Optimized extraction and partial purification of <i>S. Cerevisiae</i> invertase from peach puree

Ferreira, Marcela Vega; Rossler, Aline Farias; Toralles, Ricardo Peraça; Ruiz, Walter Augusto; Rombaldi, Cesar Valmor

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.

Index terms
Yeast; autolysis; RSM; canonical analysis

[TFN0201] ^*
Ya= valor medio de la variable respuesta en términos de actividad relativa (%AR), siendo que 100% de AR corresponde a una actividad de 14,7 U.mg-1 en pH 5 a 25ºC. Valores medios seguidos de la misma letra minúscula en la columna no difieren estadísticamente por el test de Tukey (p<0,05).

Ensayo	Variables Codificadas		Variables naturales		Y^a
	X1	X2	X1 (mM)	X2 (rpm)	Y^a
1	-1	-1	100	100	38,2 b
2	+1	-1	300	100	98,5 a
3	-1	+1	100	300	32,0 b
4	+1	+1	300	300	0,0 c
5	0	0	200	200	100 a
6	0	0	200	200	97,8 a
7	0	0	200	200	99,3 a
8	-1,414	0	50	200	99,3 a
9	0	+1,414	200	350	0,0 c
10	+1,414	0	350	200	0,0 c
11	0	-1,414	200	50	24,3b

Coeficientes	Valores^b
Intercepto (b_o)	91,5 ±3,44 **
b₁	-12,4 ±2,10**
b₂	-16,9 ±2,10**
b₁₁	-21,1 ±2,50**
b₂₂	-36,4±2,50**
b₁₂	-21,3 ±3,00**
R²	0,80

Origen de la variación	Modelo:Y = b0 + Ʃ biXi + Ʃ; bi,iXi2 + Ʃbi,jXiXj
	Suma cuadrática (SC)	Grados de libertad (GL)	Media cuadrática (MC)	F_cal
Modelo	46022,69	5	9204,54	22,9
Linear	10543,61	2
Cuadrático	30012,11	2
Interaciones	5466,97	1
Resíduo	10864.59	27	402,39
Total	51346,33	33

	ILC			ISc
	Actividad específica	Recuperación		Actividad específica	Recuperación
	(U.mg^-1)		%	(U.mg^-1)		%
EB*	216,8 ± 16,3b	1,0	100	36,2 ± 1,9c	1,0	100
Acetona	201,0 ± 2,0 b	0,93	72,1	74,8 ± 9,1 b	2,1	89,9
Etanol	275,6 ± 24,6a	1,3	69,5	108,4 ± 12, 9a	3,0	86,0

Brazil

Brazil

Optimized extraction and partial purification of S. Cerevisiae invertase from peach puree

Abstract

Código del nível	Concentración de NaHCO₃ (X₁)	Velocidad agitación (X₂)
	(mM)	(rpm)
- 1,414	50	50
-1	100	100
0	200	200
1	300	300
1,414	350	350