Cost-Effective Optimization of the Transfructosylation Activity of an Invertase Produced from Aspergillus carbonarius PC-4 Using Pineapple Crown and Determination of Its Biochemical Properties

Abstract

Fructooligosaccharides are prebiotic sugars that are widely used in the production of functional foods, which can be produced enzymatically by the transfructosylation reaction of sucrose. This work aimed to optimize the production of an invertase with high transfructosylation activity from Aspergillus carbonarius PC-4 using pineapple crown as the inducer substrate and evaluate its biochemical properties. The culture medium was optimized using a Plackett–Burman experimental design and a central composite rotatable design, resulting in a maximum transfructosylation activity of 65.33 U/mL at 72 h of cultivation. The cultivation parameters were Yp/s = 1070.75 U/g and P_P = 2771.48 U/h, which showed an increase of 5.2-fold in the enzyme produced. The optimum temperature (50 °C) and pH (5.0) for the enzymatic activity were obtained by a CCR design. The enzyme showed a half-life of 60 min at 40 °C. In conclusion, the invertase produced from A. carbonarius PC-4 using agro-industrial waste (pineapple crown) and an inorganic nitrogen source (ammonium nitrate) exhibits high transfructosylation activity that can be used as a potential source for the production of fructooligosaccharides.

1. Introduction

Invertases (β-fructofuranosidase, E.C. 3.2.1.26) are enzymes that belong to the carbohydrase class (family GH32) and catalyze the hydrolysis of the α-1,4-glycosidic bond from sucrose into glucose and fructose in equimolar proportions of inverted sugar at a concentration of approximately 10% substrate. These enzymes can be obtained from a wide variety of microorganisms, including animals, plants, bacteria, yeasts, and fungi. However, fungi stand out because they have attracted the attention of different sectors of the industry due to their biotechnological potential and because they are considered saccharolytic, that is, they ferment various carbohydrates [1,2]. The fungi that stand out in the production of invertases belong to the genera Penicillium, Aerobasidium, Fusarium, and, mainly, Aspergillus [3,4,5]. In addition, invertases can perform a transfructosylation reaction when exposed to a range of sucrose concentrations from 20 to 85%, thus also being classified as fructosyltransferases (E.C. 2.4.1.9), a subclass of transferases [1,6]. Fructosyltransferases (FTases) act on the β-(1-2) bonds of sucrose and transfer the fructosyl radical to another sucrose molecule, forming fructooligosaccharides (FOSs), or transfer the fructosyl radical to an FOS by lengthening the chain by a fructose unit, releasing glucose in both situations [3,7].

Transfructosylation carried out by invertases has wide applications in several industries. In the food industry, it is used to produce fructooligosaccharides (FOSs), which act as prebiotics and improve the texture and stability of food products [8]. In the pharmaceutical industry, the resulting oligosaccharides are incorporated into prebiotic supplements and other formulations due to their benefits for intestinal health [9,10]. In biotechnology, this enzymatic reaction is exploited to synthesize new functional sugars and modify biomolecules [11]. The cosmetics industry uses products derived from transfructosylation as humectants in moisturizing formulations. Furthermore, in the agricultural industry, FOSs can be added to animal feed to promote digestive health. These applications highlight the versatility of transfructosylation in improving processes and products in several areas [9,12].

FOSs are commonly designated as 1-kestose (GF2), 1-nystose (GF3), and 1F-β-fructofuranosylnystose (GF4) [3]. They are classified as prebiotics, defined as non-digestible food ingredients that are resistant to hydrolysis by human digestion, because of the β conformation in the anomeric carbon of fructose monomers [13,14]. These biomolecules have beneficial effects on human and animal health, since they promote good absorption of minerals in the body, regulate blood glucose and cholesterol levels, promote the proliferation of beneficial bacteria in the large intestine (particularly Bifidobacteria, which regulate the intestinal microbiota), prevent the onset of colon cancer, relieve constipation, and increase immunity [15,16]. FOSs are widely used in the food industry and can be safely consumed by diabetics, since they have about one-third of the sweetening power of sucrose and are low in calories [3,7,16]. They are commonly used in dairy products, cookies, breads, confectionery products, and functional products (to promote a symbiotic effect with probiotic microorganisms), among others. Additionally, they can be used in animal feed for the same purpose as prebiotics and can also minimize the formation of tooth decay, since they are not consumed by Streptococcus mutans [1,17].

