Inhibition Effect of Ca2+ Ions on Sucrose Hydrolysis Using Invertase

*Hargono Hargono -  Department of Chemical Engineering, Faculty of Engineering, Universitas Diponegoro , Semarang, 50275, Indonesia
Bakti Jos -  Department of Chemical Engineering, Faculty of Engineering, Universitas Diponegoro , Semarang, 50275, Indonesia
Abdullah Abdullah -  Department of Chemical Engineering, Faculty of Engineering, Universitas Diponegoro , Semarang, 50275, Indonesia
Teguh Riyanto -  Department of Chemical Engineering, Faculty of Engineering, Universitas Diponegoro , Semarang, 50275, Indonesia
Received: 4 Mar 2019; Revised: 15 Jul 2019; Accepted: 9 Aug 2019; Published: 1 Dec 2019; Available online: 30 Sep 2019.
Open Access Copyright (c) 2019 Bulletin of Chemical Reaction Engineering & Catalysis
Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.
Citation Format:
Cover Image

Fermentable sugar for bioethanol production can be produced from molasses due to its high sucrose content but Ca2+ ions found in the molasses may affect the hydrolysis. Therefore, this paper was focused to study the effect of Ca2+ ions as CaO on sucrose hydrolysis using invertase and to obtain the kinetic parameters. The kinetic parameters (KM and Vmax) were obtained using a Lineweaver-Burk plot. The value of KM and Vmax parameters were 36.181 g/L and 21.322 g/L.h, respectively. The Ca2+ ions act as competitive inhibitor in sucrose hydrolysis using invertase. Therefore, the inhibition mechanism was followed the competitive inhibition mechanism. The value of inhibition constant was 0.833 g/g. These parameters were obtained from the non-substrate inhibition process because this study used the low substrate concentrations which means the fermentable sugar production was low. Hence, there were still more challenges to studying the simultaneous effect of substrate and Ca2+ on sucrose hydrolysis to produce high fermentable sugar. Copyright © 2019 BCREC Group. All rights reserved


Sucrose; Invertase; Enzymatic Hydrolysis; Ca2+ Ions; Competitive Inhibition

Article Metrics:

