Cunninghamella echinulata PA3S12MM invertase: Biochemical characterization of a promiscuous enzyme
Letícia Mara Rasbold
Centro de Ciências Médicas e Farmacêuticas, Universidade Estadual do Oeste do Paraná, Cascavel, Brazil
Contribution: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing
Search for more papers by this authorPaulo Ricardo Heinen
Centro Universitário Fundação Assis Gurgacz, Cascavel, Brazil
Contribution: Formal analysis, Methodology, Visualization
Search for more papers by this authorJosé Luis da Conceição Silva
Centro de Ciências Médicas e Farmacêuticas, Universidade Estadual do Oeste do Paraná, Cascavel, Brazil
Contribution: Formal analysis, Investigation
Search for more papers by this authorRita de Cássia Garcia Simão
Centro de Ciências Médicas e Farmacêuticas, Universidade Estadual do Oeste do Paraná, Cascavel, Brazil
Contribution: Formal analysis, Investigation
Search for more papers by this authorMarina Kimiko Kadowaki
Centro de Ciências Médicas e Farmacêuticas, Universidade Estadual do Oeste do Paraná, Cascavel, Brazil
Contribution: Formal analysis, Investigation, Writing - original draft
Search for more papers by this authorCorresponding Author
Alexandre Maller
Centro de Ciências Médicas e Farmacêuticas, Universidade Estadual do Oeste do Paraná, Cascavel, Brazil
Correspondence
Alexandre Maller, Centro de Ciências Médicas e Farmacêuticas, Universidade Estadual do Oeste do Paraná, 2069 Universitária Street, Faculdade, 85819-110 Cascavel, Paraná, Brazil.
Email: [email protected]
Contribution: Conceptualization, Formal analysis, Investigation, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing
Search for more papers by this authorLetícia Mara Rasbold
Centro de Ciências Médicas e Farmacêuticas, Universidade Estadual do Oeste do Paraná, Cascavel, Brazil
Contribution: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing
Search for more papers by this authorPaulo Ricardo Heinen
Centro Universitário Fundação Assis Gurgacz, Cascavel, Brazil
Contribution: Formal analysis, Methodology, Visualization
Search for more papers by this authorJosé Luis da Conceição Silva
Centro de Ciências Médicas e Farmacêuticas, Universidade Estadual do Oeste do Paraná, Cascavel, Brazil
Contribution: Formal analysis, Investigation
Search for more papers by this authorRita de Cássia Garcia Simão
Centro de Ciências Médicas e Farmacêuticas, Universidade Estadual do Oeste do Paraná, Cascavel, Brazil
Contribution: Formal analysis, Investigation
Search for more papers by this authorMarina Kimiko Kadowaki
Centro de Ciências Médicas e Farmacêuticas, Universidade Estadual do Oeste do Paraná, Cascavel, Brazil
Contribution: Formal analysis, Investigation, Writing - original draft
Search for more papers by this authorCorresponding Author
Alexandre Maller
Centro de Ciências Médicas e Farmacêuticas, Universidade Estadual do Oeste do Paraná, Cascavel, Brazil
Correspondence
Alexandre Maller, Centro de Ciências Médicas e Farmacêuticas, Universidade Estadual do Oeste do Paraná, 2069 Universitária Street, Faculdade, 85819-110 Cascavel, Paraná, Brazil.
Email: [email protected]
Contribution: Conceptualization, Formal analysis, Investigation, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing
Search for more papers by this authorAbstract
The Cunninghamella echinulata PA3S12MM fungus is a great producer of invertases in a growth medium supplemented by apple peels. The enzyme was purified 4.5 times after two chromatographic processes, and it presented a relative molecular mass of 89.2 kDa. The invertase reached maximum activity at pH of 6 and at 60°C, in addition to presenting stability in alkaline pH and thermal activation at 50°C. The enzymatic activity increased in the presence of Mn2+ and dithiothreitol (DTT), while Cu2+ and Z2+ ions inhibited it. Also, DTT showed to protect enzymatic activity. The apparent values for Km, Vmáx, and Kcat for the sucrose hydrolysis were, respectively, 173.8 mmol/L, 908.7 mmol/L min−1, and 1,388.79 s−1. The carbohydrate content was of 83.13%. The invertase presented hydrolytic activity over different types of glycosidic bonds, such as α1 ↔ 2β (sucrose), α1 → 4 (polygalacturonic acid), α1 → 4 and α1 → 2 (pectin), and α1 ↔ 1 (trehalose), indicating that the enzyme is multifunctional. Thus, the biochemical properties showed by the C. echinulata PA3S12MM suggest a broad industrial application, such as in the biomass hydrolysis or in the food industry.
