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This article discusses the effect of selenium in aqueous solutions on aspects of lipid and amino acid metabolism in the cell biomass of Saccharomyces cerevisiae MYA-2200 and Candida utilis ATCC 9950 yeasts. The yeast biomass was obtained by using waste products (potato wastewater and glycerol). Selenium, at a dose of 20 mg/L of aqueous solution, affected the differentiation of cellular morphology. Yeast enriched with selenium was characterized by a large functional diversity in terms of protein and amino acid content. The protein content in the biomass of S. cerevisiae enriched with selenium (42.6%) decreased slightly as compared to that in the control sample without additional selenium supplementation (48.4%). Moreover, yeasts of both strains enriched with selenium contained a large amount of glutamic acid, aspartic acid, lysine, and leucine. Analysis of fatty acid profiles in S. cerevisiae yeast supplemented with selenium showed an increase in the unsaturated fatty acid content (e.g., C18:1). The presence of margaric acid (C17:0) and hexadecanoic acid (C17:1) was found in the C. utilis biomass enriched with selenium, in contrast to that of S. cerevisiae. These results indicate that selenium may induce lipid peroxidation, which consequently affects the loss of integrity of the cytoplasmic membrane. Yeast enriched with selenium with optimal amino acid and lipid composition can be used to prepare a novel formula of dietary supplements, which can be applied directly to various diets for both humans and animals.

A novel strain, SK26.001, which can produce mannitol from a high concentration of glucose without the addition of fructose, was isolated from sugarcane juice. This strain was identified as Candida parapsilosis based on 18S ribosomal RNA (rRNA) sequence analysis and the morphological and physiological-biochemical characteristics of the strain. Under optimized fermentation conditions, the mannitol concentration in shake flasks reached 68.5 g/L. When batch fermentation was performed, the fed glucose was completely consumed after 72 h, resulting in a final mannitol concentration of 80.3 g/L. Fed-batch fermentation was then performed with glucose feed. During the fed-batch process, ammonia water was added to maintain the pH at 4.0. The mannitol concentration in the fermenter reached 97.1 g/L after 120 h, with a total glucose consumption of 284 g/L.

Widespread interest in the use of room-temperature ionic liquids (RTILs) as solvents in anhydrous biocatalytic reactions has largely been met with underwhelming results. Enzymes are frequently inactivated in RTILs as a result of the influence of solvent on the enzyme's microenvironment, be it through interacting with the enzyme or enzyme-bound water molecules. The purpose of this chapter is to present a rational approach to mediate RTIL-enzyme interactions, which is essential if we are to realize the advantages of RTILs over conventional solvents for biocatalysis in full. The underlying premise for this approach is the stabilization of enzyme structure via multipoint covalent immobilization within a polyurethane foam matrix. Additionally, the approach entails the use of salt hydrates to control the level of hydration of the immobilized enzyme, which is critical to the activation of enzymes in nonaqueous media. Although lipase is used as a model enzyme, this approach may be effective in activating and stabilizing virtually any enzyme in RTILs.

A selection of best combination of adequate immobilization support and efficient immobilization method is still a key requirement for successful application of immobilized enzymes on an industrial level. Eupergit ® supports exhibit good mechanical and chemical properties and allow establishment of satisfactory hydrodynamic regime in enzyme reactors. This is advantageous for their wide application in enzyme immobilization after finding the most favorable immobilization method. Methods for enzyme immobilization that have been previously reported as efficient considering the obtained activity of immobilized enzyme are presented: direct binding to polymers via their epoxy groups, binding to polymers via a spacer made from ethylene diamine/glutaraldehyde, and coupling the periodate-oxidized sugar moieties of the enzymes to the polymer beads. The modification of the conventionally immobilized enzyme with ethylenediamine via the carbodiimide route seems to be a powerful tool to improve its stability and catalytic activity.

