Influence of the reaction products in the inversion of sucrose by invertase

Influence of the reaction products in the inversion of sucrose by invertase

U. C. FILHO¹ , C. E. HORI² and E. J. RIBEIRO2

¹Unit-Av. Rafael Marino Neto, 600, 384002-310, Uberlândia-MG, Brazil

²Departamento de Engenharia Química (UFU), P.O.Box 593, 38400-902, Uberlândia - MG, Brazil.

(Received: January 19, 1999; Accepted: April 6, 1999)

Abstract - In this work a kinetic model for the enzymatic conversion of sucrose to glucose and fructose using the free form of the enzyme invertase was studied. The initial rates of conversion of sucrose into glucose and fructose were evaluated for different initial concentrations of sucrose, fructose and glucose. The results of the square test showed that the partially non competitive inhibition model for glucose and for fructose can be used to describe the reaction kinetics with a degree of confidence higher than 99.9%. An analysis of the sum of squares of the residuals for several inhibition models cited in the literature showed that the partially noncompetitive inhibition model is the best one for quantifying the effect of the concentration of glucose and fructose on sucrose hydrolysis. A comparison of a simulation of this model with the experimental results showed that the model can describe well the reaction in a batch reactor using initial concentrations of 600 g/L.

Keywords: enzymatic conversion, enzyme invertase, sucrose, glucose, fructose.

INTRODUCTION

Sucrose ( glucose-1,2 fructose) is the most commonly used sweetener for human consumption, and one of the most important natural sources is probably sugar cane since it contains up to 20% sucrose by weight (Glazer and Nikaido, 1995). The hydrolysis of sucrose generates an equimolar mixture of fructose and glucose denominated invert sugar, which has applications in several industrial processes. The production of invert sugar syrups from sucrose can be achieved by acid hydrolysis or by using the enzyme invertase (D-fructofuranosidase - E.C. 3.2.1.26). Syrups produced by the enzymatic process show several advantages, such as not crystallizing as readily when present in mixtures, and they are also free of the colored by-products generated by the acid hydrolysis of sucrose (Monsan et al., 1984). The chemical process is used more often despite the fact that the enzymatic process produces a higher quality product. According to Reed and Nagodawithana (1993), the enzymatic production of glucose and fructose syrups is not larger because the process that starts from corn starch is more economical. However, this is not the case in Brazil where sugar cane is produced on a large scale and corn has more important applications in the food processing industries.

Although somewhat inhibited, invertase can catalyze sucrose hydrolysis at concentrations above 50% wt/vol. Increasing sucrose concentrations up to 80% wt/vol. significantly reduces enzyme activity, probably due to low water concentration, substrate inhibition and substrate aggregation (Somiari and Bielecki, 1995). In this work the kinetics of sucrose hydrolysis were evaluated by initial rates in order to obtain a model that can describe the reaction under conditions of high concentrations of substrate and products. A larger emphasis was places on examining the influence of product concentration on the kinetics of the reaction. The validity of the model was verified by comparing the results of a computer simulation with experimental data, both obtained under the same conditions.

MATERIALS AND METHODS

Determination of Initial Rates of Reaction

The initial rates of sucrose hydrolysis by invertase were determined for two independent reaction systems: a system formed of sucrose and fructose and another of sucrose and glucose. The study covered five levels of sucrose concentration (10, 20, 30, 40, and 50 g/L) and five levels of product concentration for both glucose and fructose (0, 5, 20, 35 and 50 g/L). The enzyme used in the hydrolysis was invertase from Novo Nordisk, and the experiments were done under the following conditions: 40ºC, pH=4.50 and 750 rpm.

The experiments used for the determination of the kinetic model were carried out in a microreactor with temperature control and mechanical agitation.

Samples were taken at appropriate time intervals in order to assure that the curve generated by plotting reducing sugar as a function of time be as close as possible to a straight line.

High sucrose concentrations in a mini-fermentator were used in order to confirm the kinetic model obtained. Reducing sugars were determined by two methods: the dinitrossalicilic acid colorimetric method and the glucose-oxidase enzymatic method.

Model Fits and Statistical Tests

The model parameters were estimated by nonlinear regression (Coutinho Filho, 1996). The discrimination of the model was done by probability calculation by the square test and by analysis of the sum of the square of the residues (Himmelblau and Edgar, 1988). Several models available in the literature (Segel, 1993; Dixon et al., 1979) were used to perform the computer simulation.

