Effect of temperature, pH and storage time on the stability of an extracellular fructosyltransferase from Aspergillus oryzae IPT-301

Efeito da temperatura, pH e tempo de armazenamento na estabilidade de frutosiltransferase extracelular de Aspergillus oryzae IPT-301

Maria Beatriz Pregnolato de Oliveira Silva Daniela Abdal José Pedro Zanetti Prado Giancarlo de Souza Dias Sergio Andres Villalba Morales Michelle da Cunha Abreu Xavier Alex Fernando de Almeida Elda Sabino da Silva Alfredo Eduardo Maiorano Rafael Firmani Perna About the authors

Abstract

In this work, it was determined the influence of temperature, pH and storage time on the enzymatic activity and stability of an extracellular fructosyltransferase (FTase E.C.2.4.1.9) from Aspergillus oryzae IPT-301 produced by submerged fermentation. The thermodynamic parameters showed a tendency for increasing enzyme denaturation with the rise in temperature. The maximum transfructosylation activity was obtained at the incubation pH 5.5. During storage at 4 °C, the transfructosylation activity decreased, whereas the hydrolytic activity increased, especially in the first nine hours, a time in which the enzyme presented 45.6% of its initial transfructosylation activity. These results contributed to the improvement of the conditions of storage, immobilization and use of the soluble fructosyltransferases (FTase) in fructooligosaccharide (FOS) production.

Keywords:
Submerged fermentation; Soluble enzyme; Enzyme stability; Thermodynamic parameters; Aspergillus; Transfructosylation activity

Resumo

Neste trabalho foi determinada a influência da temperatura, do pH e do tempo de armazenamento na estabilidade enzimática de frutosiltransferase (E.C.2.4.1.9) extracelular de Aspergillus oryzae IPT-301, produzida por fermentação submersa. Os parâmetros termodinâmicos mostraram tendência de aumento da desnaturação da enzima com o aumento da temperatura. A atividade de transfrutosilação máxima foi obtida para a enzima incubada em pH 5,5. Durante o armazenamento a 4 °C, a atividade de transfrutosilação diminuiu, enquanto a atividade hidrolítica aumentou, principalmente nas primeiras nove horas, período em que a enzima apresentou 45,6% de sua atividade inicial de transfructosilação. Esses resultados contribuem para a melhoria das condições de armazenamento, imobilização e utilização das frutosiltransferases solúveis na produção de frutooligossacarídeos.

Palavras-chave:
Fermentação submersa; Enzima solúvel; Estabilidade enzimática; Parâmetros termodinâmicos; Aspergillus; Atividade de transfrutosilação

1 Introduction

The enzyme fructosyltransferase (FTase E.C.2.4.1.9) catalyzes the reaction of sucrose transfructosylation for the production of fructooligosaccharides (FOS), fructose oligomers with degree of polymerization between two and 20, whose fructosyl units are linked to the terminal sucrose molecule by glycosidic bonds at the β-(2→1) position (Faria et al., 2021Faria, L. L., Morales, S. A. V., Prado, J. P. Z., Dias, G. S., Almeida, A. F., Xavier, M. C. A., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides. Biotechnology Letters, 43(1), 43-59. PMid:33025334. http://dx.doi.org/10.1007/s10529-020-03016-7
http://dx.doi.org/10.1007/s10529-020-030...
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http://dx.doi.org/10.1016/0141-0229(95)0...
). The FOS are functional sugars with major importance for the production of foods and pharmaceuticals, since they offer several benefits to human health. Indeed, the FOS present from 40 to 60% of the sweetening power of sucrose, being low in calories and presenting low glycemic index, as well as prebiotic activity. They participate in the inhibition of putrefactive substances, reduction of pathogenic microorganisms and the balance of the intestinal flora, thus helping to prevent colon cancer (Andersson et al., 1999Andersson, H. B., Ellegard, L. H., & Bosaeus, I. G. (1999). Nondigestibility characteristics of inulin and oligofructose in humans. The Journal of Nutrition, 129(7), 1428S-1430S. PMid:10395612. http://dx.doi.org/10.1093/jn/129.7.1428S
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; Marx et al., 2000Marx, S. P., Winkler, S., & Hartmeier, W. (2000). Metabolization of β-(2,6)-linked fructose-oligosaccharides by different bifidobacteria. FEMS Microbiology Letters, 182(1), 163-169. PMid:10612749. http://dx.doi.org/10.1016/S0378-1097(99)00584-4
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; Nobre et al., 2018Nobre, C., Alves Filho, E. G., Fernandes, F. A. N., Brito, E. S., Rodrigues, S., Teixeira, J. A., & Rodrigues, L. R. (2018). Production of fructo-oligosaccharides by Aspergillus ibericus and their chemical characterization. LWT, 89, 58-64. http://dx.doi.org/10.1016/j.lwt.2017.10.015
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; Rivero-Urgell & Santamaria-Orleans, 2001Rivero-Urgell, M., & Santamaria-Orleans, A. (2001). Oligosaccharides: Application in infant food. Early Human Development, 65(Suppl. 2), S43-S52. PMid:11755034. http://dx.doi.org/10.1016/S0378-3782(01)00202-X
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; Saulnier et al., 2007Saulnier, D. M., Molenaar, D., Vos, W. M., Gibson, G. R., & Kolida, S. (2007). Identification of prebiotic fructooligosaccharide metabolism in Lactobacillus plantarum WCFS1 through microarrays. Applied and Environmental Microbiology, 73(6), 1753-1765. PMid:17261521. http://dx.doi.org/10.1128/AEM.01151-06
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; Yun, 1996Yun, J. W. (1996). Fructooligosaccharides: Occurrence, preparation and application. Enzyme and Microbial Technology, 19(2), 107-117. http://dx.doi.org/10.1016/0141-0229(95)00188-3
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).

