AmyA, an ␣-amylase from the hyperthermophilic bacterium Thermotoga maritima, is able to hydrolyze internal ␣-1,4-glycosidic bonds in various ␣-glucans at 85°C as the optimal temperature. Like other glycoside hydrolases, AmyA also catalyzes transglycosylation reactions, particularly when oligosaccharides are used as substrates. It was found that when methanol or butanol was used as the nucleophile instead of water, AmyA was able to catalyze alcoholysis reactions. This capability has been evaluated in the past for some ␣-amylases, with the finding that only the saccharifying fungal amylases from Aspergillus niger and from Aspergillus oryzae present measurable alcoholysis activity (R. I. Santamaria, G. Del Rio, G. Saab, M. E. Rodriguez, X. Soberon, and A. Lopez, FEBS Lett. 452:346-350, 1999). In the present work, we found that AmyA generates larger quantities of alkyl glycosides than any amylase reported so far. In order to increase the alcoholytic activity observed in AmyA, several residues were identified and mutated based on previous analogous positions in amylases, defining the polarity and geometry of the active site. Replacement of residue His222 by glutamine generated an increase in the alkyl glucoside yield as a consequence of a higher alcoholysis/hydrolysis ratio. The same change in specificity was observed for the mutants H222E and H222D, but instability of these mutants toward alcohols decreased the yield of alkyl glucoside.␣-Amylases (EC 3.2.1.1) are retaining glycosidases that catalyze the hydrolysis of internal ␣-1,4-glycosidic bonds in starch through a double-displacement mechanism in which a covalent intermediate glycosyl enzyme is deglycosylated by water (43, 62). ␣-Amylases contain 5 to 11 subsites that bind glucose moieties (8, 51), with their numbers and relative affinities defining their product profiles (38). Like all retaining glycosidases, ␣-amylases can also catalyze transfer reactions, which are the result of employing molecules other than water as glucosyl acceptors, such as carbohydrates (transglycosylation reactions) or alcohols (alcoholysis reactions). When a highmolecular-weight alcohol is used as an acceptor, the product is an alkyl glycoside with surface tension activity properties that are important in several industrial applications. Therefore, the extremely laborious and inefficient chemical synthesis of alkyl glycosides presents an opportunity to develop enzymatic methods devoted to increasing the yields and specificities of these reactions.The feasibility of alcoholysis reactions using various exoglycosidases has been extensively investigated (references 57 and 65 and references therein), and although efficient processes have been developed using activated substrates, such as pnitrophenyl-glucoside or p-nitrophenyl-galactoside, with ␣-and -glucosidases and galactosidases, the use of a readily available substrate, such as starch or amylodextrins, could prove attractive if efficient reactions employing ␣-amylases are developed.For a given degree of starch depolymerization, endoa...
Aguamiel is the sap collected from agave , while pulque is the result of the natural fermentation of aguamiel . Despite its ancestral origin and numerous publications on pulque production, little is known about the evolution and concentration of sugars and fructo-oligosaccharides in aguamiel , either during its daily accumulation or through the agave production lifetime. In this study, we examined aguamiel composition in three agave plants during their productive lifetime (4 to 9 months). After each collection, the agave pine is scraped to induce aguamiel to flow into an internally created cavity ( cajete ), producing a residual bagasse ( metzal ). We found that the concentration of agave fructans and sucrose, as well as the fructan profile, change during the aguamiel production process. During the daily collection, a small amount of agave fructans released from the pine by scraping is drawn into the cajete with the first milliliters of sap where it is then diluted with the inflow of aguamiel . The main component of aguamiel is the sucrose produced in high concentration in the leaves through photosynthesis and then hydrolyzed in the cajete as aguamiel accumulates. We also describe how the fructan profile changes during the accumulation of aguamiel in the cajete . In addition to the varying amount of sucrose that is hydrolyzed in the aguamiel accumulated, we found that fructo-oligosaccharides are either diluted, consumed, or hydrolyzed, depending on the plant and its production stage, thus yielding different fructan profiles. New fructo-oligosaccharides are, in some cases, synthesized by bacteria present in aguamiel . These profiles were also observed in aguamiel collected from ten different plants in the same production region. We also found that a considerable amount of agave fructans is lost in metzal (bagasse), the agave material that is scraped and thrown away twice a day during the production process.
The use of the solvent engineering has been applied for controlling the resolution of lipase-catalyzed synthesis of β-aminoacids via Michael addition reactions. The strategy consisted of the thermodynamic control of products at equilibrium using the lipase CalB as a catalyst. The enzymatic chemo- and enantioselective synthesis of (R)-(−)-N-benzyl-3-(benzylamino)butanamide is reported, showing the influence of the solvent on the chemoselectivity of the aza-Michael addition and the subsequent kinetic resolution of the Michael adduct; both processes are catalyzed by CalB and both are influenced by the nature of the solvent medium. This approach allowed us to propose a novel one-pot strategy for the enzymatic synthesis of enantiomerically enriched β-aminoesters and β-aminoacids.
El objetivo de este estudio es demostrar la estrecha relación que media entre dos de los manuscritos que actualmente se conservan de la traducción latina del Corán de Robert de Ketton (1142-1143), y la figura de Juan de Segobia (1390/5-1458), quien desde 1437 había estudiado esta versión del Corán hasta que él mismo, con la colaboración de un alfaquí hispano, elaboró una nueva traducción. Tras exponer las noticias que Juan de Segobia ofrece sobre los ejemplares del Corán latino que poseyó, se esboza una ordenación general de la tradición manuscrita del Alchoran de Robert de Ketton, que se divide en dos familias, α (la de los códices más antiguos) y β (la de los más recientes, derivados de un manuscrito traído en 1437 desde Constantinopla a Basilea por Johannes de Ragusio). La comparación entre la redacción que ofrece un grupo de manuscritos de la familia α (muy en particular, la del códice Paris BNF lat. 3393) con las variantes presentes en los códices de la familia β y en la propia edición de Theodor Bibliander (1543) permite concluir que el códice Paris BNF lat. 3669 es una copia de un manuscrito supervisado por Juan de Segobia, donde se ofrecía una redacción del Alchoran de Robert de Ketton revisada a partir de las variantes del códice de Johannes de Ragusio.
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