Lipases form Candida rugosa and Candida antarctica were tested for their application in the enzymatic kinetic resolution of (R,S)-flurbiprofen by enantioselective esterification. Successful chromatographic separation with well-resolved peaks of (R)- and (S)-flurbiprofen and their esters was achieved in one run on chiral stationary phases by high-performance liquid chromatography (HPLC). In this study screening of enzymes was performed, and Novozym 435 was selected as an optimal catalyst for obtaining products with high enantiopurity. Additionally, the influence of organic solvents (dichloromethane, dichloroethane, dichloropropane, and methyl tert-butyl ether), primary alcohols (methanol, ethanol, n-propanol, and n-butanol), reaction time, and temperature on the enantiomeric ratio and conversion was tested. The high values of enantiomeric ratio (E in the range of 51.3-90.5) of the esterification of (R,S)-flurbiprofen were obtained for all tested alcohols using Novozym 435, which have a great significance in the field of biotechnological synthesis of drugs. The optimal temperature range for the performed reactions was from 37 to 45 °C. As a result of the optimization, (R)-flurbiprofen methyl ester was obtained with a high optical purity, eep = 96.3 %, after 96 h of incubation. The enantiomeric ratio of the reaction was E = 90.5 and conversion was C = 35.7 %.

An inexpensive, facile, and environmentally benign method was developed to improve the activity and stability of Candida rugosa lipase (triacylglycerol acylhydrolase) immobilized on microemulsion-based organogels (CRL MBGs) via the addition of additives during immobilization. The additives used were polyethylene glycol (PEG) or polysaccharides. This study is the first report on the effect of additives in CRL MBGs. Among the tested additives, PEG produced the most improvement in the immobilized CRL, enhancing its stability in organic solvents (specifically polar solvents). The results of circular dichroism and fluorescence spectra experiments indicated that exposure of the acidic CRL to electronegative additives in the buffer, such as polyethylenimine and the electropositive surfactant cetyltrimethylammonium bromide, may change the lipase secondary structure, ultimately causing enzyme inactivation. However, sodium bis(2-ethylhexyl)sulfosuccinate and PEG 2000 had minimal effects on the secondary structure of CRL. The CRL MBGs containing PEG 2000 demonstrated remarkable retention of their catalytic activity during the recycling test. No significant changes in enzymatic activity were observed, even after nine runs, and 90% of the original yield was maintained after 15 cycles.

The main purpose of this study was to prepare selenium/glutathione-enriched Candida utilis and investigate its effect on growth performance, antioxidant capacity, and immune response in rats. The preparation of the selenium/glutathione-enriched yeast was conducted using fed-batch culture for high cell density. The optimal culture conditions for increased intracellular organic selenium and glutathione contents were as follows: the concentrated medium was fed beginning at 12 h using a polynomial feeding strategy until a total glucose concentration of 150 g/l was reached, and sodium selenite was continuously added together with glucose to a total concentration of 60 mg/l. As a result, 81 % of sodium selenite was assimilated and transformed into organic selenium by C. utilis under optimal conditions, which in turn resulted in greater glutathione accumulation and lower malondialdehyde cellular content in the yeast. To investigate and compare the effects of the prepared selenized C. utilis and other dietary supplements, 40 female rats were divided into five groups of eight rats each, following a randomized block design. Experimental feeding was conducted for a period of 6 weeks. Selenium supplementation with inorganic selenium (sodium selenite) and organic selenium (selenized C. utilis) showed better results than the control and other groups supplemented with yeast with or without glutathione. The body mass of rats, selenium deposition, and oxidative enzymes activities in both serum and liver samples, and immunity responses were all significantly improved by selenium supplementation, and between the two sources, organic selenium was more effective than inorganic selenium.

The application of lipases as catalysts in the synthesis of an intermediate of alfuzosin and lapyrium chloride is described. In the first case, the one-pot procedure to obtain the intermediate involves the treatment of tetrahydrofuroic acid with ethanol in the presence of Candida antarctica lipase followed by the addition of N-methyl-1,3-diaminopropane. In the second part of the chapter, an efficient route for large-scale preparation of lapyrium chloride is developed from chloroacetic acid in four steps, three of them enzymatic. Due to the chemoselective behavior of the lipases, both products described in the present chapter were obtained in a high degree of purity and yield, applying mild reaction conditions, and following a low environmental impact methodology.