The industrial FOS production depends on the efficient and cheap production of FTases, as well as their enzymatic activities and their resistance to denaturation during the enzymatic reaction and during storage (Mutanda et al., 2014Mutanda, T., Mokoena, M. P., Olaniran, A. O., Wilhelmi, B. S., & Whiteley, C. G. (2014). Microbial enzymatic production and applications of short‑chain fructooligosaccharides and inulooligosaccharides: Recent advances and current perspectives. Journal of Industrial Microbiology & Biotechnology, 41(6), 893-906. PMid:24793124. http://dx.doi.org/10.1007/s10295-014-1452-1
http://dx.doi.org/10.1007/s10295-014-145...
; Yun, 1996Yun, J. W. (1996). Fructooligosaccharides: Occurrence, preparation and application. Enzyme and Microbial Technology, 19(2), 107-117. http://dx.doi.org/10.1016/0141-0229(95)00188-3
http://dx.doi.org/10.1016/0141-0229(95)0...
). FTases can be produced by submerged fermentation using, mainly, fungi from the species Aspergillus niger, A. oryzae, Rhodoturola diarensis and Cryptococcus sp., and they can be used either in the soluble or immobilized forms for FOS production (Antošová & Polakovič, 2001Antošová, M., & Polakovič, M. (2001). Fructosyltransferase: The enzymes catalyzing production of fructooligosaccharides. Chemical papers-slovak Academy of Sciences, 55(6), 350-358. Retrieved in 2020, December 11, from https://www.chempap.org/file_access.php?file=556a350.pdf
https://www.chempap.org/file_access.php?...
; Cuervo-Fernandez et al., 2007Cuervo-Fernandez, R., Ottoni, C. A., Silva, E. S., Matsubara, R. M. S., Carter, J. M., Magossi, L. R., Wada, M. A., Rodrigues, M. F. A., Maresma, B. G., & Maiorano, A. E. (2007). Screening of β-fructofuranosidase-producing microorganisms and effect of pH and temperature on enzymatic rate. ‎. Applied Microbiology and Biotechnology, 75(1), 87-93. PMid:17375295. http://dx.doi.org/10.1007/s00253-006-0803-x
http://dx.doi.org/10.1007/s00253-006-080...
; Ganaie & Gupta, 2014Ganaie, M. A., & Gupta, U. S. (2014). Recycling of cell culture and efficient release of intracelular fructosyltransferase by ultrasonication for the production of fructooligosaccharides. Carbohydrate Polymers, 110, 253-258. PMid:24906753. http://dx.doi.org/10.1016/j.carbpol.2014.03.066
http://dx.doi.org/10.1016/j.carbpol.2014...
; Huang et al., 2016Huang, M. P., Wu, M., Xu, Q. S., Mo, D. J., & Feng, J. X. (2016). Highly efficient synthesis of fructooligosaccharides by extracellular fructooligosaccharide-producing enzymes and immobilized cells of Aspergillus aculeatus M105 and purification and biochemical characterization of a fructosyltransferase from the fungus. Journal of Agricultural and Food Chemistry, 64(33), 6425-6432. PMid:27492129. http://dx.doi.org/10.1021/acs.jafc.6b02115
http://dx.doi.org/10.1021/acs.jafc.6b021...
; Mutanda et al., 2014Mutanda, T., Mokoena, M. P., Olaniran, A. O., Wilhelmi, B. S., & Whiteley, C. G. (2014). Microbial enzymatic production and applications of short‑chain fructooligosaccharides and inulooligosaccharides: Recent advances and current perspectives. Journal of Industrial Microbiology & Biotechnology, 41(6), 893-906. PMid:24793124. http://dx.doi.org/10.1007/s10295-014-1452-1
http://dx.doi.org/10.1007/s10295-014-145...
). Specifically, the fungus A. oryzae IPT-301 is one of the main producers of extracellular and mycelial FTases which present high transfructosylation activity and low hydrolytic activity (Cuervo-Fernandez et al., 2007Cuervo-Fernandez, R., Ottoni, C. A., Silva, E. S., Matsubara, R. M. S., Carter, J. M., Magossi, L. R., Wada, M. A., Rodrigues, M. F. A., Maresma, B. G., & Maiorano, A. E. (2007). Screening of β-fructofuranosidase-producing microorganisms and effect of pH and temperature on enzymatic rate. ‎. Applied Microbiology and Biotechnology, 75(1), 87-93. PMid:17375295. http://dx.doi.org/10.1007/s00253-006-0803-x
http://dx.doi.org/10.1007/s00253-006-080...
). In the reaction of sucrose transfructosylation, the high ratios between the transfructosylation and hydrolytic activities (AT/AH) is an essential indicator of the predominance of the transfructosylation reaction over the hydrolytic reaction, and consequently, of higher FOS production (Cuervo-Fernandez et al., 2007Cuervo-Fernandez, R., Ottoni, C. A., Silva, E. S., Matsubara, R. M. S., Carter, J. M., Magossi, L. R., Wada, M. A., Rodrigues, M. F. A., Maresma, B. G., & Maiorano, A. E. (2007). Screening of β-fructofuranosidase-producing microorganisms and effect of pH and temperature on enzymatic rate. ‎. Applied Microbiology and Biotechnology, 75(1), 87-93. PMid:17375295. http://dx.doi.org/10.1007/s00253-006-0803-x
http://dx.doi.org/10.1007/s00253-006-080...
; Ghazi et al., 2007Ghazi, I., Fernandez-Arrojo, L., Garcia-Arellano, H., Ferrer, M., Ballesteros, A., & Plou, F. J. (2007). Purification and kinetic characterization of a fructosyltransferase from Aspergillus aculeatus. Journal of Biotechnology, 128(1), 204-211. PMid:17056145. http://dx.doi.org/10.1016/j.jbiotec.2006.09.017
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).

Several studies have reported optimal parameters for the production of FTase from A. oryzae IPT-301 and the behavior of its enzymatic activity according to pH, temperature and sucrose concentration (Cunha et al., 2019Cunha, J. S., Ottoni, C. A., Morales, S. A. V., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2019). Synthesis and characterization of fructosyltransferase from Aspergillus oryzae IPT-301 for high fructooligosaccharides production. Brazilian Journal of Chemical Engineering, 36(2), 657-668. http://dx.doi.org/10.1590/0104-6632.20190362s20180572
http://dx.doi.org/10.1590/0104-6632.2019...
, Ottoni et al., 2012Ottoni, C. A., Cuervo-Fernandez, R., Piccoli, R. M., Moreira, R., Guilarte-Maresma, B., Silva, E. S., Rodrigues, M. F. D. A., & Maiorano, A. E. (2012). Media optimization for b-fructofuranosidase production by Aspergillus oryzae. Brazilian Journal of Chemical Engineering, 29(1), 49-59. http://dx.doi.org/10.1590/S0104-66322012000100006
http://dx.doi.org/10.1590/S0104-66322012...
; Perna et al., 2018Perna, R. F., Cunha, J. S., Gonçalves, M. C. P., Basso, R. C., Silva, E. S., & Maiorano, A. E. (2018). Microbial fructosyltransferase: Production by submerged fermentation and evaluation of pH and temperature effects on transfructosylation and hydrolytic enzymatic activities. International Journal of Engineering Reasearch & Science, 4(3), 43-50. http://dx.doi.org/10.5281/zenodo.1213538
https://doi.org/ ...
). Furthermore, Faria et al. (2021)Faria, L. L., Morales, S. A. V., Prado, J. P. Z., Dias, G. S., Almeida, A. F., Xavier, M. C. A., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides. Biotechnology Letters, 43(1), 43-59. PMid:33025334. http://dx.doi.org/10.1007/s10529-020-03016-7
http://dx.doi.org/10.1007/s10529-020-030...
and Gonçalves et al. (2020)Gonçalves, M. C. P., Morales, S. A. V., Silva, E. S., Maiorano, A. E., Perna, R. F., & Kieckbusch, T. G. (2020). Entrapment of glutaraldehyde-crosslinked cells from Aspergillus oryzae IPT-301 in calcium alginate for high transfructosylation activity. Journal of Chemical Technology & Biotechnology, 95(9), 2473-2482. http://dx.doi.org/10.1002/jctb.6429
http://dx.doi.org/10.1002/jctb.6429...
reported the enzymatic activity and thermal and operational stability of A. oryzae IPT-301 FTases immobilized by adsorption and encapsulation, respectively. Similarly, several authors have reported optimal pH and temperature values to maximize the enzymatic activity of extracellular FTases from Rhodotorula sp. used in the free form, as well as the thermal stability of this enzyme immobilized by adsorption in Niobium ore (Aguiar-Oliveira & Maugeri, 2010Aguiar-Oliveira, E., & Maugeri, F. (2010). Characterization of the immobilized fructosyltranferase from Rhodotorula sp. International Journal of Food Engineering, 6(3), http://dx.doi.org/10.2202/1556-3758.1894
http://dx.doi.org/10.2202/1556-3758.1894...
; Hernalsteens & Maugeri, 2008Hernalsteens, S., & Maugeri, F. (2008). Purification and characterization of a fructosyltransferase from Rhodotorula sp. Applied Microbiology and Biotechnology, 79(4), 589-596. PMid:18481059. http://dx.doi.org/10.1007/s00253-008-1470-x
http://dx.doi.org/10.1007/s00253-008-147...
).

