Resumo Estudaram-se as melhores condições de hidrólise da sacarose in vitro a partir da invertase de Candida guilliermondii (ICg), também se determinou os parâmetros cinéticos KM, Vmax e a estabilidade térmica da ICg. A levedura Candida guilliermondii (Cg) extraída, isolada e liofilizada de resíduo de pêssego foi identificada usando o método API 20C AUX. Posteriormente, a levedura Cg foi submetida a um processo de autólise, usando NaHCO3 a 200 mM sob agitação a 200 rpm e 40°C, por período de 24 horas. Os extratos enzimáticos obtidos foram recuperados por precipitação com acetona seguido de diálise e cromatografia de troca iônica. O extrato purificado por precipitação com acetona teve a atividade de 27,7 U.mg-1 e 56% de recuperação, sendo que, por cromatografia, obtiveram-se 46,5 U.mg-1 e 44,8%. As condições ótimas na hidrólise da sacarose foram pH de 5,0 e 50°C, apresentando KM de 30,5/mM e 28,7 mM de sacarose, respectivamente, a 25°C e a 50°C, ambas com comportamento Michaeliano. A inativação térmica da ICg mostrou uma cinética aparente de primeira ordem, e sua atividade residual foi tipicamente linear entre 40°C - 70°C. Por meio de eletroforese, foram detectadas três isoenzimas.

Abstract The best conditions for in vitro sucrose hydrolysis based on invertase from Candida guilliermondii (ICg) were studied and the kinetic parameters KM,Vmax, and thermal stability of ICg were determined. Candida guilliermondii (Cg) yeast isolated and lyophilized from peach solid wastes was identified using the API 20C AUX method. Subsequently, the Cg was submitted to an autolysis process using NaHCO3 at 200 mM under 200 rpm stirring and 40 °C for 24 h. The enzyme extracts obtained were recovered through precipitation with acetone followed by dialysis and ion-exchange chromatography. The extract purified through precipitation with acetone had activity of 27.7 U.mg-1 and 56% recovery whereas the chromatography process yielded 46.5 U.mg-1 and 44.8%. The optimal sucrose hydrolysis conditions were pH 5.0 and 50 °C, resulting in KM of 30.5 mM and 28.7 mM sucrose, respectively, at 25 °C and 50 °C, both with Michaelian behavior. Thermal inactivation of ICg exhibited first-order apparent kinetics and its residual activity was typically linear between 40 °C and 70 °C. Three isoenzymes were detected through electrophoresis.

Abstract The extraction was performed using an autolysis process where enzymes degrade cell structures and release the cytoplasmic contents to the extracellular medium. The autolysis process was carried out in shake flasks with different agitation speeds (50 - 350 rpm) and the concentration of sodium bicarbonate (50 -350 mM NaHCO3) at 40 ° C for 24 hours, using a central composite design adapted for two variables and five levels and response surface methodology (RSM) and canonical analysis to define the optimal conditions of extraction of invertase S. cerevisiae from peach puree (ISc). In great condition extraction, we studied the effect of the liofilization and purified in the invertase activity using colorimetric method of 3,5-dinitrosalicílico (DNS) at 490 nm to monitor their activity. One unit (U) defined as 1 mg glicose.min-1. Protein concentrations were determined by Lowry and invertase comercial yeast (ICY). The influence of concentration NaHCO3 and stirring speed to extract the enzyme invertase showed linear effect, quadratic and interactive. The polynomial model of second order could explain the extraction phenomenon of invertase with R2 of 0.80, with significantly dependent on both variables activity. The maximum extraction was observed in the experimental center point (200 mM NaHCO3 and 200 rpm) with an activity of 14.7 U.mg-1. The stationary point is a point of maximum extraction, differing by at least 10% of the experimental center point. Based on these characteristics, the optimal conditions for extraction of S. cerevisiae invertase from peach puree (ISc) are 200 mM NaHCO3 as autolysis agent, 200 rpm orbital shaking at 40ºC for 24 hours and freeze-dried using biomass. Ethanol is more effective than acetone for recovery of specific activity.

Resumen La extracción fue realizada a través de un proceso de autolisis, donde las enzimas degradan las estructuras celulares y liberan el contenido citoplasmático al medio extracelular. El proceso de autolisis fue realizado en shaker con diferentes velocidades de agitación (50 - 350 rpm) y concentraciones de bicarbonato de sodio (50 -350 mM NaHCO3) a 40 °C por 24 horas, usando un diseño experimental compuesto central adaptado para dos variables y cinco niveles, así como una metodología de superficie de respuest a (MSR) y análisis canónico para definir las condiciones óptimas de extracción de invertasa de S. cerevisiae aislada de puré de durazno (ISc). En la condición optima de extracción, se estudió el efecto de la liofilización y purificación en la actividad de la invertasa, usando el método colorimétrico del ácido 3,5-dinitrosalicílico (DNS) a 490 nm para acompañar su actividad expresada en unidades. Una unidad (U) es definida como 1 mg glucosa.min-1. El valor de proteínas fue cuantificado por el método Lowry y la invertasa de levadura comercial (ILC) como testigo. La influencia de la concentración de NaHCO3 y de la velocidad de agitación en la extracción de la enzima invertasa indicó efecto lineal, cuadrático e interactivo. El modelo de forma polinómica de segundo orden explicó el fenómeno de extracción de la invertasa con un R2 de 0,80, y con actividad significativamente dependiente de ambas variables. La máxima extracción fue observada en el punto central experimental (200 mM NaHCO3 y 200 rpm) con una actividad de 14,7 U.mg-1. El punto estacionario es un punto de máxima extracción, el cual difiere en menos de 10% del punto central experimental. De acuerdo a estas características, las óptimas condiciones para extracción de la invertasa de S. cerevisiae aislada de puré de durazno son 200 mM NaHCO3 como agente de autolisis, 200 rpm de agitación orbital a 40oC durante 24 horas y usando una biomasa de levaduras liofilizadas. El etanol es más efectivo que la acetona para la recuperación de la actividad específica.

Abstract The identification of yeasts isolated from spoiled Jubileu peach puree using the API 20C AUX method and a commercial yeast as witness were studied. Subsequently, the yeast’s growth potential using two batch culture treatments were performed to evaluate number of colonies (N), reducing sugar concentration (RS), free-invertase (FI), and culture-invertase activity (CI). Stock cultures were maintained on potato dextrose agar (PDA) slants at 4 °C and pH 5 for later use for batch-culture (150 rpm) at 30°C for 24 h, then they were stored at 4 °C for subsequent invertase extraction. The FI extract was obtained using NaHCO3 as autolysis agent, and CI activity was determined on the supernatant after batch-cultured centrifugation. The activity was followed by an increase in absorbance at 490 nm using the acid 3,5-DNS method with glucose standard. Of the four yeasts identified, Saccharomyces cerevisiae was chosen for legal reasons. It showed logarithmic growth up to 18 h of fermentation with positive correlation CI activity and inverse with RS. FI showed greater activity by the end of the log phase and an inverse correlation with CI activity. Finally, it was concluded that treatment “A” is more effective than “B” to produce invertase (EC 3.2.1.26).