RESUMEN
Stereospecific α-glucosylation of primary and secondary OH-group at carbohydrate acceptors is achieved using glucosyl N-phenyl-trifluoroacetimidate (PTFAI) donor protected with an electron-withdrawing 2,4,5-trifluorobenzoyl (TFB) group at O-6 and the participating levulinoyl (Lev) group at O-3. New factors have been revealed that might explain α-stereoselectivity in the case of TFB and pentafluorobenzoyl (PFB) groups at O-6. They are of conformational nature and confirmed by DFT calculations. The potential of this donor, as well as the orthogonality of TFB and Lev protecting groups, is showcased by the synthesis of α-(1 â 3)-linked pentaglucoside corresponding to Aspergillus fumigatus α-(1 â 3)-d-glucan and of its hexasaccharide derivative, bearing ß-glucosamine residue at the non-reducing end.
Asunto(s)
Aspergillus fumigatus , Oligosacáridos , Teoría Funcional de la Densidad , Electrones , GlucanosRESUMEN
The dexA gene encoding Penicillium funiculosum dextranase (GenBank accession MH581385) belonging to family 49 of glycoside hydrolases (GH49) was cloned and heterologously expressed in two recipient strains, P. canescens RN3-11-7 and P. verruculosum B1-537. Crude enzyme preparations with the recombinant dextranase content of 8-36% of the total secreted protein were obtained on the basis of new Penicillium strains. Both recombinant forms of the dextranase were isolated in a homogeneous state using chromatographic techniques. The purified enzymes displayed very similar properties, that is, pI 4.55, activity optima at pH 4.5-5.0 and 55-60⯰C and a melting temperature of 60.7-60.9⯰C. They were characterized by similar specific activities (1020-1340 U/mg) against dextrans with a mean molecular mass of 20, 70 and 500â¯kDa, as well as similar kinetic parameters in the hydrolysis of 70â¯kDa dextran (Kmâ¯=â¯1.10-1.11â¯g/L, kcatâ¯=â¯640-680 s-1). However, the recombinant dextranases expressed in P. canescens and P. verruculosum had different molecular masses according to the data of SDS-PAGE (â¼63 and â¼60â¯kDa, respectively); this was the result of different N-glycosylation patterns as MALDI-TOF mass spectrometry analysis showed. The main products of dextran hydrolysis at its initial phase were isomaltooligosaccharides, while after the prolonged time (24â¯h) the reaction system contained isomaltose and glucose as the major products and minor amounts of other oligosaccharides.
Asunto(s)
Dextranasa , Proteínas Fúngicas , Expresión Génica , Penicillium/enzimología , Dextranasa/sangre , Dextranasa/química , Dextranasa/genética , Dextranasa/aislamiento & purificación , Estabilidad de Enzimas , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Calor , Concentración de Iones de Hidrógeno , Penicillium/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The pretreatment of softwood and hardwood samples (spruce and hornbeam wood) with 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) was undertaken for further simultaneous enzymatic saccharification of renewable non-food lignocellulosic biomass and microbial fermentation of obtained sugars to ethanol and fumaric acid. A multienzyme cocktail based on cellulases and yeast or fungus cells producing ethanol and fumaric acid were the main objects of [Bmim]Cl influence studies. A complex effect of lignocellulosic biomass pretreatment with [Bmim]Cl on various aspects of the process (both action of cellulases and microbial conversion of hydrolysates to target products) was revealed. Positive effects of the pretreatment with [Bmim]Cl included decreasing the lignin content in the biomass, and increasing the effectiveness of enzymatic hydrolysis and microbial transformation of pretreated biomass. Immobilized cells of both yeasts and fungi possessed improved productive characteristics in the biotransformation of biomass pretreated with [Bmim]Cl to ethanol and fumaric acid.