Nevertheless, FTases in free form loose enzymatic activity rapidly by thermal denaturation as storage time increases which limits its use in FOS production. Hence, the storage step is crucial to maintain the catalytic site and enzyme conformation of FTases close to its initial state and then to extend the enzyme lifetime (Shuler & Kargi, 2002Shuler, M. L., & Kargi, F. (2002). Bioprocess enginnering: Basic concepts (2nd ed.). Hoboken, NJ: Prentice Hall PTR.). Several authors reported that the stability of free FTases during storage depends mainly on the producer microorganism and storage medium parameters such as composition, pH and temperature (Park et al., 2001Park, J. P., Oh, T. K., & Yun, J. W. (2001). Purification and characterization of a novel transfructosylating enzyme from Bacillus macerans EG-6. Process Biochemistry, 37(5), 471-476. http://dx.doi.org/10.1016/S0032-9592(01)00237-0
http://dx.doi.org/10.1016/S0032-9592(01)...
; Hernalsteens & Maugeri, 2008Hernalsteens, S., & Maugeri, F. (2008). Purification and characterization of a fructosyltransferase from Rhodotorula sp. Applied Microbiology and Biotechnology, 79(4), 589-596. PMid:18481059. http://dx.doi.org/10.1007/s00253-008-1470-x
http://dx.doi.org/10.1007/s00253-008-147...
; Yang et al., 2016Yang, H., Wang, Y., Zhang, L., & Shen, W. (2016). Heterologous expression and enzymatic characterization of fructosyltransferase from Aspergillus niger in Pichia pastoris. New Biotechnology, 33(1), 164-170. PMid:25976629. http://dx.doi.org/10.1016/j.nbt.2015.04.005
http://dx.doi.org/10.1016/j.nbt.2015.04....
).

The stability of free enzymes during storage is an important information which allows to analyze its potential application in enzymatic process, to evaluate the necessity of applying an immobilization process, and also to evaluate the effect of immobilization processes in storage stability. However, until the performance of this study, no information has been reported on the stability of the extracellular FTase from A. oryzae IPT-301 in the free form during storage, a characteristic indicated among the main limitations of the use of free enzymes in industrial processes. In this context, the present work aimed at determining the effect of temperature, pH and storage time on the stability of the extracellular FTase from A. oryzae IPT-301 used in its soluble form.

2 Materials and methods

2.1 Microorganism and production of the extracellular enzyme

Strains of the fungus A. oryzae IPT-301 were provided by the Institute for Technological Research (Instituto de Pesquisas Tecnológicas/São Paulo (IPT/SP)) and used to produce extracellular FTase. Enzyme production was performed by submerged fermentation in Erlenmeyer flasks (250 mL) containing 50 mL of culture medium with the following composition (in %, m v-1): sucrose 15.0, yeast extract 0.5, NaNO3 0.5, KH2PO4 0.2, MgSO4.7H2O 0.05, MnCl2.4H2O 0.03 and FeSO4.7H2O 0.001. The initial pH of the culture medium was adjusted to 5.5 before sterilization. The flasks were inoculated with 0.5 mL of a previously prepared suspension of 107 spores mL-1, and incubated in orbital shaker at 30 ºC, 200 rpm for 64 h (Cunha et al., 2019Cunha, J. S., Ottoni, C. A., Morales, S. A. V., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2019). Synthesis and characterization of fructosyltransferase from Aspergillus oryzae IPT-301 for high fructooligosaccharides production. Brazilian Journal of Chemical Engineering, 36(2), 657-668. http://dx.doi.org/10.1590/0104-6632.20190362s20180572
http://dx.doi.org/10.1590/0104-6632.2019...
; Faria et al., 2021Faria, L. L., Morales, S. A. V., Prado, J. P. Z., Dias, G. S., Almeida, A. F., Xavier, M. C. A., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides. Biotechnology Letters, 43(1), 43-59. PMid:33025334. http://dx.doi.org/10.1007/s10529-020-03016-7
http://dx.doi.org/10.1007/s10529-020-030...
). At the end of fermentation, the culture broth was vacuum-filtered (Whatman nº1) and the resulting supernatant containing an activity of 12.34 ± 0.23 U mL-1 for the FTase from A. oryzae IPT-301 was employed for further studies.

2.2 Thermal stability assays

The enzyme FTase (culture broth) was incubated in tris-acetate buffer at 0.2 mol L-1 and pH 5.5, in the absence of substrate, in a broad temperature range (30 ºC, 40 ºC, 50 ºC and 60 ºC). The samples were collected at specified time intervals (1 h, 2 h, 4 h and 8 h) and immediately cooled in ice bath for 5 min, and the residual activity was determined under standard conditions, according to Ottoni et al. (2012)Ottoni, C. A., Cuervo-Fernandez, R., Piccoli, R. M., Moreira, R., Guilarte-Maresma, B., Silva, E. S., Rodrigues, M. F. D. A., & Maiorano, A. E. (2012). Media optimization for b-fructofuranosidase production by Aspergillus oryzae. Brazilian Journal of Chemical Engineering, 29(1), 49-59. http://dx.doi.org/10.1590/S0104-66322012000100006
http://dx.doi.org/10.1590/S0104-66322012...
, Cunha et al. (2019)Cunha, J. S., Ottoni, C. A., Morales, S. A. V., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2019). Synthesis and characterization of fructosyltransferase from Aspergillus oryzae IPT-301 for high fructooligosaccharides production. Brazilian Journal of Chemical Engineering, 36(2), 657-668. http://dx.doi.org/10.1590/0104-6632.20190362s20180572
http://dx.doi.org/10.1590/0104-6632.2019...
and Faria et al. (2021)Faria, L. L., Morales, S. A. V., Prado, J. P. Z., Dias, G. S., Almeida, A. F., Xavier, M. C. A., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides. Biotechnology Letters, 43(1), 43-59. PMid:33025334. http://dx.doi.org/10.1007/s10529-020-03016-7
http://dx.doi.org/10.1007/s10529-020-030...
. The experiments were performed in triplicate.

The first-order thermal denaturation constant (kD, in min-1) was estimated adjusting the model of Sadana & Henley (1987)Sadana, A., & Henley, J. P. (1987). Single-step unimolecular non-first-order enzyme deactivation kinetics. Biotechnology and Bioengineering, 30(6), 717-723. PMid:18581490. http://dx.doi.org/10.1002/bit.260300604
http://dx.doi.org/10.1002/bit.260300604...
to the experimental data of residual activities versus time (Equation 1):

A A 0 = 1 α exp k D t + α (1)

in which (A/A0) is the dimensionless residual activity; α is the ratio between the enzymatic activity of the final state (A) and the enzymatic activity of the initial state (A0); and t is the incubation time of the enzymatic solution (min).