Practical applications
Invertases are hydrolytic enzymes employed in several industrial sectors. Given their great importance for the economy and several industrial sectors, there is a growing interest in microorganisms producing this enzyme. The analysis of the biochemical properties of invertase in C. echinulata PA3S12MM suggest applications in the food industry. Due to its increased hydrolytic activity, the hydrolysis process of the sucrose may employ invertase for the production of invert sugar. The stability at alkaline pH suggests an application in the development of enzymatic electrodes for the quantification of sucrose in food and beverage. The multifunctional activity may work in the biomass hydrolysis or saccharification of by-products for the extraction of fermentable sugars. The high level of invertase N-linked glycosylation of invertase grants this enzyme thermal stability at high temperatures, in addition to resistance against the action of proteases, which are desirable characteristics for the application of this enzyme in industrial processes.
CONFLICT OF INTEREST
The authors declared that they have no conflict of interest.
REFERENCES
- Almeida, M. N., Falkoski, D. L., Guimarães, V. M., Ramos, H. J. O., Visser, E. M., Maitan-Alfenas, G. P., & Rezende, S. T. (2013). Characteristics of free endoglucanase and glycosidases multienzyme complex from Fusarium verticillioides. Bioresource Technology, 143, 413–422. https://doi.org/10.1016/j.biortech.2013.06.021
- Almeida, M. N., Guimarães, V. M., Falkoski, D. L., Camargo, B. R., Fontes-Sant'Ana, G. C., Maitan-Alfenas, G. P., & Rezende, S. T. (2018). Purification and characterization of an invertase and a transfructosylase from Aspergillus terreus. Journal of Food Biochemistry, 42, Article e12551. https://doi.org/10.1111/jfbc.12551
- Arez, B. F., Alves, L., & Paixão, S. M. (2014). Production and characterization of a novel yeast extracellular invertase activity towards improved dibenzothiophene biodesulfurization. Applied Biochemistry and Biotechnology, 174(6), 2048–2057. https://doi.org/10.1007/s12010-014-1182-x
- Aung, T., Jiang, H., Liu, G. L., Chi, Z., Hu, Z., & Chi, Z. M. (2019). Overproduction of a β-fructofuranosidase1 with a high FOS synthesis activity for efficient biosynthesis of fructooligosaccharides. International Journal of Biological Macromolecules, 130, 988–996. https://doi.org/10.1016/j.ijbiomac.2019.03.039
- Ávila-Fernández, Á., Cuevas-Juárez, E., Rodríguez-Alegría, M. E., Olvera, C., & López-Munguía, A. (2016). Functional characterization of a novel β-fructofuranosidase from Bifidobacterium longum subsp. infantis ATCC 15697 on structurally diverse fructans. Journal of Applied Microbiology, 121(1), 263–276. https://doi.org/10.1111/jam.13154
- Andjelković, U., & Lah, J. (2011). Thermodynamics and structural features of the yeast Saccharomyces cerevisiae external invertase isoforms in guanidinium-chloride solutions. Journal of Agricultural and Food Chemistry, 59(2), 727–732. https://doi.org/10.1021/jf103441p
- Bagal-Kestwal, D., Karve, M. S., Kakade, B., & Pillai, V. K. (2008). Invertase inhibition based electrochemical sensor for the detection of heavy metal ions in aqueous system: Application of ultra-microelectrode to enhance sucrose biosensor's sensitivity. Biosensors and Bioelectronics, 24(4), 657–664. https://doi.org/10.1016/j.bios.2008.06027
- Chand Bhalla, T., Bansuli, Thakur, N., Savitri, & Thakur, N. (2017). Invertase of Saccharomyces cerevisiae SAA-612: Production, characterization and application in synthesis of fructooligosaccharides. LWT - Food Science and Technology, 77, 178–185. https://doi.org/10.1016/j.lwt.2016.11.034
- Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1), 248–254. https://doi.org/10.1006/abio.1976.9999
- Chávez, F. P., Rodriguez, L., Díaz, J., Delgado, J. M., & Cremata, J. A. (1997). Purification and characterization of an invertase from Candida utilis: Comparison with natural and recombinant yeast invertases. Journal of Biotechnology, 53(1), 67–74. https://doi.org/10.1016/s0168-1656(97)01663-5
- Dapper, T. B., Arfelli, V. C., Henn, C., Simões, M. R., Santos, M. F., Della Torre, C. L., Silva, J. L. C., Simão, R. C. G., & Kadowaki, M. K. (2016). β-Fructofuranosidase production by Aspergillus versicolor isolated from Atlantic forest and grown on apple pomace. African Journal of Microbiology Research, 10(25), 938–948. https://doi.org/10.5897/AJMR2016.8038.
- Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28(3), 350–356. https://doi.org/10.1021/ac60111a017
- Fernandes, M. L. P., Jorge, J. A., & Guimarães, L. H. S. (2018). Characterization of an extracellular β-d-fructofuranosidase produced by Aspergillus niveus during solid-state fermentation (SSF) of cassava husk. Journal of Food Biochemistry, 42, Article e12443. https://doi.org/10.1111/jfbc.12443
- Ghosh, K., Dhar, A., & Samanta, T. B. (2001). Purification and characterization of an invertase produced by Aspergillus ochraceus TS. Indian Journal of Biochemistry & Biophysics, 38(3), 180–185.
- Ginés, S. C., Maldonado, M. C., & Valdez, G. F. (2000). Purification and characterization of Invertase from Lactobacillus reuteri CRL 1100. Current Microbiology, 40, 181–184. https://doi.org/10.1007/s002849910036
- Giraldo, M. A., Gonçalves, H. B., Furriel, R. P. M., Jorge, J. A., & Guimarães, L. H. S. (2014). Characterization of the co-purified invertase and β-glucosidase of a multifunctional extract from Aspergillus terreus. World Journal of Microbiology and Biotechnology, 30(5), 1501–1510. https://doi.org/10.1007/s11274-013-1570-3
- Guimarães, L. H. S., Terenzi, H. F., Polizeli, M. L. T. M., & Jorge, J. A. (2007). Production and characterization of a thermostable extracellular β-d-fructofuranosidase produced by Aspergillus ochraceus with agroindustrial residues as carbon sources. Enzyme and Microbial Technology, 42(1), 52–57. https://doi.org/10.1016/j.enzmictec.2007.07.021
- Guimarães, L. H. S., Somera, A. F., Terenzi, H. F., Polizeli, M. L. T. M., & Jorge, J. A. (2009). Production of β-fructofuranosidases by Aspergillus niveus using agroindustrial residues as carbon sources: Characterization of an intracellular enzyme accumulated in the presence of glucose. Process Biochemistry, 44(2), 237–241. https://doi.org/10.1016/j.procbio.2008.10.011.
- Hanes, C. S. (1932). Studies on plant amylases: The effect of starch concentration upon the velocity of hydrolysis by the amylase of germinated barley. The Biochemical Journal, 26(5), 1406–1421. https://doi.org/10.1042/bj0261406
- Heinen, P. R., Henn, C., Peralta, R. M., Bracht, A., Simão, R. C. G., Silva, J. L. C., Polizeli, M. L. T. M., & Kadowaki, M. K. (2014). Xylanase from Fusarium heterosporum: Properties and influence of thiol compounds on xylanase activity. African Journal of Biotechnology, 13(9), 1047–1055. https://doi.org/10.5897/AJB2013.13282
10.5897/AJB2013.13282 Google Scholar
- Hult, K., & Berglund, P. (2007). Enzyme promiscuity: Mechanism and applications. Trends in Biotechnology, 25(5), 231–238. https://doi.org/10.1016/j.tibtech.2007.03.002
- Janer, C., Rohr, L. M., Peláez, C., Laloi, M., Cleusix, V., Requena, T., & Meile, L. (2004). Hydrolysis of oligofructoses by the recombinant beta-fructofuranosidase from Bifidobacterium lactis. Systematic and Applied Microbiology, 27(3), 279–285. https://doi.org/10.1078/0723-2020-00274
- Jiang, H., Ma, Y., Chi, Z., Liu, G. L., & Chi, Z. M. (2016). Production, purification, and gene cloning of a β-fructofuranosidase with a high inulin-hydrolyzing activity produced by a novel yeast Aureobasidium sp. P6 isolated from a mangrove ecosystem. Marine Biotechnology, 18(4), 500–510. https://doi.org/10.1007/s10126-016-9712-x
- Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685. https://doi.org/10.1038/227680a0
- Lahiri, S., Basu, A., Sengupta, S., Banerjee, S., Dutta, T., Soren, D., Chattopadhyay, K., & Ghosh, A. K. (2012). Purification and characterization of a trehalase-invertase enzyme with dual activity from Candida utilis. Archives of Biochemistry and Biophysics, 522(2), 90–99. https://doi.org/10.1016/j.abb.2012.03.026
- Leone, F. A., Baranauskas, J. A., & Ciancaglini, P. (1995). ENZYPLOT: A microcomputer assisted program for teaching enzyme kinetics. Biochemical Education, 23(1), 35–37. https://doi.org/10.1016/0307-4412(94)00159-M
- Lincoln, L., & More, S. S. (2018). Purification and biochemical characterization of an extracellular β-d-fructofuranosidase from Aspergillus sp. 3. Biotech, 8(2), 86–97. https://doi.org/10.1007/s13205-018-1109-2
- Lincoln, L., More, S. S., & Reddy, S. V. (2018). Purification and biochemical characterization of β-d-fructofuranosidase from Bacillus subtilis LYN12. Journal of Food Biochemistry, 42, Article e12592. https://doi.org/10.1111/jfbc.12592
- McIlvaine, T. C. (1921). A buffer solution for colorimetric comparison. Journal of Biological Chemistry, 49, 183–186. https://doi.org/10.1016/S0021-9258(18)86000-8.
