One-Pot Lipase-Catalyzed Enantioselective Synthesis of (R)-(-)-N-Benzyl-3-(benzylamino)butanamide: The Effect of Solvent Polarity on Enantioselectivity.

The use of the solvent engineering has been applied for controlling the resolution of lipase-catalyzed synthesis of ß-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 ß-aminoesters and ß-aminoacids.

Evolution of Fructans in Aguamiel (Agave Sap) During the Plant Production Lifetime.

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.

Enhancement of the alcoholytic activity of alpha-amylase AmyA from Thermotoga maritima MSB8 (DSM 3109) by site-directed mutagenesis.

AmyA, an alpha-amylase from the hyperthermophilic bacterium Thermotoga maritima, is able to hydrolyze internal alpha-1,4-glycosidic bonds in various alpha-glucans at 85 degrees 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 alpha-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.