The half-life (t1/2, in min) of the enzyme was calculated using Equation 2 (Perna et al., 2017Perna, R. F., Tiosso, P. C., Sgobi, L. M., Vieira, M. A. S., Vieira, M. F., Tardioli, P. W., Soares, C. M. F., & Zanin, G. M. (2017). Effects of triton X-100 and PEG on the catalytic properties and thermal stability of lipase from Candida rugosa free and immobilized on glyoxyl-agarose. The Open Biochemistry Journal, 11(1), 66-76. PMid:29290831. http://dx.doi.org/10.2174/1874091X01711010066
http://dx.doi.org/10.2174/1874091X017110...
):

t 1 / 2 = 1 k D ln 1 2 α 1 α (2)

The activation energy for thermal denaturation (ED) was determined using the linearized Arrhenius Equation, as shown in Equation 3.

ln k D = ln A E D R 1 T (3)

in which A is the Arrhenius frequency of the collision factor, R is the universal gas constant (8.314 J mol-1 K-1) and T is the absolute temperature (K).

On the other hand, the variations of the enthalpy of activation of denaturation (∆HD), Gibbs energy of activation of denaturation (∆GD) and entropy of activation of denaturation (∆SD) were calculated using Equation 4, 5 and 6, respectively (Faria et al., 2021Faria, L. L., Morales, S. A. V., Prado, J. P. Z., Dias, G. S., Almeida, A. F., Xavier, M. C. A., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides. Biotechnology Letters, 43(1), 43-59. PMid:33025334. http://dx.doi.org/10.1007/s10529-020-03016-7
http://dx.doi.org/10.1007/s10529-020-030...
; Gonçalves et al., 2020Gonçalves, M. C. P., Morales, S. A. V., Silva, E. S., Maiorano, A. E., Perna, R. F., & Kieckbusch, T. G. (2020). Entrapment of glutaraldehyde-crosslinked cells from Aspergillus oryzae IPT-301 in calcium alginate for high transfructosylation activity. Journal of Chemical Technology & Biotechnology, 95(9), 2473-2482. http://dx.doi.org/10.1002/jctb.6429
http://dx.doi.org/10.1002/jctb.6429...
; Saqib et al., 2010Saqib, A. A. N., Hassan, M., Khan, N. F., & Baig, S. (2010). Thermostability of crude endoglucanase from Aspergillus fumigatus grown under solid state fermentation (SSF) and submerged fermentation (SmF). Process Biochemistry, 45(5), 641-646. http://dx.doi.org/10.1016/j.procbio.2009.12.011
http://dx.doi.org/10.1016/j.procbio.2009...
; Souza et al., 2015Souza, P. M., Aliakbarian, B., Filho, E. X. F., Magalhães, P. O., Junior, A. P., Converti, A., & Perego, P. (2015). Kinetic and thermodynamic studies of a novel acid protease from Aspergillus foetidus. International Journal of Biological Macromolecules, 81, 17-21. PMid:26210038. http://dx.doi.org/10.1016/j.ijbiomac.2015.07.043
http://dx.doi.org/10.1016/j.ijbiomac.201...
).

Δ H D = E D R . T (4)
Δ G D = R . T . ln k D . h k b . T (5)
Δ S D = Δ H D Δ G D T (6)

in which T is the experimental absolute temperature (K), R is the universal gas constant (8.314 J mol-1 K-1), h is the Planck constant (11.04 x 10-36 J min-1) and kb is the Boltzmann constant (1.38 x 10-23 J K-1).

2.3 pH stability assays

Stability at different pH values was determined by the incubation of the FTase in tris-acetate buffer at 0.2 mol L-1 for a pH interval of 3.5-8.5, in the absence of substrate, at 4 ºC, for 24 h, and measuring the residual activity under standard conditions. The experiments were performed in triplicate.

2.4 Storage stability assays

The assays were performed with the extracellular FTase for different storage periods (1 h, 2 h, 3 h, 5 h, 7 h, 9 h, 24 h, 18 h, 72 h and 96 h). For this, the culture broth was maintained under refrigeration at 4 ºC and the transfructosylation (AT) and hydrolytic (AH) activities were monitored under standard conditions. The experiments were performed in triplicate.

2.5 Analytical methods

2.5.1 Enzymatic activity assays

The transfructosylation (AT) and hydrolytic (AH) activities were determined according to Ottoni et al. (2012)Ottoni, C. A., Cuervo-Fernandez, R., Piccoli, R. M., Moreira, R., Guilarte-Maresma, B., Silva, E. S., Rodrigues, M. F. D. A., & Maiorano, A. E. (2012). Media optimization for b-fructofuranosidase production by Aspergillus oryzae. Brazilian Journal of Chemical Engineering, 29(1), 49-59. http://dx.doi.org/10.1590/S0104-66322012000100006
http://dx.doi.org/10.1590/S0104-66322012...
, Cunha et al. (2019)Cunha, J. S., Ottoni, C. A., Morales, S. A. V., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2019). Synthesis and characterization of fructosyltransferase from Aspergillus oryzae IPT-301 for high fructooligosaccharides production. Brazilian Journal of Chemical Engineering, 36(2), 657-668. http://dx.doi.org/10.1590/0104-6632.20190362s20180572
http://dx.doi.org/10.1590/0104-6632.2019...
and Faria et al. (2021)Faria, L. L., Morales, S. A. V., Prado, J. P. Z., Dias, G. S., Almeida, A. F., Xavier, M. C. A., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides. Biotechnology Letters, 43(1), 43-59. PMid:33025334. http://dx.doi.org/10.1007/s10529-020-03016-7
http://dx.doi.org/10.1007/s10529-020-030...
. For this, 0.1 mL of the soluble enzyme (culture broth) was incubated with 3.7 mL of sucrose solution (47%, m v-1) and 1.2 mL of tris-acetate buffer at 0.2 mol L-1, at pH 5.5. The reaction was conducted in Dubnoff bath at 50 ºC, 190 rpm for 1 h and interrupted by the immersion of the reaction medium in boiling water for 10 min, followed by cooling in water and ice bath for 5 min for enzyme inactivation. Subsequently, the reaction medium was vacuum-filtered and the concentration of reducing sugars and glucose was quantified. One unit (1 U) of transfructosylation activity was defined as the amount of enzyme that transfers one micromol (1 µmol) of fructose per minute, under the established experimental conditions. One unit (1 U) of hydrolytic activity was defined as the amount of enzyme which releases one micromol (1 µmol) of fructose per minute, under the established experimental conditions (Cunha et al., 2019Cunha, J. S., Ottoni, C. A., Morales, S. A. V., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2019). Synthesis and characterization of fructosyltransferase from Aspergillus oryzae IPT-301 for high fructooligosaccharides production. Brazilian Journal of Chemical Engineering, 36(2), 657-668. http://dx.doi.org/10.1590/0104-6632.20190362s20180572
http://dx.doi.org/10.1590/0104-6632.2019...
; Gonçalves et al., 2020Gonçalves, M. C. P., Morales, S. A. V., Silva, E. S., Maiorano, A. E., Perna, R. F., & Kieckbusch, T. G. (2020). Entrapment of glutaraldehyde-crosslinked cells from Aspergillus oryzae IPT-301 in calcium alginate for high transfructosylation activity. Journal of Chemical Technology & Biotechnology, 95(9), 2473-2482. http://dx.doi.org/10.1002/jctb.6429
http://dx.doi.org/10.1002/jctb.6429...
; Faria et al., 2021Faria, L. L., Morales, S. A. V., Prado, J. P. Z., Dias, G. S., Almeida, A. F., Xavier, M. C. A., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides. Biotechnology Letters, 43(1), 43-59. PMid:33025334. http://dx.doi.org/10.1007/s10529-020-03016-7
http://dx.doi.org/10.1007/s10529-020-030...
).