Enzymes can be produced by submerged cultivation or solid-state cultivation; nevertheless, most industrial enzymes are produced by submerged cultivation, since it is an efficient method for enzyme production and due to the greater ease in the control of the physicochemical parameters [18,19]. The use of agro-industrial waste for the production of microbial enzymes has been widely adopted as a substitute for pure carbon sources, since it represents a valuable and rich source of energy and other nutrients (lignocellulose, proteins, carbohydrates, lipids, etc.), and it is also a low-cost biomass, which would otherwise be discarded into the environment and cause major problems of solid waste pollution [15,20]. One of the residues that causes major environmental issues is the waste from pineapple cultivation. Brazil is the second largest producer of pineapple (Ananas comosus) in the world, producing around 1.56 billion pineapples per year [21]. The by-products generated from pineapple correspond to residual pulp, peels, crowns, stems, and leaves [22].

Using a statistical approach to optimize enzyme production is of the utmost importance, since it involves the analysis of different variables, such as carbon, nitrogen, salts, and physicochemical factors, in the search for optimal conditions in the production process. In this sense, Plackett and Burman designs allow for the screening of the most important parameters and the response surface for the achievement of optimal conditions [16,18,23]. Previous studies have shown that a strain of Aspergillus sp. isolated from canned peach syrup was able to produce high amounts of fructosyltransferase when grown in a submerged medium, using pineapple crown and raffinose as the carbon source and soy protein and ammonium nitrate as the nitrogen source. Thus, the present work aimed to optimize the production of an invertase with high transfructosylation activity produced from A. carbonarius PC-4 under submerged conditions using Plackett and Burman and DCCR experimental designs and to characterize the biochemical properties of the enzyme produced in the crude extract.

2. Materials and Methods

2.1. Materials

The pineapple crown was purchased from local commerce (Gurupi, Brazil). Potato dextrose agar (PDA) was purchased from Merck (Rahway, NJ, USA). The glucose oxidase–peroxidase kit was purchased from Sigma-Aldrich (St. Louis, MO, USA). Citric acid*H₂O, ZnSO₄*7H₂O, Fe(NH₄)₂(SO₄)₂*6H₂O, CuSO₄*5H₂O, MnSO₄*H₂O, H₃BO₃, Na₂MoO₄*2H₂O, sodium citrate*5H₂O, KH₂PO₄, NH₄NO₃, MgSO₄*7H₂O, CaCl₂*2H₂O, 0,1 mg/mL biotin solution, glucose, sucrose, and sodium acetate were purchased from Labynth^® (Diadema, Brazil). All chemicals used were analytical grade.

2.2. Methods

2.2.1. Microorganism and Maintenance

A black Aspergillus carbonarius strain PC-4 was isolated from peach syrup and maintained in the Laboratory of Biotechnology, Food Analysis and Products (LABAP), Habite—Biotechnology Companies Incubator, Federal University of Tocantins—UFT, Gurupi, Tocantins, Brazil [17]. A. carbonarius PC-4 was cultivated on PDA for 3 days at 28 °C and then stored at 4 °C in a PDA slant. A. carbonarius PC-4 was also preserved in sterile distilled water [24]. The dishes (approximately 5 mm) containing a small portion of the culture medium and sporulated mycelium were aseptically transferred into sterile 6 mL flasks filled with 4 mL of sterile distilled water and sealed with a rubber stopper. The flasks were stored at 4 °C, and the viability of the strains was verified every six months.

2.2.2. Submerged Culture Conditions

A liquid medium was prepared using Vogel’s medium (Merck, Rahway, NJ, USA) [25]. The trace element solution (solution A) was prepared containing (g/L) the following ingredients: citric acid*H₂O, 50; ZnSO₄*7H₂O, 50; Fe(NH₄)₂(SO₄)₂*6H₂O, 10; CuSO₄*5H₂O, 2.5; MnSO₄*H₂O, 0.05; H₃BO₃, 0.05; and Na₂MoO₄*2H₂O, 0.05. The salt solution (solution B) was prepared containing (g/L) the following ingredients: sodium citrate*5H₂O, 150; KH₂PO₄, 250; NH₄NO₃, 100; MgSO₄*7H₂O, 10; CaCl₂*2H₂O, 5 and biotin solution (0.1 mg/mL), 5 mL; and solution A, 5 mL. The solutions were maintained at 4 °C. The medium preparation consisted of a 50-fold dilution of solution B, replacing glucose with other carbon sources, and adjusting the final pH to 6.0.