The chemical acylation of natural antioxidants may improve their oxidative and thermal stability, as well as modify their hydrophile-lipophile balance (HLB). These processes are generally carried out under harsh conditions using strongly corrosive acids. In contrast, lipase-catalyzed acylation is characterized by mild reaction conditions, low energy requirements, and a minimization of side reactions. We report the one-step enzymatic acylation of a phenolic antioxidant (α-tocopherol) and a polyphenol (resveratrol) by lipase-catalyzed transesterification. In particular, the regioselectivity of resveratrol acylation can be controlled by an adequate selection of the biocatalyst.

In this article, we describe the application of lipases in acylation and alcoholysis reactions on steroids and nucleosides. In the field of steroids, a variety of acetyl and fatty acid derivatives of androstanes, pregnanes, and cholestanes have been prepared through lipase-catalyzed acylation and alcoholysis reactions taking advantage of the high regio- and stereoselectivity of these enzymes. The substrates as well as the products show a high degree of biological activity as neurosteroids, hormones, and glucocorticoids. The regioselective preparation of diacylated nucleosides by means of an enzymatic alcoholysis allowed the synthesis of nucleosides prodrugs or modified nucleosides. The quantitative full deacylation and dealkoxycarbonylation of nucleosides and steroids is a mild synthetic method for the deprotection of these labile compounds. Some of the reported steroid and nucleoside products are novel, and it is not possible to obtain them satisfactorily by following traditional synthetic procedures. The advantages presented by this methodology, such as selectivity, mild reaction conditions, and low environmental impact, make the lipases an important tool in the application of the principles of Green Chemistry, offering a convenient way to prepare derivatives of natural compounds with a great potential in the pharmaceutical industry.

The production of heterologous lipases is one of the most promising strategies to increase the productivity of the bioprocesses and to reduce costs, with the final objective that more industrial lipase applications could be implemented. In this chapter, an overview of the most common microbial expression systems for the overproduction of microbial lipases is presented. Prokaryotic system as Escherichia coli and eukaryotic systems as Saccharomyces cerevisiae and Pichia pastoris are analyzed and compared in terms of productivity, operational, and downstream processing facilities. Finally, an overview of heterologous Candida rugosa and Rhizopus oryzae lipases, two of the most common lipases used in biocatalysis, is presented. In both cases, P. pastoris has been shown as the most promising host system.

The global shortages of fossil fuels, significant increase in the price of crude oil, and increased environmental concerns have stimulated the rapid growth in biodiesel production. Biodiesel is generally produced through transesterification reaction catalyzed either chemically or enzymatically. Enzymatic transesterification draws high attention because that process shows certain advantages over the chemical catalysis of transesterification and it is "greener." This paper reviews the current status of biodiesel production with lipase-biocatalysis approach, including sources of lipases, kinetics, and reaction mechanism of biodiesel production using lipases, and lipase immobilization techniques. Factors affecting biodiesel production and economic feasibility of biodiesel production using lipases are also covered.

Interactions of lipases with hydrophobic substrate-water interfaces are of great interest to design improved lipase variants and engineer reaction conditions. This chapter describes the necessary steps to carry out molecular dynamics simulations of Candida antarctica lipase B at tributyrin-water interface using the GROMACS simulation software. Special attention is drawn to the preparation of the protein and the substrate-water interface and to the analysis of the obtained trajectory.

This study investigated the optimization of the enzymatic processing conditions for polylactic acid (PLA) fibers using enzymes consisting of lipases originating from different sources. The hydrolytic activity was evaluated taking into consideration the pH, temperature, enzyme concentration, and treatment time. The structural change of the PLA fibers was measured in the optimal treatment conditions. PLA fiber hydrolysis by lipases was maximized for lipase from Aspergillus niger at 40 °C for 60 min at pH 7.5 with 60% (owf) concentration, for lipase from Candida cylindracea at 40 °C for 120 min at pH 8.0 with 70% (owf) concentration, and for lipase from Candida rugosa at 45 °C for 120 min at pH 8.0 with 70% (owf) concentration. There was a change in protein absorbance of the treatment solution before and after all lipase treatments. The analyses of the chemical structure change and structural properties of the PLA due to lipase treatment was confirmed by tensile strength, differential scanning calorimetry, wide-angle X-ray scattering diffractometry, Fourier transform infrared spectroscopy, and scanning electron microscopy.

