A new kinetic model proposed for enzymatic hydrolysis of lactose by a β-galactosidase from Kluyveromyces fragilis

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Abstract

We study the enzymatic hydrolysis of lactose by a commercial enzyme from a selected strain of Kluyveromyces fragilis. The variables analyzed were: temperature (25–40 °C), enzyme concentration (0.1–3.0 g l−1), lactose concentration (0.0278–0.208 M), and initial galactose concentration (0.0347 M). On the basis of the data analyzed, both published and in the present work, we propose a Michaelis–Menten kinetic model with inhibition by the product (galactose), which reveals that the substrate (lactose) and the product (galactose) present similar affinity for the active site of the enzyme.

Introduction

Enzymatic hydrolysis of lactose is one of the most important biotechnological processes in the food industry because of the potentially beneficial effects on the assimilation of foods containing lactose, as well as the possible technological and environmental advantages of industrial application, including:

  • 1.

    Elimination of lactose intolerance (3–70% depending on the populational group [1]), encouraging the utilization of lactose as an energy source, as well as calcium and magnesium assimilation from milk.

  • 2.

    Formation of galacto-oligosaccharides during lactose hydrolysis to favor the growth of intestinal bacterial microflora. The presence of these compounds is considered desirable in foods [2], [3].

  • 3.

    Improvement in the technological and sensorial characteristics of foods containing hydrolyzed lactose from milk or whey [4], [5], [6], [7] such as: increased solubility (avoidance of lactose crystallization and the grainy aspect of ice creams and condensed or powdered products); greater sweetening power and thus lower caloric content of the products (glucose and galactose monosaccharides have greater sweetening power than does lactose); formation of monosaccharides, which are easier to ferment in certain products such as yogurt [8]; lower freezing point of ice creams (increasing softness and creaminess); and reduction of the Maillard reaction.

  • 4.

    Greater biodegradability of whey in which the lactose has been hydrolyzed [9].

The commercial enzymes used for lactose hydrolysis are β-galactosidases of diverse origins [5], [6], [10]. Yeast and fungal enzymes have the greatest commercial interest. Many studies have been made with β-galactosidases obtained from Escherichia coli, although their use is not viable for products intended for human consumption [11], [12], [13], [14], [15].

The optimal operating conditions are described in Table 1. Fungal enzymes are usually used to hydrolyze lactose from products with acidic pH values, such as whey. Yeast enzymes are habitually used for products with neutral pH values [16] such as milk and sweet whey.

The mechanism of enzymatic hydrolysis of lactose by β-galactosidase applied to different substrates (lactose solutions, whey and skim-milk) under different experimental conditions has been studied by several authors. Table 2 presents the kinetic models proposed by different researchers, showing that most propose a Michaelis–Menten kinetic model, with competitive inhibition by galactose. However, there is great dispersion of the values of the kinetic constants proposed (see Table 3).

The present work provides a survey of the models proposed by different authors, presents an experimental study of enzymatic hydrolysis in a stirred tank with β-galactosidase from Kluyveromyces fragilis, and proposes a simplified kinetic model for the action of the enzyme.

Section snippets

Materials and methods

The chemical products used (PRS quality) are glucose, citric acid, K2HPO4, KCl, trichloroacetic acid (supplied by Panreac), MgCl2·6H2O (Prolabo), monohydrate lactose (Scharlau), and galactose (Across).

The enzyme used was a commercial β-galactosidase, lactozym 3000L HP-G [EC.3.2.1.23], which has a protein content of 35 g l−1, supplied by Novo Nordisk, derived from a selected strain of the yeast K. fragilis, ρ=1.2 g ml−1, with a declared activity of 3000 LAU ml−1 (1 LAU=commercial enzyme which can

Kinetic model of enzymatic hydrolysis with competitive inhibition of galactose

The widely accepted kinetic model to explain enzymatic lactose hydrolysis is competitive inhibition by the product (galactose):E+L↔ELELkEGa+GlEGaKIE+Ga

The lactose, galactose, and glucose concentrations were defined as a function of the conversion:L=L0(1−x)Ga=Ga0+L0xGl=Gl0+L0xconsidering the concentrations of the enzymatic complexes EL and EGa to be negligible.

The variation in the glucose concentration over time can be defined as:L0dxdt=(k/KM)Le1+(L/KM)+(Ga/KI)=kL0(1−x)eKM+L0(1−x)+(KM/KI)(Ga0+L

Conclusions

The conclusions of the present study can be summarized as follows:

  • The kinetic mechanism of lactose hydrolysis by the enzyme β-galactosidase responds to a model of inhibition by the product (galactose).

  • It has been verified that the enzyme has similar affinity both for the substrate (lactose) and for the product (galactose) because both compounds are capable of occupying the active site of the enzyme with equal probability, so that the constants that govern the equilibrium semi-reactions have

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