The cultures were performed in Erlenmeyer flasks (100 mL) containing 20 mL of culture medium. All media were autoclaved at 121 °C for 20 min. The inocula were prepared using 3-day-old cultures. The media were inoculated with 1 mL of a conidia suspension (1 × 10⁷ spores or cells per mL) and incubated at 28 °C, 180 rpm, for 3 and 5 days. The biomass was separated from the fermentation broth by filtration in muslin. The cell-free broth was used for fructosyltransferase activity assays. The results were assayed for fructosyltransferase activity (TFA), fructosyltransferase yield on the substrate (Yp/s), and fructosyltransferase productivity (Pp). The parameters Yp/s (U/g) and Pp (U/h) were calculated using Equations (1) and (2), respectively.

$$Y_{p/s}=P_f-\left({\displaystyle \frac{P_0}{S_0}}\right)$$

(1)

$$Pp=TFA/t$$

(2)

where P_f is the final product, P₀ is the initial product, S₀ is the initial substrate (pineapple crown waste), and t is the time of cultivation.

2.2.3. The Transfructosylation Activity of the Invertase from A. carbonarius PC-4

The transfructosylation activity was determined according to Rawat et al. [26]. A total of 100 microliters of culture filtrate with 400 µL of sucrose (20% w/v in 0.1 M sodium acetate buffer pH 5.0) was heated at 50 °C for 1 h in a water bath. The reaction was stopped by boiling the mixture in a water bath at 100 °C for 10 min. The transfructosylation activity was estimated by taking 20 µL of the appropriately diluted reaction mixture and mixing it with 2 mL of the test reagent (Glucose oxidase–peroxidase kit, Sigma-Aldrich, St. Louis, MO, USA). The glucose released was measured at 505 nm. One unit of transfructosylation activity was defined as the amount of enzyme required to produce 1 mol of glucose per minute under the assay conditions.

2.2.4. Optimization of the Production of Transfructosylation Activity

Plackett–Burman Design

The Plackett–Burman experimental design [27] was used to evaluate the relative importance of carbon and nitrogen in the production of an invertase with high transfructosylation activity from A. carbonarius PC-4 after 72 h and 120 h of cultivation. Raffinose and pineapple crown were used as carbon sources, while ammonium nitrate and soybean protein were used as nitrogen sources (

Table 1. Experimental variables and levels used for the production of an invertase with high transfructosylation activity from A. carbonarius PC-4 using the Plackett–Burman design.

Factors	Code	−1	0	1
Pineapple crown	X1	0	5	10
Raffinose	X2	0	5	10
Soy protein	X3	0	1	2
Ammonium nitrate	X4	0	1	2

Note: the values are expressed in g/L.

). Each variable represented three levels—high concentration (1), low concentration (−1), and intermediate concentration (0)—in eight trials (Table 1). The response variable analyzed was enzyme activity.

Central Composite Rotatable Design

After the identification of the carbon and nitrogen sources affecting enzyme production in the Plackett–Burman design, a response surface methodology of a central composite rotatable design (CCRD) was used to discover the optimal combination of the independent variables for invertase production. The two main independent variables chosen for this experiment were pineapple crown (X₁) and ammonium nitrate (X₂) with 4 trials under the axial conditions and 3 repetitions at the central point, totaling 11 trials (

Table 2. Experimental variables and levels using a CCRD for the production of an invertase with high transfructosylation activity from A. carbonarius PC-4.

Factors	Code	−1.41	−1	0	1	1.41
Pineapple crown	X1	6	10	20	30	34
Ammonium nitrate	X2	0.5	3	9	15	17.5

Note: the values are expressed in g/L.

). The response variable analyzed was enzyme activity.

