. The developed method is also useful for improving medium composition in specific applications. Moreover, it was also adapted to use alternative carbon sources, such as lignocellulosic materials and glycerol. The developed method was applied to screen 124 recently isolated nonpigmented yeasts on three different carbon sources, namely, glucose, glycerol, and agave bagasse hydrolysate. Five strains were selected as good lipid producers on all tested carbon sources and accumulated over 48% lipids. Furthermore, the assay was adapted to screen reddish-pigmented yeasts. Considering all the above, the developed method has a wide range of applications in the field of microbial oils.

Engineering Yarrowia lipolytica to enhance lipid production from lignocellulosic materials.

BACKGROUND: Yarrowia lipolytica is a common biotechnological chassis for the production of lipids, which are the preferred feedstock for the production of fuels and chemicals. To reduce the cost of microbial lipid production, inexpensive carbon sources must be used, such as lignocellulosic hydrolysates. Unfortunately, lignocellulosic materials often contain toxic compounds and a large amount of xylose, which cannot be used by Y. lipolytica. RESULTS: In this work, we engineered this yeast to efficiently use xylose as a carbon source for the production of lipids by overexpressing native genes. We further increased the lipid content by overexpressing heterologous genes to facilitate the conversion of xylose-derived metabolites into lipid precursors. Finally, we showed that these engineered strains were able to grow and produce lipids in a very high yield (lipid content = 67%, titer = 16.5 g/L, yield = 3.44 g/g sugars, productivity 1.85 g/L/h) on a xylose-rich agave bagasse hydrolysate in spite of toxic compounds. CONCLUSIONS: This work demonstrates the potential of metabolic engineering to reduce the costs of lipid production from inexpensive substrates as source of fuels and chemicals.

Functionalization of natural compounds by enzymatic fructosylation.

Enzymatic fructosylation of organic acceptors other than sugar opens access to the production of new molecules that do not exist in nature. These new glycoconjugates may have improved physical-chemical and bioactive properties like solubility, stability, bioavailability, and bioactivity. This review focuses on different classes of acceptors including alkyl alcohols, aromatic alcohols, alkaloids, flavonoids, and xanthonoids, which were tested for the production of fructoderivatives using enzymes from the glycoside hydrolase (GH) families 32 and 68 that use sucrose as donor substrate. The enzymatic strategies and the reaction conditions required for the achievement of these complex reactions are discussed, in particular with regard to the type of acceptors. The solubility and pharmacokinetic and antioxidant activity of some of these new ß-D-fructofuranosides in comparison is reviewed and compared with their glucoside analogs to highlight the differences between these molecules for technological applications.

High Throughput Screening: Developed Techniques for Cellulolytic and Xylanolytic Activities Assay.

High throughput screening (HTS) is a powerful tool in biotechnology. The search for new or improved enzymes with suitable biochemical properties for industrial processes, has resulted in high efforts and research activities to develop new methodologies for activity screening. In this context, important advances have been achieved for the screening of cellulases and xylanases activities from wild and recombinant microorganisms, and from sequence databases. These enzymes have a wide range of industrial applications, including food, animal feed, textile, pulp and paper industries and detergents. Cellulases and xylanases along with pectinases, represent 20% of the world enzyme market. Recently, cellulases and xylanases have been used on fermentable sugars recovered from lignocellulosic biomass for second-generation biorefineries, aimed to produce chemical and biofuel platforms. As a result, HTS methods for biomass or biomass-degrading enzymes are gaining importance. This article presents evidence of the studies carried out for HTS of cellulase and xylanase activities.

Synthesis and emulsifying properties of carbohydrate fatty acid esters produced from Agave tequilana fructans by enzymatic acylation.

Carbohydrate fatty acid esters are non-ionic surfactants with a broad spectrum of applications. These molecules are generally synthesized using short carbohydrates or linear fructans; however in this research carbohydrate fatty acid esters were produced for the first time with branched fructans from Agave tequilana. Using immobilized lipases we successfully acylated A. tequilana fructans with vinyl laurate, obtaining products with different degrees of polymerization (DP). Lipozyme 435 was the most efficient lipase to catalyze the transesterification reaction. HPLC and ESI-MS analysis proved the presence of a mixture of acylated products as a result of the chemical complexity of fructans in the A. tequilana. The ESI-MS spectra showed a molecular mass shift between 183 and 366g/mol for fructooligosaccharides with a DP lower than 6, which indicated the presence of Agave fructans that had been mono- and diacylated with lauric acid. The carbohydrate fatty acid esters (CFAE) obtained showed good emulsifying properties in W/O emulsions.

Proteases and their Inhibitors: From Basic to High Throughput Screening.

Proteases constitute one of the most important groups of industrial enzymes with a worldwide value expected to reach 2.7 billion US dollars by 2019. Proteases represent a group of enzymes that hydrolyze the peptide bonds of proteins, releasing polypeptides or free amino acids. These enzymes are used in cleaning products, production of leathers, textiles, food and dairy products, in the pharmaceutical and diagnostic industries and for water treatment. Another area of interest regarding proteases is the development of drugs that act as protease inhibitors. This review will briefly describe the general methods used in the detection of proteases and the few studies in the development of high throughput screening methods of proteases and protease inhibitors.