2.5.2 Analysis of carbohydrates

The concentrations of glucose (G) and reducing sugars (RS) were quantified by the colorimetric methods GOD/PAP® (glucose-oxidase enzyme kit) and DNS (3,5 dinitrosalicylic acid) (Miller, 1959Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3), 426-428. http://dx.doi.org/10.1021/ac60147a030
http://dx.doi.org/10.1021/ac60147a030...
), respectively. The concentrations of released fructose (F) and transferred fructose (FT) in the reaction medium were determined by Equation 7 and 8 (Chen & Liu, 1996Chen, W. C, & Liu, C. H. (1996). Production of β-fructofuranosidase by Aspergillus japonicus. Enzyme and Microbial Technology, 18(2), 153-160. http://dx.doi.org/10.1016/0141-0229(95)00099-2
http://dx.doi.org/10.1016/0141-0229(95)0...
, Cunha et al., 2019Cunha, J. S., Ottoni, C. A., Morales, S. A. V., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2019). Synthesis and characterization of fructosyltransferase from Aspergillus oryzae IPT-301 for high fructooligosaccharides production. Brazilian Journal of Chemical Engineering, 36(2), 657-668. http://dx.doi.org/10.1590/0104-6632.20190362s20180572
http://dx.doi.org/10.1590/0104-6632.2019...
; Gonçalves et al., 2020Gonçalves, M. C. P., Morales, S. A. V., Silva, E. S., Maiorano, A. E., Perna, R. F., & Kieckbusch, T. G. (2020). Entrapment of glutaraldehyde-crosslinked cells from Aspergillus oryzae IPT-301 in calcium alginate for high transfructosylation activity. Journal of Chemical Technology & Biotechnology, 95(9), 2473-2482. http://dx.doi.org/10.1002/jctb.6429
http://dx.doi.org/10.1002/jctb.6429...
; Faria et al., 2021Faria, L. L., Morales, S. A. V., Prado, J. P. Z., Dias, G. S., Almeida, A. F., Xavier, M. C. A., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides. Biotechnology Letters, 43(1), 43-59. PMid:33025334. http://dx.doi.org/10.1007/s10529-020-03016-7
http://dx.doi.org/10.1007/s10529-020-030...
).

F = R S G (7)
F T = G F (8)

3 Results and discussion

3.1 Determination of thermodynamic parameters from thermal stability tests

The thermal stability analysis of the extracellular FTase was performed at a temperature range between 30 ºC and 60 ºC over an incubation period of 8 h. Figure 1 shows the relative transfructosylation activities after thermal pretreatment of the extracellular FTase. The maximum enzyme activity (initial time) was of 13.15 ± 1.47 U mL-1 (relative activity, 100%). The highest retention of AT was observed for the enzyme incubated at 30 ºC, whose residual activity was of approximately 80% (10.17 ± 0.94 U mL-1) after 8 h of incubation. For the same period, it was also observed that the FTase presented residual activity around 40% (5.26 ± 1.71 U mL-1), 38% (5.12 ± 1.33 U mL-1) and 27% (3.55 ± 0.91 U mL-1) for the temperatures of 40 ºC, 50 ºC and 60 ºC, respectively.

Figure 1
Thermal stability at different temperatures (30 ºC, 40 ºC, 50 ºC and 60 ºC) and pH 5.5 for soluble extracellular FTase from Aspergillus oryzae IPT-301. Reactions conditions: 47% (w v-1) sucrose solution, 190 rpm, 0,2 mol L-1 of tris-acetate buffer, pH 5.5, and 50 ºC. The maximum enzyme activity (initial time) was of 13.15 ± 1.45 U mL-1 (relative activity, 100%).

Faria et al. (2021)Faria, L. L., Morales, S. A. V., Prado, J. P. Z., Dias, G. S., Almeida, A. F., Xavier, M. C. A., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides. Biotechnology Letters, 43(1), 43-59. PMid:33025334. http://dx.doi.org/10.1007/s10529-020-03016-7
http://dx.doi.org/10.1007/s10529-020-030...
reported that the extracellular enzyme of A. oryzae IPT-301 showed thermostability with AT retention higher than 70% after 17 h of incubation at 30 ºC. The results obtained by Cunha et al. (2019)Cunha, J. S., Ottoni, C. A., Morales, S. A. V., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2019). Synthesis and characterization of fructosyltransferase from Aspergillus oryzae IPT-301 for high fructooligosaccharides production. Brazilian Journal of Chemical Engineering, 36(2), 657-668. http://dx.doi.org/10.1590/0104-6632.20190362s20180572
http://dx.doi.org/10.1590/0104-6632.2019...
for the FTase produced by the same microorganism showed that thermal stability occurred only for temperatures below 35 ºC, reaching 96% of AT retention after 1 h of incubation. For temperatures above 40 ºC, a marked reduction in enzymatic activity was observed, with only 44.26% of the initial activity retained at 65 ºC. Han et al. (2020)Han, S., Ye, T., Leng, S., Pan, L., Zeng, W., Chen, G., & Liang, Z. (2020). Purification and biochemical characteristics of a novel fructosyltransferase with a high FOS transfructosylation activity from Aspergillus oryzae S719. Protein Expression and Purification, 167, 105549. PMid:31805395. http://dx.doi.org/10.1016/j.pep.2019.105549
http://dx.doi.org/10.1016/j.pep.2019.105...
showed that the FTase from A. oryzae S719 remained stable, presenting AT retention higher than 80% at 25 ºC after 12 h of enzyme incubation in acetate buffer at 0.2 mol L-1 and pH 5.5. Xu et al. (2015)Xu, Q., Zheng, X., Huang, M., Wu, M., Yan, Y., Pan, J., Yang, Q., Duan, C. J., Liu, J. L., & Feng, J. X. (2015). Purification and biochemical characterization of a novelfructofuranosidase from Penicillium oxalicum with transfructosylating activity producing neokestose. Process Biochemistry, 50(8), 1237-1246. http://dx.doi.org/10.1016/j.procbio.2015.04.020
http://dx.doi.org/10.1016/j.procbio.2015...
reported that the FTase produced by Penicillium oxalicum retained 80% of AT for a temperature range between 25 ºC and 55 ºC. Finally, Madlová et al. (2000)Madlová, A., Antošová, M., Polakovič, M., & Báleš, V. (2000). Thermal stability of fructosyltransferase from Aureobasidium pullulans. Chemical Papers, 54(6a), 339-344. Retrieved in 2020, December 11, from https://www.chempap.org/?id=7&paper=431
https://www.chempap.org/?id=7&paper=431...
demonstrated that Aureobasidium pullulans FTase was rapidly inactivated when incubated at temperatures higher than 60 ºC.

The parameters used for the determination of the thermal stability of the enzyme were calculated from the thermal inactivation curves (Figure 1) and their values are shown in Table 1. The thermal inactivation constant (kD) was estimated according to the nonlinear decay model proposed by Sadana & Henley (1987)Sadana, A., & Henley, J. P. (1987). Single-step unimolecular non-first-order enzyme deactivation kinetics. Biotechnology and Bioengineering, 30(6), 717-723. PMid:18581490. http://dx.doi.org/10.1002/bit.260300604
http://dx.doi.org/10.1002/bit.260300604...
(Figure 1) and the activation energy for thermal denaturation (ED) of the enzyme was obtained by linear adjustment of the Arrhenius Equation (Figure 2).