All experiments were conducted as previously described, and invertase production was considered to be the independent variable or response (Y). The second-order polynomial coefficients were obtained and evaluated statistically by analysis of variance (ANOVA). The model’s accuracy in predicting the maximum transfructosylation activity was confirmed by a triplicate experiment in the optimized conditions. The predicted and experimental values of transfructosylation activity were confirmed using the optimal values for the significant variables generated by CCRD.

2.2.5. The Biochemical Properties of the Invertase

Effect of pH and Temperature on Invertase Activity

The optimal conditions for transfructosylation activity in different ranges of pH and temperature were determined in a test tube immersed in a water bath, containing 20% (w/v) sucrose, in McIlvaine buffer, with a reaction time of up to 60 min. This experiment was carried out using the response surface methodology of CCRD, in which pH was defined as X₁ and temperature as X₂ (

Table 3. Experimental variables and levels using the CCRD for the influence of pH and temperature on the activity of the invertase with high transfructosylation activity from A. carbonarius PC-4.

Factors	Code	−1.41	−1	0	1	1.41
pH	X1	2.17	3	5	7	7.83
Temperature (°C)	X2	14.64	25	50	75	85.36

). Each independent variable was studied at five coded levels (Table 3). The response variable analyzed was enzyme activity.

All experiments were conducted as previously described, and invertase production was considered to be the independent variable or response (Y). The second-order polynomial coefficients were obtained and evaluated statistically by analysis of variance (ANOVA). The model’s accuracy in predicting the maximum transfructosylation activity was confirmed by a triplicate experiment in the optimized conditions. The predicted and experimental values of transfructosylation activity were confirmed using the optimal values for the significant variables generated by CCRD.

The Effects of pH and Temperature on the Stability of the Transfructosylation Activity

The effect of pH on the enzyme stability was tested using the McIlvaine buffer with pH values ranging from 3.0 to 8.0. The enzyme preparation was diluted in each buffer (1:2, v/v) and incubated for 7 h and 24 h at 15 °C, without substrate.

Thermal stability assays were conducted by incubating the crude extract, without substrate, at the temperatures of 40 °C, 50 °C, and 60 °C for up to 150 min, at the previously determined pH value.

Aliquots were withdrawn at specific time intervals and maintained in an ice bath for 5 min. The residual activity was determined as previously described, using the McIlvaine buffer with 20% of sucrose (w/v) for 60 min and at 50 °C. Results were expressed as relative activity, considering the initial activity (t = 0) to be 100%.

2.2.6. Statistical Analysis

The experimental design was evaluated using the software “Protimiza Experimental Design” (https://experimental-design.protimiza.com.br, accessed on 9 August 2024) [28]. The polynomial models with 90% and 95% confidence levels were evaluated by analysis of variance (ANOVA), and Student’s t-test and Fischer’s test (F) were used to determine the statistical significance of the regression coefficients and to obtain the second-order model equation, respectively. In turn, the quality of the fit polynomial model was assessed by the coefficient of determination (R²).

3. Results and Discussion

3.1. Screening of Carbon and Nitrogen Sources by the Plackett–Burman Design

A. carbonarius PC-4 has been previously identified as the producer of an invertase with high transfructosylation activity under submerged conditions using pure and complex carbon and nitrogen sources [17]. Under these conditions, the maximum enzyme production was obtained using Vogel’s salt medium supplemented with raffinose and pineapple crown as carbon sources and ammonium nitrate and soybean protein as nitrogen sources (44.40 U/mL) after 168 h of cultivation. Sequential optimization approaches were applied in this study, initially by screening the nutritional factors that affected the production of the invertase with transfructosylation activity using the Plackett–Burman design after three and five days of cultivation (

Table 4. CCRD for the transfructosylation activity of the invertase produced from A. carbonarius PC-4.

