Biotechnological processes are widely used to obtain products with high added value [
The enzyme beta-galactosidase is one among other enzymes with industrial potential used in the hydrolysis of lactose in milk and cheese whey, generating food with low levels of lactose, which results in a better solubility and digestibility of milk and dairy products, making them ideal for consumers intolerant to this sugar [
When a metabolite is intracellular, the rupture of the cell wall is the first step performed in the downstream process. This allows separating the substance for later purification. Various methods may be used, but the process will depend on the location and stability of the metabolite. There are mechanical methods of cell disruption such as high-pressure homogenizers, ultrasonic waves, and glass beads. Chemical disruption methods use alkalis, detergents, and organic solvents. Enzymatic methods, however, consist of enzymatic lysis or inhibition of cell wall synthesis [
The use of organic solvents for cellular permeabilization and enzymatic extraction is the most common methodology, which was applied after the cell fermentation process. The permeabilization is a simple and fast method that allows measuring the enzymatic activity [
Thus, the aim of this work was to cultivate the filamentous fungus
The
For culture, 1% v/v of the inoculum was added and the fermentation was performed on an orbital shaker (Tecnal®, TE-420) at 28°C and 120 rpm for 5 days. The biomass produced after five days of fermentation was ground with the homogenizer (IKA®, T10 Basic), and 5 mL of the suspension was collected and transferred to falcon tubes, followed by centrifugation (Quimis®, Q222G) for 10 min at 1100 rpm, for separation of the biomass and supernatant. The Biomass was used for the two methods: cell permeabilization and extraction of beta-galactosidase.
After fermentation of
The Box–Behnken design used to determine the effects of ethanol, temperature, and time on the enzymatic activity of permeabilized cells of
Run | Variables | Beta-galactosidase activity (U·mL−1) | |||
---|---|---|---|---|---|
|
|
|
|||
Ethanol (%) | Temperature (°C) | Time (min) | Experimental | Predicted | |
1 | −1 (15) | −1 (20) | −1 (30) | 0.22 | 0.17 |
2 | −1 (15) | 0 (30) | 1 (90) | 0.36 | 0.40 |
3 | −1 (15) | 1 (40) | 0 (60) | 0.29 | 0.29 |
4 | 0 (25) | −1 (20) | 1 (90) | 0.38 | 0.38 |
5 | 0 (25) | 0 (30) | 0 (60) | 0.44 | 0.39 |
6 | 0 (25) | 1 (40) | −1 (30) | 0.17 | 0.22 |
7 | 1 (35) | −1 (20) | 0 (60) | 0.25 | 0.29 |
8 | 1 (35) | 0 (30) | −1 (30) | 0.23 | 0.23 |
9 | 1 (35) | 1 (40) | 1 (90) | 0.40 | 0.35 |
After fermentation of
Analysis of variance (ANOVA) of the regression parameters for the Box–Behnken design used to determine the effect of ethanol, temperature, and time on the enzymatic activity of permeabilized cells of
Seq. SS | df | MS |
|
| |
---|---|---|---|---|---|
Ethanol (%) (L + Q) | 0.00324 | 2 | 0.001619 | 0.251141 | 0.799271 |
Temperature (°C) (L + Q) | 0.00617 | 2 | 0.003084 | 0.478510 | 0.676356 |
Time (min) (L + Q) | 0.0451 | 2 | 0.022552 | 3.499239 | 0.222260 |
Error | 0.01289 | 2 | 0.006445 | — | — |
Total SS | 0.06739 | 8 | — | — | — |
Seq. SS: sequential sums of squares; df: the degrees of freedom; MS: adjusted mean square;
The enzymatic activity was determined by the initial rates of the lactose hydrolysis reaction through the glucose dosage produced by the enzymatic-colorimetric glucose oxidase kit (Bioliquid®) method. The unit of activity used in the work was the glucose unit produced per minute, per milliliter of enzymatic suspension (U·mL−1), defined as
Taking into account that permeabilized cells are biocatalysts, that is, they function as a source of enzymes that naturally remains immobilized. The Box–Behnken design (BBD) was used to evaluate the effects among the significant variables and to determine their optimal values. BBD was developed to reduce the number of experimental runs and increase the efficiency. BBD has been applied and considered a very efficient statistical experimental design tool in several areas including chemical engineering optimization [
The determination of the enzymatic activity for permeabilized cells of
The coefficient of determination
Based on the results for high beta-galactosidase activity from permeabilized cells of
In order to determine the ranges of variables that influence beta-galactosidase activity from permeabilized cells of
Response surface plot representing the effect of the variables on beta-galactosidase activity (U·mL−1) of permeabilized cells from
Based on the results obtained, the temperature and time are fundamental factors in the process of cell permeabilization and can be explained by the fact that the cell walls of the fungi are more rigid than others, requiring a lower concentration of ethanol with more reaction time and a higher temperature for cellular disorganization.
The determination of the enzymatic activity for extracted beta-galactosidase of
The Box–Behnken design used to determine the effect of chloroform and temperature on the enzymatic activity of extracted beta-galactosidase of
Run | Variables | Beta-galactosidase activity (U·mL−1) | ||
---|---|---|---|---|
|
|
|||
Chloroform (%) | Temperature (°C) | Experimental | Predicted | |
1 | −1 (4.0) | −1 (40) | 0.09 | 0.08 |
2 | −1 (4.0) | 0 (45) | 0.12 | 0.13 |
3 | −1 (4.0) | 1 (50) | 0.15 | 0.14 |
4 | 0 (5.0) | −1 (40) | 0.10 | 0.11 |
5 | 0 (5.0) | 0 (45) | 0.17 | 0.15 |
6 | 0 (5.0) | 1 (50) | 0.16 | 0.16 |
7 | 1 (6.0) | −1 (40) | 0.12 | 0.12 |
8 | 1 (6.0) | 0 (45) | 0.16 | 0.16 |
9 | 1 (6.0) | 1 (50) | 0.15 | 0.15 |
Analysis of variance (ANOVA) of the regression parameters for the Box–Behnken design used to determine the effects of chloroform and temperature on the enzymatic activity of extracted beta-galactosidase of
SS | df | MS |
|
| |
---|---|---|---|---|---|
Chloroform (%) (L + Q) | 0.001089 | 2 | 0.000544 | 2.63677 | 0.218346 |
Temperature (°C) (L + Q) | 0.004689 | 2 | 0.002344 | 11.35426 | 0.039863 |
|
0.0002225 | 1 | 0.000225 | 1.08969 | 0.373253 |
Error | 0.000619 | 3 | 0.000206 | — | — |
Total SS | 0.006622 | 8 | — | — | — |
Seq. SS: sequential sums of squares; df: the degrees of freedom; MS: adjusted mean square;
Based on the results for high beta-galactosidase activity from extracted beta-galactosidase of Aspergillus oryzae CCT 0977, the effect of temperature (L + Q) was significant (
In order to determine the ranges of variables that influence enzymatic activity of beta-galactosidase extracted from
Response surface plot representing the effect of the variables on enzymatic activity (U mL−1) of beta-galactosidase extracted from
Several authors have studied the effect of solvents on the extraction of enzymes [
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
The authors would like to thank Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior–CAPES/Brazil for the financial support and KROTON/UNOPAR for school and masterships.