Table 1
Thermodynamic parameters of extracellular FTase from A. oryzae IPT-301 incubated at different temperatures.
Figure 2
Determination of the activation energy for thermal denaturation (ED). Arrhenius plot of ln (kD) versus (1/T) was used for the ED of soluble extracellular FTase from Aspergillus oryzae IPT-301.

The half-life (t1/2) is defined as the time necessary for the enzymatic activity to decrease around 50% from its initial activity at a given temperature (Saqib et al., 2010Saqib, A. A. N., Hassan, M., Khan, N. F., & Baig, S. (2010). Thermostability of crude endoglucanase from Aspergillus fumigatus grown under solid state fermentation (SSF) and submerged fermentation (SmF). Process Biochemistry, 45(5), 641-646. http://dx.doi.org/10.1016/j.procbio.2009.12.011
http://dx.doi.org/10.1016/j.procbio.2009...
). Thus, higher half-life values indicate that the biocatalyst can withstand the thermal effect for a longer period and, therefore, present higher thermostability (Griffin et al., 1984Griffin, H., Dintzis, F. R., Krull, L., & Baker, F. L. (1984). A microfibril generating factor from the enzyme complex of Trichoderma reesei. Biotechnology and Bioengineering, 26(3), 296-300. PMid:18551740. http://dx.doi.org/10.1002/bit.260260315
http://dx.doi.org/10.1002/bit.260260315...
; Marangoni, 2003Marangoni, A. G. (2003). Enzyme kinetics: a modern approach. Hoboken, NJ: Wiley-Interscience.; Saqib et al., 2010Saqib, A. A. N., Hassan, M., Khan, N. F., & Baig, S. (2010). Thermostability of crude endoglucanase from Aspergillus fumigatus grown under solid state fermentation (SSF) and submerged fermentation (SmF). Process Biochemistry, 45(5), 641-646. http://dx.doi.org/10.1016/j.procbio.2009.12.011
http://dx.doi.org/10.1016/j.procbio.2009...
; Souza et al., 2015Souza, P. M., Aliakbarian, B., Filho, E. X. F., Magalhães, P. O., Junior, A. P., Converti, A., & Perego, P. (2015). Kinetic and thermodynamic studies of a novel acid protease from Aspergillus foetidus. International Journal of Biological Macromolecules, 81, 17-21. PMid:26210038. http://dx.doi.org/10.1016/j.ijbiomac.2015.07.043
http://dx.doi.org/10.1016/j.ijbiomac.201...
). The results presented in Table1 showed that the t1/2 of the extracellular FTase decreased progressively with the rise in temperature, leading to an irreversible denaturation of the biocatalyst. The half-life of the FTase incubated at 40 ºC (303.99 min) was around five times higher than when incubated at 60 ºC (63.59 min). A similar behavior was reported by Faria et al. (2021)Faria, L. L., Morales, S. A. V., Prado, J. P. Z., Dias, G. S., Almeida, A. F., Xavier, M. C. A., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides. Biotechnology Letters, 43(1), 43-59. PMid:33025334. http://dx.doi.org/10.1007/s10529-020-03016-7
http://dx.doi.org/10.1007/s10529-020-030...
, estimating half-life times for the extracellular FTase from A. oryzae IPT-301 at different incubation temperatures. The authors verified a reduction of approximately 30% in the biocatalyst half-life for a temperature range from 40 ºC to 60 ºC, proving the irreversible denaturation of the microbial FTase.

The activation energy of denaturation (ED) can be defined as the minimum amount of energy which must be provided to the soluble enzyme (native state) to be denatured (irreversible modification of the conformation). Therefore, high values of this parameter indicate higher enzyme thermostability (Ferreira et al., 2018Ferreira, M. M., Santiago, F. L. B., Silva, N. A. G., Luiz, J. H. H., Fernández-Lafuente, R., Mendes, A. A., & Hirata, D. B. (2018). Different strategies to immobilize lipase from Geotrichum candidum: Kinetic and thermodynamic studies. Process Biochemistry, 67, 55-63. http://dx.doi.org/10.1016/j.procbio.2018.01.028
http://dx.doi.org/10.1016/j.procbio.2018...
; Saqib et al., 2010Saqib, A. A. N., Hassan, M., Khan, N. F., & Baig, S. (2010). Thermostability of crude endoglucanase from Aspergillus fumigatus grown under solid state fermentation (SSF) and submerged fermentation (SmF). Process Biochemistry, 45(5), 641-646. http://dx.doi.org/10.1016/j.procbio.2009.12.011
http://dx.doi.org/10.1016/j.procbio.2009...
). According to Figure 2, the linear regression for the temperature range evaluated (30 ºC to 60 ºC) presented a good adjustment (R2 = 0.86) and the ED estimated was of 49.75 kJ mol-1, a value which is very close to that obtained by Faria et al. (2021)Faria, L. L., Morales, S. A. V., Prado, J. P. Z., Dias, G. S., Almeida, A. F., Xavier, M. C. A., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides. Biotechnology Letters, 43(1), 43-59. PMid:33025334. http://dx.doi.org/10.1007/s10529-020-03016-7
http://dx.doi.org/10.1007/s10529-020-030...
(39.8 kJ mol-1) for the FTase from A. oryzae IPT-301, proving the thermostability of the soluble enzyme.

The enthalpy of activation of denaturation (∆HD) is an important thermodynamic parameter associated to the total amount of energy necessary to denature the enzyme (Marangoni, 2003Marangoni, A. G. (2003). Enzyme kinetics: a modern approach. Hoboken, NJ: Wiley-Interscience.; Souza et al., 2015Souza, P. M., Aliakbarian, B., Filho, E. X. F., Magalhães, P. O., Junior, A. P., Converti, A., & Perego, P. (2015). Kinetic and thermodynamic studies of a novel acid protease from Aspergillus foetidus. International Journal of Biological Macromolecules, 81, 17-21. PMid:26210038. http://dx.doi.org/10.1016/j.ijbiomac.2015.07.043
http://dx.doi.org/10.1016/j.ijbiomac.201...
). For this parameter, Table 1 showed high and positive values (around 47 kJ mol-1) for the whole range of incubation temperatures investigated, thus indicating that the soluble FTase from A. oryzae IPT-301 presented a thermostable behavior. Close ∆HD values (around 37 kJ mol-1) were also obtained by Faria et al. (2021)Faria, L. L., Morales, S. A. V., Prado, J. P. Z., Dias, G. S., Almeida, A. F., Xavier, M. C. A., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides. Biotechnology Letters, 43(1), 43-59. PMid:33025334. http://dx.doi.org/10.1007/s10529-020-03016-7
http://dx.doi.org/10.1007/s10529-020-030...
when evaluating the biocatalyst produced by the same microorganism.