Run	Pineapple Crown (X1, g/L)	Ammonium Nitrate (X2, g/L)	TFA (U/mL)	Yp/s (U/g)	Pp (U/h)
1	10	3	35.18 ± 1.50	2526.17	7.02
2	30	3	16.73 ± 0.35	409.49	3.41
3	10	15	29.88 ± 5.42	1711.80	4.75
4	30	15	40.83 ± 7.61	941.26	7.84
5	6	9	24.92 ± 3.23	2891.45	4.82
6	34	9	16.73 ± 3.58	309.41	2.92
7	20	0.5	32.76 ± 0.69	1443.03	8.02
8	20	17.5	65.86 ± 0.12	2958.73	16.44
9	20	9	68.98 ± 3.92	2916.05	16.20
10	20	9	69.44 ± 3.00	2657.67	14.76
11	20	9	52.48 ± 3.35	2233.18	12.41

). The results of enzyme production were given in units per mL (U/mL), production yield (Y_P/S), and productivity (P_P). In this sense, a large variation was observed, from 0.02 to 46.26 U/mL after 72 h of cultivation, and from 0 to 52.20 U/mL after 120 h of cultivation. The mean effects of the factors examined for enzymatic activity are presented in the Pareto plot (Figure 1).

The analysis of the cultivation parameters of the transfructosylation activity of the invertase produced using A. carbonarius PC-4 with the Plackett–Burman design showed that the enzyme production (46.26 U/mL) was not reduced after three days of cultivation. As a comparison, the initial cultivations carried out using the parametric technique of culture optimization with carbon and nitrogen sources showed maximal production after 168 h of cultivation [17]. According to the results obtained using the Plackett–Burman design, the variables pineapple crown, ammonium nitrate, and soy protein had a significant positive effect (p < 0.10), while raffinose had a significant negative effect, at the 72 h time point. At the 120 h time point, only the variables pineapple crown and ammonium nitrate had a significant positive effect (p < 0.10) (Figure 1). These results suggest that higher concentrations of these components lead to increased enzymatic activity for the variables with a positive effect. Conversely, for the variable with a negative effect, lower concentrations result in higher enzymatic activity. Furthermore, a medium of the following composition is expected to lead to similar results: 20 g/L pineapple crown, 4 g/L ammonium nitrate, and 4 g/L soy protein. Under these conditions, Y_P/S was 3700.44 U/g of the substrate and P_P was 10.28 U/h. The results showed a 1.59-fold increase in enzyme productivity while maintaining the production yield. In this study, a decrease in the duration of the cultivation to 72 h was achieved, which represents an important reduction in energy costs to produce the invertase from A. carbonarius PC-4 under submerged conditions using agro-industrial waste and a mineral nitrogen source.

Organic and inorganic nitrogen sources are essential for enzyme synthesis. Inorganic nitrogen can be utilized quickly, while organic sources provide various cell growth factors and amino acids that are essential for cell metabolism and enzyme production [29]. Ademakinwa et al. [18] observed that ammonium ions can influence fructosyltransferase production from Aureobasidium pullulans using the Plackett–Burman design. Ammonium nitrate has been used as a nitrogen source for the production of xylanase from Aspergillus candidus [30], amylase from Aspergillus niger [31], and lipase from Candida viswanathii [32].

Agro-industrial waste is economically feasible for enzyme production, with a reduction in operational costs, and it is environmentally friendly. Pineapple waste contains a considerable amount of soluble sugars, such as sucrose, which makes it suitable for use as a substrate in microbial fermentation [33,34]. Therefore, agro-industrial waste, such as pineapple crown, are potentially suitable substrates for the production of value-added biomolecules when used in the composition of microbial culture media [17,28]. This substrate has been used to produce β-glucosidase and xylanase from Trichoderma viride [22,35], cellulases from Trichoderma ressei [36], and invertase from A. niger [34].

3.2. Medium Composition Optimization by Central Composite Rotational Design (CCRD)

For the optimization experiment using CCRD and RSM methodologies, and according to previous results from the Plackett–Burman design, at the 72 h time point, despite the soy protein having a significant positive effect on the enzymatic activity, only ammonium nitrate was selected as the nitrogen source due to its more pronounced positive effect. Pineapple crown was chosen as the sole carbon source, because raffinose had a negative effect on the enzymatic activity. The response data based on the independent variables were obtained from the experiments and are depicted in Table 4.

The factors analyzed showed that the concentration of ammonium nitrate had a positive linear effect and a negative quadratic effect, indicating a tendency for maximum FTase production near the central point, while the concentration of the pineapple crown had a negative quadratic effect, indicating that an increase or decrease in the value of this variable would lead to a reduction in FTase production [37] (

Table 5. Estimates of the coefficient of independent variables on invertase with transfructosylation activity production using the CCRD to determine the transfructosylation activity of invertase.