Comparison Between Simulated and Experimental Data

The conditions adopted for the model simulation were similar to those used for the industrial hydrolysis of sucrose in syrup production. The sucrose concentration used in the experiments was 600 g/L and the invertase concentration was 0.1308 g/L (100 g of enzyme per ton. of sucrose solution). The reaction was carried out in a batch reactor at a temperature of 40°C and a pH of 4.50. Results of the simulation were obtained by solving the following system of differential equations:

where I represents inhibitor concentration (fructose or glucose) and v represents the partially noncompetitive inhibition model for glucose or fructose, as given by Equation 2:

RESULTS AND DISCUSSION

Models for hydrolysis inhibition by the products glucose and fructose were modeled by competitive, noncompetitive, uncompetitive, linearly mixed and partially noncompetitive inhibition. All these models could describe the reaction kinetics with a degree of confidence higher than 99.9% according to the square test. Analysis of the standard square deviations showed that the smallest sum of deviations for both glucose and fructose was obtained with the partially noncompetitive inhibition model. For this reason this model was chosen for this work. These results are in agreement with other data in the literature such as the data obtained by Combes and Monsan (1983).

The adjustment of experimental points to the partially noncompetitive inhibition model allowed the determination of the kinetic parameters given in Table ¹.

The experimental points as well as the curve obtained by computer fitting using the fructose inhibition are shown in Figure ¹. Figure ² shows the same results using glucose inhibition model.

The value of parameter V_m depends on the enzymatic concentration used. In the experiments performed, this value was much smaller than the value usually associated with high sucrose concentrations.

Figure 1: Adjustment to the partially noncompetitive inhibition model for fructose.

Figure 2: Adjustment to the partially noncompetitive inhibition model for glucose.

A comparison between the experimental data shown in Figures ¹ and ² and the model from Equation 2 showed that the model represents the data well with a degree of confidence higher than 99.9% for the square test.

The analytical solutions of the equation presented in (1) for fructose and for glucose are represented by Equations (3) and (4), respectively :

The value of V_m used during the simulation was corrected to account for the effect of invertase concentration in the experiments conducted at high sucrose concentrations. A comparison between the experimental data and the computer-generated curves is shown in Figure 3. Notice that the model can represent the end of the hydrolysis very well.

Figure 3: Experimental (symbols) and computer-generated (lines) data for sucrose hydrolysis.

The model that presented the best fit for the experimental results was also the partially noncompetitive inhibition model. A comparison between the experimental and simulated results showed that the model represented the sucrose hydrolysis well.

CONCLUSIONS

This study of sucrose hydrolysis by invertase showed that both the products and the substrate act as inhibition factors in the reaction. When considering the inhibition effect caused by the products, the inhibition model that provided the best fit for both glucose and fructose was the partially noncompetitive. A comparison between the experimental results and the data generated by computer simulation of several inhibition models led to the choice of the partially noncompetitive model as the one that provided the best fit. This model could correctly predict the reaction time required to reach total conversion.

NOMENCLATURE

V_m – Maximum rate of reaction (g/L.min)

v - Reaction rate (g/L.min)

S - Sucrose concentration (g/L)

I - Inhibitor concentration (g/L)

F - Fructose concentration (g/L)

G - Glucose concentration (g/L)

T - Time (min)

K_i - Inhibition constant (g/L)

K_m – Michaelis-Menten constant (g/L)

b - Constant dimensionless

REFERENCES

Combes, D. and Monsan, P., Sucrose Hydrolysis by Invertase. Characterisation of Products and Substrate Inhibition. Carbohydrates Research, vol. 117, 215 (1983).

Coutinho Filho, U., Contribuição ao Estudo da Cinética da Síntese de Hidrólise de Sacarose por Invertase Livre. Masters thesis, Univ. Federal de Uberlândia, 1996.

Dixon, M., Webb, E. C., Thorne, C. J. R., and Tipton, K. F., Enzymes. 3^rd ed. Longman Group Limited (1979).

Glazer, A. N. and Nikaido, H., Microbial Technology – Fundamentals of Applied Microbiology. W. H. Freeman and Company, New York (1995).

Himmelblau, D. M. and Edgar, T. F., Optimisation of Chemical Processes. McGraw-Hill (1988).

Monsan, P., Combes, D., and Alemzadeh, E., Biotechnology and Bioengineering, vol. XXVI, 658 (1984).

Reed, G. and Nagodawithana, T., Enzymes in Food Processing. 3^rd ed. New York Academic Press (1993).

Segel, I., Enzyme Kinetics. 2^nd ed. New York, John Wiley (1993).

Somiari, R. I. and Bielecki, S., Biotechnology Letters, vol. 17, n° 5 (1995).

Table 1: Kinetic parameters of the partially noncompetitive inhibition model for fructose and glucose.

Publication Dates

• Publication in this collection
  15 Sept 1999
• Date of issue
  June 1999

History

• Accepted
  06 Apr 1999
• Received
  19 Jan 1999