Gibbs energy of activation of denaturation (∆GD) is the most precise and reliable thermodynamic parameter to evaluate enzyme thermostability, in other words, its resistance to denaturation, since it includes the enthalpic and entropic contributions (Saqib et al., 2010Saqib, A. A. N., Hassan, M., Khan, N. F., & Baig, S. (2010). Thermostability of crude endoglucanase from Aspergillus fumigatus grown under solid state fermentation (SSF) and submerged fermentation (SmF). Process Biochemistry, 45(5), 641-646. http://dx.doi.org/10.1016/j.procbio.2009.12.011
http://dx.doi.org/10.1016/j.procbio.2009...
; Souza et al., 2015Souza, P. M., Aliakbarian, B., Filho, E. X. F., Magalhães, P. O., Junior, A. P., Converti, A., & Perego, P. (2015). Kinetic and thermodynamic studies of a novel acid protease from Aspergillus foetidus. International Journal of Biological Macromolecules, 81, 17-21. PMid:26210038. http://dx.doi.org/10.1016/j.ijbiomac.2015.07.043
http://dx.doi.org/10.1016/j.ijbiomac.201...
). According to Marangoni (2003)Marangoni, A. G. (2003). Enzyme kinetics: a modern approach. Hoboken, NJ: Wiley-Interscience. and Damodaran (2005)Damodaran, S. (2005). Protein: denaturation. In: Y. H. Hi (Ed.), Handbook of food science, technology and engineering, (1st ed., pp. 1–14). Boca Raton, FL: CRC Press., higher ∆GD values indicate higher enzyme thermostability, as shown in this work for the soluble FTase (Table 1). It is also noted that all ∆GD values obtained were positive, since, under the equilibrium condition, the concentration of the protein in the native state is higher than in the denatured state. Consequently, the transition from the native to the denatured states was not a spontaneous process (Fonseca et al., 2006Fonseca, L. C., Corrêa, N. C. R., Garrote-Filho, M. S., Cunha, C. C., & Penha-Silva, N. (2006). Effects of the solvent composition on the stability of proteins in aqueous solutions. Química Nova, 29(3), 543-548. http://dx.doi.org/10.1590/S0100-40422006000300024
http://dx.doi.org/10.1590/S0100-40422006...
; Gonçalves et al., 2020Gonçalves, M. C. P., Morales, S. A. V., Silva, E. S., Maiorano, A. E., Perna, R. F., & Kieckbusch, T. G. (2020). Entrapment of glutaraldehyde-crosslinked cells from Aspergillus oryzae IPT-301 in calcium alginate for high transfructosylation activity. Journal of Chemical Technology & Biotechnology, 95(9), 2473-2482. http://dx.doi.org/10.1002/jctb.6429
http://dx.doi.org/10.1002/jctb.6429...
).

Enzyme thermal denaturation causes the rupture of the protein structure and generates a disorganized system, leading to the rise in entropy. Therefore, denaturation depends on the entropy of activation of denaturation (∆SD) (Gonçalves et al., 2020Gonçalves, M. C. P., Morales, S. A. V., Silva, E. S., Maiorano, A. E., Perna, R. F., & Kieckbusch, T. G. (2020). Entrapment of glutaraldehyde-crosslinked cells from Aspergillus oryzae IPT-301 in calcium alginate for high transfructosylation activity. Journal of Chemical Technology & Biotechnology, 95(9), 2473-2482. http://dx.doi.org/10.1002/jctb.6429
http://dx.doi.org/10.1002/jctb.6429...
). This thermodynamic parameter expresses the amount of energy per degree of disorder involved in the transition from a native state to a denatured state of the biocatalyst (Marangoni, 2003Marangoni, A. G. (2003). Enzyme kinetics: a modern approach. Hoboken, NJ: Wiley-Interscience.; Souza et al., 2015Souza, P. M., Aliakbarian, B., Filho, E. X. F., Magalhães, P. O., Junior, A. P., Converti, A., & Perego, P. (2015). Kinetic and thermodynamic studies of a novel acid protease from Aspergillus foetidus. International Journal of Biological Macromolecules, 81, 17-21. PMid:26210038. http://dx.doi.org/10.1016/j.ijbiomac.2015.07.043
http://dx.doi.org/10.1016/j.ijbiomac.201...
). As observed in Table 1, negative ∆SD values were obtained for the soluble FTase at temperatures from 30 ºC to 60 ºC, indicating a more orderly FTase transition state (Rashid & Siddiqui 1998Rashid, M. H., & Siddiqui, K. S. (1998). Thermodynamic and kinetic study of stability of the native and chemically modified β-glucosidases fromAspergillus niger. Process Biochemistry, 33(2), 109-115. http://dx.doi.org/10.1016/S0032-9592(97)00036-8
http://dx.doi.org/10.1016/S0032-9592(97)...
). Furthermore, the negative ∆SD values can be explained by the rise in incubation temperature, which can weaken the polar interactions of the enzyme and strengthen its hydrophobic interactions (Siddiqui et al., 1997Siddiqui, K. S., Saqib, A. A. N., Rashid, M. H., & Rajoka, M. I. (1997). Thermostabilization of carboxymethylcellulase from Aspergillus niger by carboxyl group modification. Biotechnology Letters, 19(4), 325-330. http://dx.doi.org/10.1023/A:1018342632083
http://dx.doi.org/10.1023/A:101834263208...
; Faria et al., 2021Faria, L. L., Morales, S. A. V., Prado, J. P. Z., Dias, G. S., Almeida, A. F., Xavier, M. C. A., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides. Biotechnology Letters, 43(1), 43-59. PMid:33025334. http://dx.doi.org/10.1007/s10529-020-03016-7
http://dx.doi.org/10.1007/s10529-020-030...
).

3.2 Effect of pH on enzyme stability

Samples of the enzyme solution (culture broth) were incubated for 24 h at 4 ºC in six different tris-acetate buffer solutions, all at 0.2 mol L‑1, with pH values ranging from 3.5 to 8.5 (Figure 3). The enzyme FTase presented notably higher stability at pH 5.5, with AT equal to 8.87 ± 1.45 U mL-1, corresponding to a relative activity of 100%. At pH 6.5, a drop to 65% (5.78 ± 1.45 U mL-1) in relative activity was observed. Under extreme pH conditions (3.5 and 8.5), the relative activity was inferior to 25% (2.14 ± 1.18 U mL‑1), whereas for pH 4.5 and 7.5, residual activities around 40% (3.55 ± 1.95 U mL-1) were obtained. These results are in agreement with previous studies performed for the extracellular FTase of A. oryzae IPT-301, whose best stability profiles were verified for a pH range between 5.0 and 6.5 (Cunha et al., 2019Cunha, J. S., Ottoni, C. A., Morales, S. A. V., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2019). Synthesis and characterization of fructosyltransferase from Aspergillus oryzae IPT-301 for high fructooligosaccharides production. Brazilian Journal of Chemical Engineering, 36(2), 657-668. http://dx.doi.org/10.1590/0104-6632.20190362s20180572
http://dx.doi.org/10.1590/0104-6632.2019...
; Faria et al., 2021Faria, L. L., Morales, S. A. V., Prado, J. P. Z., Dias, G. S., Almeida, A. F., Xavier, M. C. A., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides. Biotechnology Letters, 43(1), 43-59. PMid:33025334. http://dx.doi.org/10.1007/s10529-020-03016-7
http://dx.doi.org/10.1007/s10529-020-030...
).

Figure 3
Stability of the soluble extracellular FTase after 24 h of incubation at 4 ºC in tris-acetate buffer solutions, at different pH values. Reaction conditions: 47% (w v-1) sucrose solution, 190 rpm, 0.2 mol L-1 of tris-acetate buffer, and 50 ºC. The maximum enzyme activity (8.87 ± 1.45 U mL-1) at pH 5.5 was defined as 100% of relative activity.

