Ultrasound assisted enzymatic hydrolysis of sucrose catalyzed by invertase: Investigation on substrate, enzyme and kinetics parameters
Introduction
Invertase/β-fructofuranosidase (EC.3.2.1.26) catalyzes the hydrolysis of sucrose, producing an equimolar mixture of glucose and fructose, which is called “invert sugar” (Kulshrestha, Tyagi, Sindhi, & Yadavilli, 2013; Nadeem et al., 2015). Fructose is sweeter than sucrose, and both glucose and fructose are more soluble than sucrose. Therefore, when compared with sucrose, invert sugar can increase sweetness without crystallization. Consequently, this syrup is widely used in sweets and beverages manufacture, and invertase is the most used enzyme in the confectionery, sweets, chocolates and cookies areas. Moreover, invertase is also used for the manufacture of artificial honey, plasticizers with application in the cosmetics, pharmaceutical and paper industries, as enzyme electrodes for sucrose detection, as well as for production of fructo-oligosaccharides (FOS) (Chand Bhalla, Bansuli, Thakur, Savitri, & Thakur, 2017; Nadeem et al., 2015).
Sucrose may also be hydrolyzed in acidic medium (pH 2–3) at elevated temperatures (70–85 °C) to produce invert sugar. However, acid hydrolysis has the disadvantages of undesirable product formation, high energy, low efficiency and high corrosive power of the equipment (Vitolo, 2004) In this way, the production of invert sugar by enzymatic action is replacing the acid hydrolysis, since the reaction is conducted under milder conditions of temperature and pH (Vitolo, 2004), resulting in a product of enhanced quality. Therefore, due to several applications, invertase is an enzyme of great industrial importance.
However, the use of enzymes as catalysts for large-scale industrial processes can be limited by their high production cost and low stability (Patel, Singhania, & Pandey, 2016). In fact, different alternatives are being developed to minimize these limitations, such as enzymes immobilization, use of genetic engineering (Patel et al., 2016), and use of different emerging technologies. In this context, emerging technologies such as high hydrostatic pressure, microwave and ultrasound began to be studied for enzyme activation and stabilization (Dalagnol, Silveira, Baron, Manfroi, & Rodrigues, 2017; Rejasse, Lamare, Legoy, & Besson, 2007; Tribst & Cristianini, 2012).
Ultrasound (US) is an emerging technology that relies on the propagation of sound waves with frequencies higher than the audible limit of human hearing (>20 kHz). US has been proposed to improve heat and mass transfer operations and to assist food processing and preservation (Huang et al., 2017). One of the emerging applications of US process is the modification of enzyme performance, which may cause enzymatic activation or inactivation (Huang et al., 2017; Nadar & Rathod, 2017). There are many works available in the literature that focus on the efficiency of ultrasound to promote enzymatic inactivation (Arroyo, Kennedy, Lyng, & Sullivan, 2017; Saeeduddin et al., 2015; Sulaiman, Soo, Farid, & Silva, 2015; Vercet, Burgos, Crelier, & Lopez-buesa, 2001). However, further studies are needed to demonstrate the ability of the ultrasound to potentiate different enzymes of industrial interest. More specifically, there is a lack of information regarding the effect of ultrasound on invertase activity and its reaction.
Ultrasound can be used in three approaches in relation to the enzymatic reactions: as a pre-treatment to the enzyme, as a pre-treatment to the substrate or assisting the reaction, i.e., in the mixed reaction system (Wang et al., 2018). The majority of studies available in the literature focus on analyzing the assisted reaction. Some examples are the works with amyloglucosidase (Oliveira, Pinheiro, Fonseca, Cabrita, & Maia, 2018), alpha-amylase (Oliveira, Correia, Segundo, Fonseca, & Cabrita, 2017), cellulase (Szabó & Csiszár, 2013), xylanase (Sun, Zhang, Xiao, & Jin, 2015) and pectinase (Ma et al., 2016). All observed that ultrasound accelerated the reaction rate. However, by evaluating only the reaction, it is impossible to fully explain the effects of ultrasound, since this technology can present a simultaneous effect on both the enzyme and the substrate. In addition, most of those studies use an ultrasound probe. Although positive, the ultrasound probe application at industrial level (scale up) is difficult, since the intensity decreases exponentially in moving away from the horn (Gogate & Kabadi, 2009). In addition, it presents a high degree of wearing, limiting its relevance for this purpose.
Only one work evaluated the effect of ultrasound on invertase. Sakakibara, Wang, Takahashi, Takahashi, and Mori (1996) evaluated the ultrasound assisted enzymatic reaction of invertase and showed a higher formation of products during the hydrolysis. However, the authors have not evaluated the effect of ultrasound on the sucrose inversion, as well on the isolated enzyme. As the reaction and possible modifications of the enzyme and the substrate occur simultaneously, it is difficult to describe how ultrasound increased the enzymatic reaction. Furthermore, it is interesting to evaluate the effect of this technology on the enzyme isolated, once this can be an approach to desirably change the activity and/or control microorganisms in commercial enzymatic solutions. In addition, the reaction conducted by Sakakibara et al. (1996) was carried out at fixed temperature (25 °C) and pH (4.0), limiting the available information. Temperature and pH are factors that influence enzymatic activity, and temperature is a limiting factor in the effect of ultrasound (Patist & Bates, 2008).
Therefore, the objective of this work was to evaluate the effect of ultrasound as a pre-treatment to the invertase activity under optimal and non-optimal conditions of pH and temperature, as well as the effect of this technology assisting the enzymatic hydrolysis of sucrose by invertase. To describe the obtained results, the effect of US technology on sucrose hydrolysis were also evaluated.
Section snippets
Material and methods
Fig. 1 present a flow chart of the present work. Different approaches were performed to elucidate the effect of ultrasound on reaction and invertase activity.
Firstly, the objective of the work was to evaluate the effect of ultrasound during the enzymatic reaction. Thus, the reaction was conducted at the optimum temperature of the enzyme (55 °C), previously determined, a condition in which the industries produce invert sugar. In addition, the reaction was conducted at 40 °C and 30 °C, since
Enzyme characterization: determination of invertase activity at different pH and temperatures
Fig. 2 shows the invertase activity at different pH and temperatures. The enzyme optimal condition (the highest activity) was obtained at pH 5.0 and 55 °C. In this condition, the activity was 12.08 kat/g of enzyme, which was considered as 100% residual activity (REA). These results are similar to those found by Bergamasco, Bassetti, de Moraes, and Zanin (2000).
Temperature and pH variation resulted in significant changes in enzyme activity, promoting a reduction of 40.3% at 45 °C (pH 4.0) and
Conclusions
This work was the first to evaluate the effect of ultrasound in the invertase reaction, as well as a pre-treatment in the substrate and enzyme. We demonstrated the ultrasound did not favor sucrose hydrolysis and invertase activity. We can thus affirm that the positive effects of ultrasound are related with the turbulence and mass transfer during the reaction. Ultrasound enhanced the sucrose enzymatic hydrolysis. The reaction temperature was an important parameter, once high temperatures reduced
Acknowledgments
The authors are grateful to the Coordination of Improvement of Higher Education Personnel - Brazil (CAPES) - Financing Code 001; the São Paulo Research Foundation (FAPESP, Brazil) for funding the project 2016/18052-5; the Minas Gerais Research Foundation (FAPEMIG, Brazil) for funding the project CAG APQ-01716-15 and the National Council for Scientific and Technological Development for funding the project 401004/2014-7 and the productivity grants of P.E.D. Augusto (306557/2017-7) and A.M. Ramos
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