- Michaelis, L., & Menten, M. L. (1913). Die Kinetik Der Invertinwirkung. Biochemische Zeitschrift, 49, 333–369.
- Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3), 426–428. https://doi.org/10.1021/ac60147a030
- Manoochehri, H., Hosseini, N. F., Saidijam, M., Taheri, M., Rezaee, H., & Nouri, F. (2020). A review on invertase: Its potentials and applications. Biocatalysis and Agricultural Biotechnology, 25, 101599. https://doi.org/10.1016/j.bcab.2020.101599.
- Moreno, S., Sanchez, Y., & Rodriguez, L. (1990). Purification and characterization of the invertase from Schizosaccharomyces pombe. A comparative analysis with the invertase from Saccharomyces cerevisiae. The Biochemical Journal, 267(3), 697–702. https://doi.org/10.1042/bj2670697
- Nadeem, H., Rashid, M. H., Siddique, M. H., Azeem, F., Muzammil, S., Javed, M. R., Ali, M. A., Rasul, I., & Riaz, M. (2015). Microbial invertases: A review on kinetics, thermodynamics, physiochemical properties. Process Biochemistry, 50(8), 1202–1210. https://doi.org/10.1016/j.procbio.2015.04.015
- Nascimento, G. C., Batista, R. D., Santos, C., Silva, E. M., Paula, F. C., Mendes, D. B., Oliveira, D. P., & Almeida, A. F. (2019). β-fructofuranosidase and β-D-fructosyltransferase from new Aspergillus carbonarius PC-4 strain isolated from canned peach syrup: Effect of carbon and nitrogen sources on enzyme production. The Scientific World Journal, 2019, Article 6956202. https://doi.org/10.1155/2019/6956202.
10.1155/2019/6956202 Google Scholar
- Nguyen, Q. D., Rezessy-Szabó, J. M., Bhat, M. K., & Hoschke, Á. (2005). Purification and some properties of β-fructofuranosidase from Aspergillus niger IMI303386. Process Biochemistry, 40(7), 2461–2466. https://doi.org/10.1016/j.procbio.2004.09012
- Oda, Y., & Tonomura, K. (1994). Purification and characterization of invertase from Torulaspora pretoriensis YK-1. Bioscience, Biotechnology, and Biochemistry, 58(6), 1155–1157. https://doi.org/10.1271/bbb.58.1155
- Oliveira, R. L., Silva, M. F., Converti, A., & Porto, T. S. (2020). Production of β-fructofuranosidase with transfructosylating activity by Aspergillus tamarii URM4634 solid-state fermentation on agroindustrial by-products. International Journal of Biological Macromolecules, 144, 343–350. https://doi.org/10.1016/j.ijbiomac.2019.12084
- Ozcan, O., Yildirim, R. M., Toker, O. S., Akbas, N., Ozulku, G., & Yaman, M. (2019). The effect of invertase concentration on quality parameters of fondant. Journal of Food Science and Technology, 56(9), 4242–4250. https://doi.org/10.1007/s13197-019-03894-4
- Park, J. K., Ro, H. S., & Kim, H. S. (1991). A new biosensor for specific determination of sucrose using an oxidoreductase of Zymomonas mobilis and invertase. Biotechnology and Bioengineering, 38(3), 217–223. https://doi.org/10.1002/bit.260380302
- Pek, H. B., Lim, P. Y., Liu, C., Lee, D. Y., Bi, X., Wong, F. T., & Ow, D. S. W. (2017). Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis. Biotechnology Letters, 39(5), 759–765. https://doi.org/10.1007/s10529-017-2295-4
- Pérez de los Santos, A. I., Cayetano-Cruz, M., Gutiérrez-Antón, M., Santiago-Hernández, A., Plascencia-Espinosa, M., Farrés, A., & Hidalgo-Lara, M. E. (2016). Improvement of catalytical properties of two invertases highly tolerant to sucrose after expression in Pichia pastoris. Effect of glycosylation on enzyme properties. Enzyme and Microbial Technology, 83, 48–56. https://doi.org/10.1016/j.enzmictec.2015.11.008
- Quiroga, E. N., Vattuone, M. A., & Sampietro, A. R. (1995). Purification and characterization of the invertase from Pycnoporus sanguineus. Biochimica Et Biophysica Acta, 1251(2), 75–80. https://doi.org/10.1016/0167-4838(95)00070-B
- Rehm, J., Willmitzer, L., & Heyer, A. G. (1998). Production of 1-kestose in transgenic yeast expressing a fructosyltransferase from Aspergillus foetidus. Journal of Bacteriology, 180(5), 1305–1310. https://doi.org/10.1128/JB.180.5.1305-1310.1998.