  1. Najafpour, G.D., Shan, C.P. (2003). Enzymatic hydrolysis of molasses. Bioresource Technology, 86(1): 91–94.
  2. Chen, J.C.P., Chou, C.C. (1993). Cane Sugar Handbook: A Manual for Cane Sugar Manufacturers and Their Chemists (12th ed.). New York: John Wiley & Sons Inc.
  3. Higginbotham, J.D., Mc Carthy, J. (1998). Quality and storage of molasses. In P.W. van der Poel, T.K. Schwartz, & H.M. Schiweck (Eds.), Sugar Technology Beet and Cane Manufacture (pp. 973–992). Berlin: Verlag Dr Albert Bartens KG.
  4. Doherty, W.O.S., Greenwood, J., Pilaski, D., Wright, P.G. (2002). The Effect of Liming Conditions in Juice Clarification. In Proceedings of the Australian Society of Sugar Cane Technology (Vol. 24, pp. 1–12).
  5. Chotineeranat, S., Wansuksri, R., Piyachomkwan, K., Chatakanonda, P., Weerathaworn, P., Sriroth, K. (2010). Effect of calcium ions on ethanol production from molasses by Saccharomyces cerevisiae. Sugar Tech, 12(2): 120–124.
  6. Takeshige, K., Ouchi, K. (1995). Factors affecting the ethanol productivity of yeast in molasses. Journal of Fermentation and Bioengineering, 79(5): 449–452.
  7. Ettalibi, M., Baratti, J.C. (2001). Sucrose hydrolysis by thermostable immobilized inulinases from Aspergillus ficuum. Enzyme and Microbial Technology, 28(7–8): 596–601.
  8. Onishi, V.C., Olivo, J.E., Zanin, G.M., Moraes, F.F. (2014). Enzymatic hydrolysis of sugarcane molasses as pre-treatment for bioethanol production. International Sugar Journal, 16(1392): 906–910.
  9. Bhalla, T.C., Bansuli, B., Thakur, N., Savitri, S., Thakur, N. (2017). Invertase of Saccharomyces cerevisiae SAA-612: Production, characterization and application in synthesis of fructo-oligosaccharides. LWT - Food Science and Technology, 77: 178–185.
  10. 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.
  11. Essel, K.K., Osei, Y.D. (2014). Investigation of Some Kinetic Properties of Commercial Invertase from Yeast. Natural Products Chemistry & Research, 2(6): 152–156.
  12. Plascencia-Espinosa, M., Santiago-Hernández, A., Pavón-Orozco, P., Vallejo-Becerra, V., Trejo-Estrada, S., Sosa-Peinado, A., Benitez-Cardoza, C.G., Hidalgo-Lara, M.E. (2014). Effect of deglycosylation on the properties of thermophilic invertase purified from the yeast Candida guilliermondii MpIIIa. Process Biochemistry, 49(9): 1480–1487.
  13. de Ginés, S.C., Maldonado, M.C., de Valdez, G.F. (2000). Purification and Characterization of Invertase from Lactobacillus reuteri CRL 1100. Current Microbiology, 40(3): 181–184.
  14. Kaur, N., Sharma, A.D. (2005). Production, optimization and characterization of extracellular invertase by an actinomycete strain. Journal of Scientific & Industrial Research, 64(7): 515–5019.
  15. Maruyama, Y., Onodera, K. (1979). Production and Some Properties of Invertase Isozymes of Fusarium oxyporum. The Journal of General and Applied Microbiology, 25: 361–366.
  16. Yu, J., Chou, C.C., Saska, M. (2011). The Behavior of Invert Sugar in Sugar Processing. In Technical Proceedings of the Sugar Industry Technologists (Vol. 4, pp. 1–26). Montreal: Curran Associates, Inc.
  17. El-Sayed, E.-S. M., El-Sayed, S.T., Elmallah, M.I.Y., Shehata, A.N. (2015). Immobilization, Optimization and Properties of Pea Invertase within Sodium Alginate Gel. Research Journal of Pharmaceutical Biological and Chemical Sciences, 6(6): 1213–1222.
  18. Miller, G.L. (1959). Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry, 31(3): 426–428.
  19. Somogyi, M. (1952). Notes on Sugar Determination. Journal of Biological Chemistry, 195: 19–23.
  20. Nelson, N. (1944). A photometric adaptation of the Somogyi method for the determination of glucose. Journal of Biological Chemistry, 153: 375–380.
  21. Nelson, D.L., Cox, M.M. (2005). Lehninger - Principles of Biochemistry (Fourth Edition.). New York: W.H. Freeman and Company.
  22. Lineweaver, H., Burk, D. (1934). The Determination of Enzyme Dissociation Constants. Journal of The American Chemical Society, 56(3): 658–666.
  23. Fange, D., Lovmar, M., Pavlov, M.Y., Ehrenberg, M. (2011). Identification of enzyme inhibitory mechanisms from steady-state kinetics. Biochimie, 93(9): 1623–1629.
