Lipase activity in vesicular systems: Characterization of candida cylindracea lipase and its activity in polymerizable dialkylammonium surfactant vesicles

Lipase from Candida cylindracea (CCL) was incorporated into polymerizable positively charged dialkylammonium bromide surfactant vesicles. The enzyme was incorporated by the use of the dehydration-rehydration method or by incubation. In the latter case, trapping efficiencies of up to 100% could be obtained. Activities of free and vesicle-incorporated CCL were tested for three triglycerides: triacetin, tributyrin, and tricaprylin. Enzyme activity was lowest in homogeneous mixtures (triacetin and small concentrations of tributyrin) and highest in heterogeneous mixtures (tricaprylin and high concentrations of tributyrin). Entrapment in vesicular systems is advantageous, especially in homogeneous reaction mixtures and in the case of the production of insoluble fatty acid (caproate), because inhibition by the acid can be suppressed. The influence of several surface-active additives, including vesicles, on the activity of lipase in triglyceride assays was tested. Vesicles have a positive influence on the activity, whereas other positively charged additives act as inhibitors. In the case of tricaprylin assays, the positively charged additives increase the activity. Finally, tryptic digestion for free and incorporated CCL were compared. Free CCL is readily inactivated, whereas incorporated enzyme is protected from proteolytic degradation

Imaging surface plasmon resonance for multiplex microassay sensing of mycotoxins

A prototype imaging surface plasmon resonance-based multiplex microimmunoassay for mycotoxins is described. A microarray of mycotoxin–protein conjugates was fabricated using a continuous flow microspotter device. A competitive inhibition immunoassay format was developed for the simultaneous detection of deoxynivalenol (DON) and zearalenone (ZEN), using a single sensor chip. Initial in-house validation showed limits of detection of 21 and 17 ng/mL for DON and 16 and 10 ng/mL for ZEN in extracts, which corresponds to 84 and 68 µg/kg for DON and 64 and 40 µg/kg for ZEN in maize and wheat samples, respectively. Finally, the results were critically compared with data obtained from liquid chromatography-mass spectrometry confirmatory analysis method and found to be in good agreement. The described multiplex immunoassay for the rapid screening of several mycotoxins meets European Union regulatory limits and represents a robust platform for mycotoxin analysis in food and feed sample

Studies on the use of haloperoxidases in organic synthesis

The subject of this thesis is the use of haloperoxidases in synthetic organic chemistry. Haloperoxidases are enzymes capable of halogenating a variety of organic compounds. They require hydrogen peroxide and halide ions as cosubstrates. The enzymes operate under mild conditions, compared to conventional halogenating agents, resulting in increased yields and purity of products. This thesis presents some new substrates for a chloroperoxidase from the mold Caldariomyces fumago (CPO) and a bromoperoxidase from the brown alga Ascophyllum nodosum (BPO). The reaction mechanisms of both enzymes and their natural function are discussed. The immobilization of CPO is studied.CPO catalyzes the smooth halogenation of various heterocyclic compounds. Barbituric acid and some of its derivatives give the corresponding 5-chloro or 5,5-dichloro compounds in very high yields (Ch. 2). 5-Monochlorobarbituric acid is obtained in high yield when the CPO-mediated chlorination reaction is combined with an electrochemical process. The 5,5-dichloro compound is reduced to its 5-monochloro analogue at an electrode, which is simultaneously used for the production of the cosubstrate hydrogen peroxide. The system is very efficient, as shown by the high turnover obtained for CPO (10 6-10 7) (Ch.3). Pyrazoles are also good substrates for the enzyme, giving their 4-chloro derivatives in high yields. CPO further converts 2-aminopyridine into 2-amino-3-chloropyridine and 8-hydroxyquinoline into the corresponding 5,7-dibromo compound (Ch. 4). Kinetic studies indicate that there are two possible reaction routes for CPO: I) the organic substrate binds to an oxidized enzyme intermediate and direct transfer of halogen takes place; II) the enzyme produces hypohalous acid as the active halogenating agent. The available data do not as yet permit a definitive choice of reaction mechanism. (Ch. 2).BPO smoothly brominates barbituric acid and some of its derivatives giving the corresponding 5-bromo or 5,5-dibromo compounds in good yields. Kinetic measurements show that BPO produces free hypobromous acid which either brominates the organic halogen acceptor or reacts with hydrogen peroxide giving singlet oxygen (Ch. 5).Relatively apolar substrates like monochlorodimedon and resorcinol are efficiently halogenated by CPO entrapped in reversed micelles. The system is composed of cetyltrimethyl ammonium halide (CTAX, X=Br or Cl), pentanol, octane and a small amount of aqueous buffer containing the enzyme and hydrogen peroxide. The CTAX serves a dual function: i) as a surfactant, it stabilizes the reversed micelle, and ii) as a supplier of the halide substrate. The reaction rates obtained are twice as high as in water (Ch. 6).CPO can be immobilized on various solid supports, but the low stability of the enzyme-support bond and the support itself at the optimal pH of the enzyme is a serious problem. The best way to immobilize CPO seems to be the crosslinking of a mixture of the enzyme and a water-soluble acrylamide- N -acryloxysuccinimide copolymer with an α-ω-diamine. In this way CPO is bound by means of both entrapment and covalent bonds, in 40% yield (Ch.7).Six new halometabolites have been detected by GC/MS analysis of the dichloromethane extract of the culture medium of Caldariomyces fumago. The compounds are chlorinated derivatives of ethanol, propanol-2 and 2-(4-hydroxyphenyl)ethanol. Their possible biosynthetic routes and the implications for the reaction mechanism of CPO are discussed (Ch.8).In short, the two haloperoxidases studied here are biocatalysts which are potentially useful in synthetic organic methodology, because they perform their halogenation reactions in a very smooth and mild way. However, haloperoxidases with greater specificity in their reactions would certainly be of more advantage to the organic chemist.</p