Dissolved air flotation (DAF) is a well-established separation process employing micro bubbles as a carrier phase. The application of this technique in the treatment of acid mine drainage, using three yeast biosurfactants as alternative collectors, is hereby analyzed. Batch studies were carried out in a 50-cm high acrylic column with an external diameter of 2.5 cm. High percentages (above 94%) of heavy metals Fe(III) and Mn(II) were removed by the biosurfactants isolated from Candida lipolytica and Candida sphaerica and the values were found to be similar to those obtained with the use of the synthetic sodium oleate surfactant. The DAF operation with both surfactant and biosurfactants, achieved acceptable turbidity values, in accordance with Brazilian standard limits. The best ones were obtained by the biosurfactant from C. lipolytica, which reached 4.8 NTU. The results obtained with a laboratory synthetic effluent were also satisfactory. The biosurfactants removed almost the same percentages of iron, while the removal percentages of manganese were slightly higher compared with those obtained in the acid mine drainage effluent. They showed that the use of low-cost biosurfactants as collectors in the DAF process is a promising technology for the mining industries.

In this study, the immobilized lipase was prepared by fabric membrane adsorption in fermentation broth. The lipase immobilization method in fermentation broth was optimized on broth activity units and pH adjustments. The viscose fermentation broth can be used with a certain percentage of dilution based on the original broth activity units. The fermentation broth can be processed directly without pH adjustment. In addition, the oleic acid ethyl ester production in solvent-free system catalyzed by the immobilized lipase was optimized. The molar ratio of ethanol to oil acid, the enzyme amount, the molecular amount, and the temperature were 1:1, 12% (w/w), 9% (w/w)(based the total amount of reaction mixture), and 30 °C, respectively. Finally, the optimal condition afforded at least 19 reuse numbers with esterification rate above 80% under stepwise addition of ethanol. Due to simple lipase immobilization preparation, acceptable esterification result during long-time batch reactions and lower cost; the whole process was suitable for industrial ethyl oleate production.

Development of liquid biofuels has entered a new phase of large scale pilot demonstration. A number of plants that are in operation or under construction face the task of addressing the engineering challenges of creating a viable plant design, scaling up and optimizing various unit operations. It is well-known that separation technologies account for 50-70% of both capital and operating cost. Additionally, reduction of environmental impact creates technological challenges that increase project cost without adding to the bottom line. Different technologies vary in terms of selection of unit operations; however, solid-liquid separations are likely to be a major contributor to the overall project cost. Despite the differences in pretreatment approaches, similar challenges arise for solid-liquid separation unit operations. A typical process for ethanol production from biomass includes several solid-liquid separation steps, depending on which particular stream is targeted for downstream processing. The nature of biomass-derived materials makes it either difficult or uneconomical to accomplish complete separation in a single step. Therefore, setting realistic efficiency targets for solid-liquid separations is an important task that influences overall process recovery and economics. Experimental data will be presented showing typical characteristics for pretreated cane bagasse at various stages of processing into cellulosic ethanol. Results of generic material balance calculations will be presented to illustrate the influence of separation target efficiencies on overall process recoveries and characteristics of waste streams.

The dilute-acid hydrolysis of sugarcane bagasse was optimized using a statistical experimental design resulting in hydrolysates containing 57.25 g/L of xylose, which were fermented with a high inoculum concentration (10 g/L of the yeast Candida guilliermondii IM/UFRJ 50088). The addition of urea reduced the time of conversion (tC) to 75 h (without nitrogen source addition tC > 127 h), and, consequently, improving the rates of xylitol bioproduction. Fermentator experiments, using the optimized conditions, resulted in enhanced conversion rates, reducing tC to 30 h. The stability of the yeast in the hydrolysate was also verified in a 480-h cultivation.