  24. Yoshino, M., Murakami, K. (2009). A graphical method for determining inhibition constants. Journal of Enzyme Inhibition and Medicinal Chemistry, 24(6): 1288–1290.
  25. Pál, G. (2013). Enzyme kinetics. In G. Hegyi, J. Kardos, M. Kovács, A. Málnási-Csizmadia, L. Nyitray, G. Pál, L. Radnai, A. Reményi, I. Venekei (Eds.), Introduction to Practical Biochemistry. Budapest: Eötvös Loránd University.
  26. Szymańska, K., Pudło, W., Mrowiec-Białoń, J., Czardybon, A., Kocurek, J., Jarzębski, A. B. (2013). Immobilization of invertase on silica monoliths with hierarchical pore structure to obtain continuous flow enzymatic microreactors of high performance. Microporous and Mesoporous Materials, 170: 75–82.
  27. Marquez, L.D.S., Cabral, B.V., Freitas, F.F., Cardoso, V.L., Ribeiro, E.J. (2008). Optimization of invertase immobilization by adsorption in ionic exchange resin for sucrose hydrolysis. Journal of Molecular Catalysis B: Enzymatic, 51(3–4): 86–92.
  28. Copeland, R.A. (2000). Enzymes: a practical introduction to structure, mechanism, and data analysis (2nd ed.). New York: Wiley.
  29. German, D.P., Weintraub, M.N., Grandy, A.S., Lauber, C.L., Rinkes, Z.L., Allison, S.D. (2011). Optimization of hydrolytic and oxidative enzyme methods for ecosystem studies. Soil Biology and Biochemistry, 43(7):1387–1397.
  30. Baks, T., Bruins, M.E., Janssen, A.E.M., Boom, R.M. (2008). Effect of Pressure and Temperature on the Gelatinization of Starch at Various Starch Concentrations. Biomacromolecules, 9(1): 296–304.
  31. Permanasari, A.R., Yulistiani, F., Purnama, R.W., Widjaja, T., Gunawan, S. (2018). The effect of substrate and enzyme concentration on the glucose syrup production from red sorghum starch by enzymatic hydrolysis. IOP Conference Series: Earth and Environmental Science, 160: 1–6.
  32. Weber, B.A., Nielsen, S.S. (1991). Isolation and Partial Characterization of a Native Serine-Type Protease Inhibitor from Bovine Milk. Journal of Dairy Science, 74(3): 764–771.
  33. Corazza, F.C., Calsavara, L.P.V., Moraes, F.F., Zanin, G.M., Neitzel, I. (2005). Determination of inhibition in the enzymatic hydrolysis of cellobiose using hybrid neural modeling. Brazilian Journal of Chemical Engineering, 22(1): 19–29.
  34. Salwanee, S., Wan Aida, W.M., Mamot, S., Maskat, M.Y., Ibrahim, S. (2013). Effects of Enzyme Concentration, Temperature, pH and Time on the Degree of Hydrolysis of Protein Ectract from Viscera of Tuna (Euthynnus affinis) by Using Alcalase. Sains Malaysia, 42(3): 279–287.
  35. Kharlamova, A.D., Lushchekina, S.V., Petrov, K.A., Kots, E.D., Nachon, F., Villard-Wandhammer, M., Zueva, I.V., Krejci, E., Reznik, V.S., Zobov, V.V., Nikolsky, E.E., Masson, P. (2016). Slow-binding inhibition of acetylcholinesterase by an alkylammonium derivative of 6-methyluracil: mechanism and possible advantages for myasthenia gravis treatment. Biochemical Journal, 473(9): 1225–1236.
  36. Rohatgi, N., Nielsen, T.K., Bjørn, S.P., Axelsson, I., Paglia, G., Voldborg, B.G., Palsson, B.O., Rolfsson, Ó. (2014). Biochemical Characterization of Human Gluconokinase and the Proposed Metabolic Impact of Gluconic Acid as Determined by Constraint Based Metabolic Network Analysis. PLoS ONE, 9(6): 1–9.
  37. Rubio, M.C., Runco, R., Navarro, A.R. (2002). Invertase from a strain of Rhodotorula glutinis. Phytochemistry, 61(6): 605–609.
  38. Azodi, M., Falamaki, C., Mohsenifar, A. (2011). Sucrose hydrolysis by invertase immobilized on functionalized porous silicon. Journal of Molecular Catalysis B: Enzymatic, 69(3–4): 154–160.
  39. Martínez, D., Menéndez, C., Echemendia, F.M., Pérez, E.R., Trujillo, L.E., Sobrino, A., Ramírez, R., Quintero, Y., Hernández, L. (2014). Complete sucrose hydrolysis by heat-killed recombinant Pichia pastoris cells entrapped in calcium alginate. Microbial Cell Factories, 13(1): 87–95.
  40. Whidden, M., Ho, A., Ivanova, M.I., Schnell, S. (2014). Competitive inhibition reaction mechanisms for the two-step model of protein aggregation. Biophysical Chemistry, 193–194: 9–19.
  41. Dutta, R. (2008). Fundamentals of Biochemical Engineering. Berlin, Heidelberg: Springer Berlin Heidelberg.