In general, enzymes are active at a limited pH range, because the enzyme has several ionizable groups, and changes in pH affect the catalytic site and enzyme conformation, essential for the maintenance of the catalytic capacity (Shuler & Kargi, 2002Shuler, M. L., & Kargi, F. (2002). Bioprocess enginnering: Basic concepts (2nd ed.). Hoboken, NJ: Prentice Hall PTR.). Park et al. (2001)Park, J. P., Oh, T. K., & Yun, J. W. (2001). Purification and characterization of a novel transfructosylating enzyme from Bacillus macerans EG-6. Process Biochemistry, 37(5), 471-476. http://dx.doi.org/10.1016/S0032-9592(01)00237-0
http://dx.doi.org/10.1016/S0032-9592(01)...
investigated the stability of the extracellular FTase purified from Bacillus macerans EG-6, incubated at 25 ºC, in sodium acetate buffer solution, obtaining higher AT values for a pH range of 5.0-8.0. On the other hand, Hernalsteens & Maugeri (2008)Hernalsteens, S., & Maugeri, F. (2008). Purification and characterization of a fructosyltransferase from Rhodotorula sp. Applied Microbiology and Biotechnology, 79(4), 589-596. PMid:18481059. http://dx.doi.org/10.1007/s00253-008-1470-x
http://dx.doi.org/10.1007/s00253-008-147...
studied the stability of an extracellular FTase from Rhodotorula sp., whose enzyme presented the highest transfructosylation activity and good stability in acetate buffer pH 5.0 at 50 ºC. Finally, Yang et al. (2016)Yang, H., Wang, Y., Zhang, L., & Shen, W. (2016). Heterologous expression and enzymatic characterization of fructosyltransferase from Aspergillus niger in Pichia pastoris. New Biotechnology, 33(1), 164-170. PMid:25976629. http://dx.doi.org/10.1016/j.nbt.2015.04.005
http://dx.doi.org/10.1016/j.nbt.2015.04....
, using the recombinant A. niger YZ59 expressed in Pichia pastoris GS115, detected stability of the recombinant FTase for a pH range of 3.0-10.0, incubating the enzyme at 25 ºC for 24 h. Recombinant enzyme has been genetically modified to be more effective at the desired pH range. In addition, recombinant enzymes are more robust and have greater pH stability when compared to natural enzymes, which improves their use and efficiency in industrial applications (Amid, 2015Amid, A. (Ed.). (2015). Recombinant enzymes – from basic science to commercialization. New York, NY: Springer.).

3.3 Stability during storage

Figure 4 presents the behavior of the enzymatic activity of the enzyme FTase according to storage time. It can be observed that the transfructosylation activity decreases, whereas the hydrolytic activity increases over storage time, consequently producing a reduction in the AT/AH ratio. This change in activity is more accentuated mainly at the first nine hours of storage time, a time in which the enzyme presented around 45.6% of its initial transfructosylation activity. After this period, the transfructosylation activity continued decreasing at a lower speed until it reached approximately 35% of its initial value after 96 h of storage. The increase in hydrolytic activity over time can be attributed to the loss of transfructosylation activity in the fermented broth (Faria et al., 2021Faria, L. L., Morales, S. A. V., Prado, J. P. Z., Dias, G. S., Almeida, A. F., Xavier, M. C. A., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides. Biotechnology Letters, 43(1), 43-59. PMid:33025334. http://dx.doi.org/10.1007/s10529-020-03016-7
http://dx.doi.org/10.1007/s10529-020-030...
). These results indicated the existence of a high loss of enzymatic activity of the enzyme FTase in the soluble form in a few hours of storage, suggesting it must be used for FOS production preferably in the first hours after its production. These results also suggested the need to improve the use of immobilization techniques for FTase enzymes, such as adsorption and encapsulation, aiming at increasing their thermal and operational stability (Aguiar-Oliveira & Maugeri, 2010Aguiar-Oliveira, E., & Maugeri, F. (2010). Characterization of the immobilized fructosyltranferase from Rhodotorula sp. International Journal of Food Engineering, 6(3), http://dx.doi.org/10.2202/1556-3758.1894
http://dx.doi.org/10.2202/1556-3758.1894...
; Gonçalves et al., 2020Gonçalves, M. C. P., Morales, S. A. V., Silva, E. S., Maiorano, A. E., Perna, R. F., & Kieckbusch, T. G. (2020). Entrapment of glutaraldehyde-crosslinked cells from Aspergillus oryzae IPT-301 in calcium alginate for high transfructosylation activity. Journal of Chemical Technology & Biotechnology, 95(9), 2473-2482. http://dx.doi.org/10.1002/jctb.6429
http://dx.doi.org/10.1002/jctb.6429...
; Faria et al., 2021Faria, L. L., Morales, S. A. V., Prado, J. P. Z., Dias, G. S., Almeida, A. F., Xavier, M. C. A., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides. Biotechnology Letters, 43(1), 43-59. PMid:33025334. http://dx.doi.org/10.1007/s10529-020-03016-7
http://dx.doi.org/10.1007/s10529-020-030...
).

Figure 4
Storage stabilities of soluble extracellular FTase at 4 ºC: transfructosylation activity – AT (●), hydrolytic activity – AH (○), and (AT/AH) ratio (□). Reaction conditions: 47% (w v-1) sucrose solution, 190 rpm, 0.2 mol L-1 of tris-acetate buffer, pH 5.5, and 50 ºC.

4 Conclusion

In this work, the extracellular enzyme fructosyltransferase (FTase, E.C.2.4.1.9) from A. oryzaeIPT-301 was produced by submerged fermentation, and the effects of storage, temperature and pH on enzyme stability were evaluated. The soluble biocatalyst, for a storage period of 5 h, was active with retention of 50% of the transfructosylation activity, whereas for storage times greater than 24 h, the enzymatic activity remained below 30%. The thermodynamic parameters obtained showed the thermostability of the microbial FTase when submitted to different incubation temperatures. Regarding the effects of pH, the enzyme was stable at pH 5.5, in which the highest value of transfructosylation activity was obtained. Therefore, the results presented in this work were necessary to explore both the viability of the use of soluble FTase in industrial processes for FOS production and the improvement of the immobilization processes for this microbial enzyme.

Acknowledgements

The authors gratefully acknowledge the financial support from the National Council for Scientific and Technological Development – Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Proc. 421540/2018-4), Foundation for Research of the State of Minas Gerais (Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG)), Proc. APQ-02131-14) and Coordination for the Improvement of Higher Education Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES)).

  • Cite as: Silva, M. B. P. O., Abdal, D., Prado, J. P. Z., Dias, G. S., Morales, S. A. V., Xavier, M. C. A., Alemida, A. F., Silva, E. S., Maiorano, A. E., & Perna, R. F. (2021). Effect of temperature, pH and storage time on the stability of an extracellular fructosyltransferase from Aspergillus oryzae IPT-301. Brazilian Journal of Food Technology, 24, e2020283. https://doi.org/10.1590/1981-6723.28320
  • Funding: Ministério da Ciência, Tecnologia e Inovação/Conselho Nacional de Desenvolvimento Científico e Tecnológico - Proc. 421540/2018-4 e Fundação de Amparo à Pesquisa do Estado de Minas Gerais - Proc. APQ-02131-14

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Publication Dates

  • Publication in this collection
    16 July 2021
  • Date of issue
    2021

History

  • Received
    11 Dec 2020
  • Accepted
    27 Apr 2021
Instituto de Tecnologia de Alimentos - ITAL Av. Brasil, 2880, 13070-178 Campinas - SP / Brasil, Tel 55 19 3743-1762 - Campinas - SP - Brazil
E-mail: bjftsec@ital.sp.gov.br