Factors	Coefficient	Error	t	p-Value
Média	63.63	4.66	13.65	0.000
X1	−2.38	2.85	−0.84	0.441
X12	−22.51	3.40	−6.63	0.001
X2	8.20	2.85	2.87	0.035
X22	−8.26	3.40	−2.43	0.059
X1.X2	7.35	4.04	1.82	0.128

).

A second-order equation was fitted to the data by multiple regression analysis, which generated the reparametrized model that describes the measured responses for the significant independent variables (pineapple crown and ammonium nitrate). The mathematical model was expressed with the following equation:

$$\mathrm{TFA}\left(U/mL\right)=63.63-22.51X_1^2+8.20X_2-8.26X_2^2$$

(3)

The experimental model was validated by the analysis of variance (ANOVA) with a coefficient of determination R² = 0.8533, an F value calculated from the regression to be 13.6 (greater than the tabulated F value of 5.27), and an F lack of adjustment value of 0.9 (not significant), making the model valid in the 90% confidence interval (

Table 6. Analysis of variance (ANOVA) for transfructosylation activity of the invertase.

Variation Source	Sum of Squares	Degree of Freedom	Mean Square	Fcal	p-Value
Regression	3415.5	3	1138.5	13.6	0.00265
Residue	587.4	7	83.9
Lack of fit	400.7	5	80.1	0.9	0.61566
Pure error	186.7	2	93.4

F_{tab regression/residue} (3; 7; 0.10): 3.07; F_{tab lack of fit/pure error} (5; 2; 0.10): 9.29. R²: 85.33%.

).

A response surface plot (Figure 2) was generated from the reparametrized model equation, indicating that the maximum transfructosylation activity of the invertase was achieved at the midpoint of the experimental design (pineapple crown, 2.0%, w/v, and ammonium nitrate, 1.0%, w/v) after 72 h of cultivation. The increase in pineapple crown causes a decrease in the enzymatic activity. This fact was explained by Amim et al. [38], according to whom the gradual increase in substrate concentration will cause the enzyme production to increase at a directly proportional rate until the medium becomes saturated with the substrate. After reaching the saturation point, the addition of extra substrate will no longer make a difference.

Park et al. [39] stated that increasing the amount of nitrate has no significant effect on the enzymatic activity of fructosyltransferase. This fact can be observed, using other nitrogen sources, as it was in the study conducted by Ademakinwa et al. [23], who performed a response surface experiment to evaluate the variables sucrose, NH₄Cl, and yeast extract in the production of FTase from Aureobasidium pullulans. By increasing the sucrose concentration and maintaining the NH₄Cl concentration, there was an increase in the enzymatic activity of fructosyltransferase; on the other hand, the increase in yeast extract had a negative effect on the activity of fructosyltransferase. The same behavior was observed when the concentration of yeast extract was kept constant and the concentration of NH₄Cl varied, resulting in a decline in the enzymatic activity.

In this study, a 5-fold increase in enzyme production was observed compared to the initial production of 13.38 U/mL during 72 h of cultivation [17], and there was also an increase in yield from 1070.75 U/g to 2771.48 U/g and a 5.19-fold increase in productivity. Although many researchers have investigated various effects on fructosyltransferase production [17,23,37], as far as we know, no optimization study has used the combination of ammonium nitrate and pineapple crown, which successfully increased enzyme production.

3.3. Validation

The results predicted by the model suggested that the maximum transfructosylation activity of the invertase produced from A. carbonarius PC-4 would be 65.33 ± 4.62 U/mL with the supplementation of 0.40 g of pineapple crown and 0.21 g of ammonium nitrate. The obtained results yielded an average FTase production of 66.46 ± 2.67 U/mL (

Table 7. Validation of the culture conditions for the production of the invertase with transfructosylation activity from A. carbonarius PC-4.