- Roitsch, T., & González, M. C. (2004). Function and regulation of plant invertases: Sweet sensations. Trends in Plant Science, 9(12), 606–613. https://doi.org/10.1016/j.tplants.2004.10.009
- Rubio, M. C., & Maldonado, M. C. (1995). Purification and characterization of invertase from Aspergillus niger. Current Microbiology, 31(2), 80–83. https://doi.org/10.1007/BF00294280.
- Rustiguel, C. B., Oliveira, A. H. C., Terenzi, H. F., Jorge, J. A., & Guimarães, L. H. S. (2011). Biochemical properties of an extracellular β-D-fructofuranosidase II produced by Aspergillus phoenicis under Solid-Sate Fermentation using soy bran as substrate. Electronic Journal of Biotechnology, 14(2), 1–10. https://doi.org/10.2225/vol14-issue2-fulltext-1
- Rustiguel, C. B., Jorge, J. A., & Guimarães, L. H. S. (2015). Characterization of a thermo-tolerant mycelial β-fructofuranosidase from Aspergillus phoenicis under submerged fermentation using wheat bran as carbon source. Biocatalysis and Agricultural Biotechnology, 4(3), 362–369. https://doi.org/10.1016/j.bcab.2015.05.004
- Singla, P., & Bhardwaj, R. D. (2020). Enzyme promiscuity – A light on the “darker” side of enzyme specificity. Biocatalysis and Biotransformation, 38(2), 81–92. https://doi.org/10.1080/10242422.2019.1696779
- Uma, C., Gomathi, D., Muthulakshmi, C., & Gopalakrishnan, V. K. (2010). Production, purification and characterization of invertase by Aspergillus flavus using fruit peel waste as substrate. Advances in Biological Research, 4(1), 31–36.
- Vogel, H. F. (1964). Distribution of lysine pathway among fungi: Evolutionary implications. The American Naturalist, 98(903), 435–446.
- Warchol, M., Perrin, S., Grill, J. P., & Schneider, F. (2002). Characterization of a purified beta-fructofuranosidase from Bifidobacterium infantis ATCC 15697. Letters in Applied Microbiology, 35(6), 462–467. https://doi.org/10.1046/j.1472-765x.2002.01224.x
- Xiang, Y., & Lu, Y. (2011). Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets. Nature Chemistry, 3(9), 697–703. https://doi.org/10.1038/nchem.1092
- Xu, L., Wang, D., Lu, L., Jin, L., Liu, J., Song, D., Guo, Z., & Xiao, M. (2014). Purification, cloning, characterization, and N-glycosylation analysis of a novel β-fructosidase from Aspergillus oryzae FS4 synthesizing levan- and neolevan-type fructooligosaccharides. PLoS ONE, 9(12), Article e114793. https://doi.org/10.1371/journal.pone.0114793.
- Yapo, B. M., & Gnakri, D. (2015). Pectic Polysaccharides and their functional properties. In K. Ramawat, & J. M. Mérillon (Eds.), Polysaccharides (pp. 1729–1749). Springer. https://doi.org/10.1007/978-3-319-16298-0_62.
10.1007/978-3-319-16298-0_62 Google Scholar