Biocatalytic production of flavors and fragrances

The preparation of flavors and fragrances has to be done with great care. When these compounds are meant for consumption, no toxic chemicals are allowed during their preparation. For use in non-food additives, a high purity is required. Both constraints can be easily fulfilled when enzymes are used as catalyst during the production of these compounds. This paper summarizes the work that we have done regarding the preparation of branchedchain fatty acids (sheep flavors) using lipases, the production of glucosides (controlled-release flavors) using glucosidases, the formation of terpene alcohols and grapefruit flavor using enzymes from chicory, and the formation of phenolic antioxidants using lipases

Marine sponges as pharmacy

Marine sponges have been considered as a gold mine during the past 50 years, with respect to the diversity of their secondary metabolites. The biological effects of new metabolites from sponges have been reported in hundreds of scientific papers, and they are reviewed here. Sponges have the potential to provide future drugs against important diseases, such as cancer, a range of viral diseases, malaria, and inflammations. Although the molecular mode of action of most metabolites is still unclear, for a substantial number of compounds the mechanisms by which they interfere with the pathogenesis of a wide range of diseases have been reported. This knowledge is one of the key factors necessary to transform bioactive compounds into medicines. Sponges produce a plethora of chemical compounds with widely varying carbon skeletons, which have been found to interfere with pathogenesis at many different points. The fact that a particular disease can be fought at different points increases the chance of developing selective drugs for specific target

Immobilised enzymes in biorenewable production

Oils, fats, carbohydrates, lignin, and amino acids are all important raw materials for the production of biorenewables. These compounds already play an important role in everyday life in the form of wood, fabrics, starch, paper and rubber. Enzymatic reactions do, in principle, allow the transformation of these raw materials into biorenewables under mild and sustainable conditions. There are a few examples of processes using immobilised enzymes that are already applied on an industrial scale, such as the production of High-Fructose Corn Syrup, but these are still rather rare. Fortunately, there is a rapid expansion in the research efforts that try to improve this, driven by a combination of economic and ecological reasons. This review focusses on those efforts, by looking at attempts to use fatty acids, carbohydrates, proteins and lignin (and their building blocks), as substrates in the synthesis of biorenewables using immobilised enzymes. Therefore, many examples (390 references) from the recent literature are discussed, in which we look both at the specific reactions as well as to the methods of immobilisation of the enzymes, as the latter are shown to be a crucial factor with respect to stability and reuse. The applications of the renewables produced in this way range from building blocks for the pharmaceutical and polymer industry, transport fuels, to additives for the food industry. A critical evaluation of the relevant factors that need to be improved for large-scale use of these examples is presented in the outlook of this review

Alcohol production from cheese whey permeate using genetically modified flocculent yeast cells

Alcoholic fermentation of cheese whey permeate was investigated using a recombinant flocculating Saccharomyces cerevisiae, expressing the LAC4 (coding for β-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus enabling for lactose metabolization. Data on yeast fermentation and growth on cheese whey permeate from a Portuguese dairy industry is presented. For cheese whey permeate having a lactose concentration of 50 gLˉ¹, total lactose consumption was observed with a conversion yield of ethanol close to the expected theoretical value. Using a continuously operating 5.5-L bioreactor, ethanol productivity near 10 g Lˉ¹ hˉ¹ (corresponding to 0.45 hˉ¹ dilution rate) was obtained, which raises new perspectives for the economic feasibility of whey alcoholic fermentation. The use of 2-times concentrated cheese whey permeate, corresponding to 100 gLˉ¹ of lactose concentration, was also considered allowing for obtaining a fermentation product with 5% (w/v) alcohol.Fundação para a Ciência e a Tecnologia (FCT) – PRAXIS XXI/BD/11306/97.Instituto de Biotecnologia e Química Fina (IBQF)

Biosynthesis of Sesquiterpene Lactones in Pyrethrum (Tanacetum cinerariifolium)

The daisy-like flowers of pyrethrum (Tanacetum cinerariifolium) are used to extract pyrethrins, a botanical insecticide with a long history of safe and effective use. Pyrethrum flowers also contain other potential defense compounds, particularly sesquiterpene lactones (STLs), which represent problematic allergenic residues in the extracts that are removed by the pyrethrum industry. The STLs are stored in glandular trichomes present on the pyrethrum achenes, and have been shown to be active against herbivores, micro-organisms and in the below-ground competition with other plants. Despite these reported bioactivities and industrial significance, the biosynthetic origin of pyrethrum sesquiterpene lactones remains unknown. In the present study, we show that germacratrien-12-oic acid is most likely the central precursor for all sesquiterpene lactones present in pyrethrum. The formation of the lactone ring depends on the regio- (C6 or C8) and stereo-selective (a or ß) hydroxylation of germacratrien-12-oic acid. Candidate genes implicated in three committed steps leading from farnesyl diphosphate to STL and other oxygenated derivatives of germacratrien-12-oic acid were retrieved from a pyrethrum trichome EST library, cloned, and characterized in yeast and in planta. The diversity and distribution of sesquiterpene lactones in different tissues and the correlation with the expression of these genes are shown and discussed