Variable	Experimental Conditions	Predicted Value	Experimental Value
Pineapple crown	20 g/L	65.33 ± 4.62 U/mL	66.46 ± 2.67 U/mL
Ammonium nitrate	10.5 g/L

). According to the predicted and experimental results, the model was validated based on the existence of the optimum point. There was a 1.77-fold increase compared to the best result for enzymatic activity (3 days of cultivation) of the Plackett and Burman design and 3.82-fold compared to the result obtained by Nascimento et al. [5] (17.36 U/mL).

3.4. The Physical and Chemical Properties of the Transfructosylation Activity

3.4.1. Influence of Temperature and pH on Enzyme Activity

The effect of pH and temperature on the enzymatic activity was analyzed using a central composite rotational design (

Table 8. Relative activity at different pH values and temperatures of transfructosylation activity in the invertase using CCRD.

Run	pH X1	Temperature (°C) X2	TFA (U/mL)	Relative Activity (%)
1	3	25	0.00	0.00
2	7	25	2.01	3.49
3	3	75	0.00	0.00
4	7	75	0.86	1.47
5	2.17	50	0.01	0.01
6	7.83	50	1.36	2.35
7	5	14.64	2.09	3.62
8	5	85.36	0.09	0.15
9	5	50	54.55	89.48
10	5	50	57.65	94.62
11	5	50	51.59	100

). The relative activity values were presented in relation to the maximum activity value, considered to be 100%. Thus, it was observed that pH and temperature values at different levels strongly influenced the transfructosylation activity of the enzyme produced from A. carbonarius PC-4. According to Ghazi et al. [40], there was no significant fructosyltransferase activity at pH values below 3.5 or above 9.5, and temperatures higher than 65 °C inactivated the fructosyltransferase.

The factors analyzed showed that the pH and temperature are significant variables in quadratic terms, and both have negative effects, indicating that an increase in the value of these variables above the studied limits would lead to a decrease in invertase production (

Table 9. Estimates of the coefficients of independent variables on invertase production with transfructosylation activity using the CCRD for transfructosylation activity.

Factors	Coefficient	Error	t	p-Value
Mean	54.60	1.12	48.64	0.000
X1	0.60	0.69	0.87	0.4243
X12	−27.00	0.82	−33.00	0.0000
X2	−0.50	0.69	−0.73	0.4997
X22	−26.80	0.82	−32.75	0.0000
X1.X2	−0.29	0.97	−0.30	0.7778

Factors are in bold when p < 0.05.

). A second-order equation was fitted to the data by multiple regression analysis, generating a reparametrized model that describes the measured responses for the significant independent variables (pH and temperature). The mathematical model was expressed with the following equation:

$$TFA\left(U/mL\right)=54.60-27X_1^2-26.80X_2^2$$

(4)

The experimental model was validated by analysis of variance (ANOVA) with a coefficient of determination R² = 99.62%, an F value calculated from the regression as 1048.5 (greater than the tabulated F value of 4.46), and an F value for lack of adjustment (0) that was not significant, making the model valid in the 95% confidence interval (

Table 10. Analysis of variance (ANOVA) for transfructosylation activity of the invertase.

Variation Source	Sum of Squares	DF	Mean Square	Fcal	p-Value
Regression	6314.5	2	3157.3	1048.5	0.00000
Residue	24.1	8	3.0
Lack of fit	5.7	6	1.0	0.0	0.99683
Pure error	18.4	2	9.2
Total	6338.6	10

F_{tab regression/residue} (2; 8; 0.05): 4.46; F_{tab lack of fit/pure error} (6; 2; 0.05): 19.33. R²: 99.62%.

).

The maximum transfructosylation activity was reached at the central point of the experimental design (pH of 5 and temperature of 50 °C) (Figure 3), where the ionization state and the established temperature stabilize the conformation of the enzyme, as well as the adequate binding of the substrate to the active site. Similar results were observed by Nemukula et al. [41], who characterized the fructosyltransferase from Aspergillus aculeatus and found that it has optimal activity at a pH of 6 and temperature of 60 °C. Regarding temperature, Aguiar-Oliveira and Maugeri [42] conducted a study on fructosyltransferase and stated that the highest transfructosylation rates occur at temperatures between 45 °C and 65 °C. Kashyap et al. [43] found that the relative activity of the fructosyltransferase from A. aculeatus increased as the pH increased to 4.5 at the temperature of 55 °C and subsequently declined and became constant.

