ABSTRACT
Background: Aspergillus ochraceus was isolated from coffee pulp and selected as an interesting hydroxycinnamoyl esterase strain producer, using an activity microplate high-throughput screening method. In this work, we purified and characterized a new type C A. ochraceus feruloyl esterase (AocFaeC), which synthesized specifically butyl hydroxycinnamates in a ternary solvent system. Results: AocFaeC was produced by solid state fermentation, reaching its maximal activity (1.1 U/g) after 48 h of culture. After purification, the monomeric protein (34 kDa) showed a specific activity of 57.9 U/mg towards methyl ferulate. AocFaeC biochemical characterization confirmed its identity as a type C feruloyl esterase and suggested the presence of a catalytic serine in the active site. Its maximum hydrolytic activity was achieved at 40°C and pH 6.5 and increased by 109 and 77% with Ca2+ and Mg2+, but decreased by 90 and 45% with Hg2+ and Cu2+, respectively. The initial butyl ferulate synthesis rate increased from 0.8 to 23.7 nmol/min after transesterification condition improvement, using an isooctane:butanol:water ternary solvent system, surprisingly the synthesis activity using other alcohols was negligible. At these conditions, the synthesis specific activities for butyl p-coumarate, sinapinate, ferulate, and caffeate were 87.3, 97.6, 168.2, and 234 U/µmol, respectively. Remarkably, AocFaeC showed 5 folds higher butyl caffeate synthesis rate compared to type B Aspergillus niger feruloyl esterase, a well-known enzyme for its elevated activity towards caffeic acid esters. Conclusions: Type C feruloyl esterase from A. ochraceus is a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system
Subject(s)
Aspergillus ochraceus/enzymology , Carboxylic Ester Hydrolases/metabolism , Coumaric Acids/chemical synthesis , Solvents , Spectrophotometry , Carboxylic Ester Hydrolases/isolation & purification , Chromatography , Coffee , Butanols , Electrophoresis , FermentationABSTRACT
La riboflavina (B2) es una vitamina hidrosoluble del complejo B, cuyo aporte nutritional es imprescindible para el buen funcionamiento del organismo. La leche es una de las principales fuentes de riboflavina en la dieta humana, sin embargo, en la mayoría de los alimentos se degrada durante los procesos térmicos convencionales y en el almacenamiento. En el presente trabajo se determinó el contenido de riboflavina en productos lácteos comerciales, leches para consumo directo y yogurt, por cromatografía líquida de alta resolución (HPLC) con detección por fluorescencia. La preparación de la muestra incluye una hidrólisis acida (HC10. IN), seguida por una digestión enzimática, y por último, la precipitación de proteínas (TCA100 por ciento). En el análisis cromatográfico se utilizó un sistema isocrático (acetato de amonio 5 mM-metanol, 72:28 v/v). El contenido de riboflavina de las muestras de yogurt estuvo en el rango de 0,289 - 3,078 u/g BS, mientras que en la leche ultrapasteurizada y pasteurizada de 0,61-13,64 ug/gBS y 11,73-15,41 ug/ g BS, respectivamente. Las leches para consumo directo y el yogurt comercial son fuentes de riboflavina, y su consumo regular puede ayudar a satisfacer los requerimientos diarios.
Riboflavin is a water-soluble vitamin of the B complex, and its contribution is essential for the good functioning of the body. Milk is one of the main sources of riboflavin in the human diet; however, in most foods is degraded during conventional thermal processes and storage. In this paper the content of riboflavin in commercial dairy products, milk for direct consumption and yogurt, was determined by high performance liquid chrornato graphic analysis (HPLC) with fluorescence detection. Sample preparation includedan acid hydrolysis (HCL 0.1 N) followed by an enzymatic digestion and a protein precipitation at the end (TCA100 percent). For the HPLC analysis an isocratic bomb program was used (0.005 M ammonium acetate-methanol, 72:28 vlv). Riboflavin concentrations in yogurt samples were 0.289-3.078 ugB2/gDW, while in the ultra-high-temperature pasteurized milk and pasteurized milk was 0.61 -13.64 ugB2/g DW and 11.73-15.41 ugB2/g DW, respectively. Milk for direct consumption and yogurt are good sources of riboflavin; its regular consumption helps to satisfy daily requirements.