For the purified enzyme, L’Hocine et al. [44] demonstrated that the best pH and temperature of fructosyltransferase from A. niger are 5.8 and 50 °C, respectively. Zeng et al. [45] reported that different microbial species can produce different molecular structures and active sites of the enzyme, leading to different optimal initial reaction pH values.

3.4.2. Validation

To confirm the model’s accuracy in predicting the maximum transfructosylation activity, an additional experiment was performed in triplicate under the optimized conditions. The model suggested that the maximum activity is 54.60 ± 1.12 U/mL, which can be achieved when the process conditions include a pH = 5.0 and a temperature of = 50 °C. The results obtained yielded a maximum transfructosylation activity of 53.85 U/mL. According to the predicted and experimental results, the model was validated based on the existence of the optimum point.

3.5. Thermal Stability and pH Stability

The FTase produced from A. carbonarius PC-4 was stable at 40 °C, where 50% of its activity was verified after 60 min of incubation and remained at around 30% after 155 min. On the other hand, at the temperatures of 50 °C and 60 °C, the half-life of the enzyme was reached after 15 min of incubation. The enzyme was not stable at 60 °C, and only 17% of its activity was verified after 60 min of incubation, while at 50 °C, the FTase retained 35% of its activity. After 155 min, both temperatures led to activity below 10% (Figure 4A).

Similar results were achieved by Yang et al. [46], who found that fructosyltransferase purified from A. niger was more stable at 40 °C for 60 min; at 50 °C, it retained 60% of its activity, and at 60 °C, the enzyme was not stable, with a rapid decline in its activity. The fructosyltransferase from A. aculeatus was stable at the temperatures of 25 to 50 °C; nevertheless, it decreased sharply when incubated at 60 °C and was completely inactivated when subjected to the temperature range from 65 °C to 70 °C for 60 min [40]. The invertase with transfructosylation activity was more stable in the temperature range from 25 to 30 °C for 60 min [37].

In this work, the transfructosylation activity of the invertase from A. carbonarius PC-4 was more stable at pH 5, reaching a maximum of 144.40% of its activity at 24 h of incubation and 156.134% of its activity when incubated at pH 8 for 7 h. This increase may be related to the ionization state of the enzyme, which, when incubated at pH 5 for 24 h and at pH 8 for 7 h, was better at stabilizing the protein conformation [47]. On the other hand, this fact may be related to the enzyme being present in the crude extract and being influenced by factors such as metal ions, the presence of other enzymes, cofactors, and others.

For the pH range of 3–5, only at pH 4 was the relative activity below 50%. This fact may be related to the pH influencing the kinetic parameters of the enzymatic reaction and being able to change the stability of the enzyme–substrate complex [48]. For the pH ranges 5–5.5 and 6.5–8, the activity was maintained above 90% (Figure 4B). According to Yang et al. [46], a fructosyltransferase from A. aculeatus reached the ideal stability at pH 6.0 and retained more than 90% of its activity in the pH range of 4.0–9.0 after 24 h of incubation. Furthermore, it also retained approximately 80% of activity in pH values below 2.0 or above 11.0, indicating that FTase is stable in a wide pH range, favoring its production, storage, and industrial applications.

4. Conclusions

This study demonstrates the cost-effective optimization of an invertase from A. carbonarius PC-4 with high transfructosylation activity using pineapple crown as a carbon inducer source and ammonium nitrate as an inorganic nitrogen source. The experimental design of this study allowed a 1.77-fold increase in the transfructosylation activity after 72 h of cultivation. However, this combination is not found in the literature and may serve as a model for further studies. A partial characterization of the enzyme in the crude extract showed that the reaction conditions of the enzyme for the maximum transfructosylation activity occurred at pH 5.0 and a temperature of 50 °C. The enzyme showed thermal stability at 40 °C after 1 h of incubation and was stable over the wide pH range of 3.0–8.0. These parameters are important for the development of a successful bioprocess, which may favor its production, storage, and industrial applications. Low-cost agro-industrial waste, in conjunction with ammonium nitrate, has the potential to be used in the production of invertases with the aim of optimizing their transfructosylation activity for application in the production of fructooligosaccharides.