Original PapersKinetic Modeling of Lactose Hydrolysis by a β-Galactosidase from Kluyveromices Fragilis
Introduction
The hydrolysis of lactose, the sugar of milk and whey, has been the subject of intense research during the last two decades1., 2., 3. due to several reasons, but mainly the fact that lactose is a sugar scarcely digestible by non-Caucasian people.4., 5. The other reason is the environmental pollution caused by the low biodegradability of dairy industry wastes with a high content of lactose as is the case for large whey quantities from cheese production discharge.6., 7., 8. Furthermore, lactose has a low solubility and edulcorant power; the first characteristic causes crystallization and therefore technological troubles in some processes of the dairy industry, and the second makes the hydrolysis of lactose an attractive possibility to get sweeter syrup containing galactose and glucose.3
Two methods for the hydrolysis of lactose have been applied in the literature. The first is the use of acids and high temperatures (150°C) and the second is the use of enzymes (4–40°C temperature range).3 The use of enzymes allows milder conditions of temperature and pH and does not cause several factors such as the denaturalization of the protein which can be present in the lactose solution, the production of a brown color in the solution, and the yield of undesirable by-products as the acid methods does; therefore, and mainly for applications in the food industry, the better way is the enzymatic one.
There are several sources of enzymes with β-galactosidase activity. Animal and vegetable sources are not taken into account for their high cost and low production rates. The β-galactosidase enzymes from microorganisms (bacteria, yeast, and fungi) are plentiful. Among the enzymes obtained from bacteria,9., 10. the most studied is the enzyme from Escherichia coli.10., 11., 12. This enzyme is not considered safe for use in a soluble form in the food industry, because of the digestive malfunctions related to this bacteria.13 Suitable enzymes to be used in the food industry are produced by microorganisms considered safe (GRAS). For the hydrolysis of lactose in milk and dairy products, enzymes from Kluyveromyces yeasts14., 15., 16. and Aspergillus fungi17., 18., 19., 20. are acceptable. The enzymes from fungi can be used in acid wheys since their optimum pH is 3.5–4.5. The enzymes from yeasts can be used in milk and sweet wheys since their optimum pH is between 6.5–7.0.21., 22.
The hydrolysis of lactose to glucose and galactose has been modeled by several authors using enzymes from bacteria, yeast, and fungi. Some representative works which determine the kinetic model of lactose by using soluble enzymes are summarized in Table 1. The rate equations for the models proposed in Table 1 and the mechanisms which these models are obtained from are shown in Table 2. As can be seen from Table 1, more studies have been accomplished for fungal enzymes than for yeast or bacterial enzymes, and very few studies have been done fitting the data obtained at several temperatures. The enzymes from fungi, which are only available for the hydrolysis of lactose in acid wheys, are most inhibited by galactose. This type of inhibition is quite different from enzyme to enzyme, and relatively less active than enzymes from bacteria and yeasts; however, for lactose hydrolysis in milk and dairy products which have a neutral pH, enzymes from yeasts and bacteria seem to be more suitable.23
As can be seen in Table 1, the work of Carrara and Rubiolo24 is the only one studying the kinetics of lactose hydrolysis by the β-galactosidase from Kluyveromices fragilis commercialized as Lactozym, that is, the same enzyme studied in this work. These authors studied the hydrolysis reaction only at one temperature, 43°C. This is quite a high value; therefore, the enzyme is liable to be affected by some deactivation event. Moreover, the application of this enzyme in a soluble form to hydrolyze the lactose in milk is usually carried out at low temperatures (around 5°C) due to aseptic conditions or by a combination of low (5°C) and high (35°C) temperatures.23 Also, a phosphate buffer is used in the work of Carrara and Rubiolo24 which is quite different from the composition of milk.
In this work, the kinetic model of the hydrolysis of lactose using a β-galactosidase enzyme from K. fragilis supplied by Novo Nordisk Ltd. (Danbury, CT) Lactozym 3000 L, HP-G is determined. This enzyme is a semipurified preparation, so there is not an exhaustive purification of the enzyme. The enzyme is 20% of the total protein content; thus, it is interesting its application as a tree enzyme. A buffer whose salt composition is similar to the composition of milk, called BM, has been used. A wide temperature range (5–40°C) has been studied and different initial amounts of monosaccharides have been employed to get a better analysis of the product inhibition effect. Five kinetic models have been considered based on several enzymatic mechanisms. Discrimination of the kinetic model has been done applying statistical and physical criteria as performed in previous works.25., 26.
Section snippets
Materials
The enzyme, Lactozym, is a semipurified preparation supplied by Novo Nordisk Ltd. Some features of the enzyme are shown in Table 3. The enzyme preparation with an activity of 3,000 LAU has a protein content of about 35 mg ml−1. It was determined that the β-galactosidase enzyme is about 20% of this protein content. Lactose, glucose, and galactose are reagent grade and supplied by Riedel-de-Haen (Spelze, Germany). o-Nitrophenol-β-d-galactopyranoside (ONPG) employed in the reaction test to
Experimental results
A set of preliminary runs were performed to analyze the influence of pH and the thermal stability of the enzyme. This study is necessary to determine the optimal pH and the maximum temperature allowed to eliminate the deactivation of the enzyme during the kinetic study. To determine the effect of pH, enzyme activity was tested with ONPG (CONPG = 0.5 g l−1, CE = 0.7 mg l−1), and lactose (Clactose = 50 g l−1, CE = 11.33 mg l−1) as substrates using both buffers described above (called BP and BM)
Conclusions
From the results obtained in this work, the optimum temperature can be fixed at 40°C (over this value, the deactivation effect becomes important), but the enzyme keeps a significant activity even at temperatures as low as 5°C. For example, for a typical composition of 50 g l−1 of lactose and using an enzyme concentration of 7 mg l−1, the time required to achieve a conversion of lactose of 0.8 is about 70 min and 650 min for 40 and 5°C, respectively. There is an advantage in using the enzyme at
List of symbols
- CE
Enzyme concentration (mg l−1)
- CGl
Glucose concentration (m)
- CGa
Galactose concentration (m)
- CL
Lactose concentration (m)
- CM
Monosaccharide concentration
- CONPG
ONPG concentration
- CP
Product concentration
- CS
Substrate concentration
- Ea
Activation energy (J mol−1)
- k
Kinetic constant (1 mg−1 min−1)
- k0
Kinetic constant at T = ∞ K (mol mg−1 min−1)
- Km
Michaelis-Menten constant (mol l−1)
- Km0
Michaelis-Menten constant at T = ∞ K (mol l−1)
- Ki
Inhibition equilibrium constant (mol l−1)
- Ki0
Inhibition constant at T = ∞ K (mol l−1)
- r
Acknowledgements
The authors wish to thank Novo Nordisk who has supplied the enzyme and gave us various information for the characterization of the enzyme. Also, the financial support from C.A.M. is gratefully recognized.
References (38)
- et al.
Beta-galactosidaseReview of recent research related to technological application, nutritional concerns, and immobilization
J. Dairy Sci.
(1981) Review of processes and products for utilization of lactose in deproteinized milk serum
J. Dairy Sci.
(1984)- et al.
Immobilization of β-galactosidase on an insoluble carrier with a polyisocianate polymer. I. Preparation and properties
J. Dairy Sci.
(1973) - et al.
Reduction of lactose content of milk by entrapped β-galactosidase. I. Characteristics of β-galactosidase from yeast and Escherichia coli
J. Dairy Sci.
(1973) - et al.
Determination of kinetics parameters for free and immobilized β-galactosidase
Process Biochem.
(1996) - et al.
Product inhibition of the enzymatic hydrolysis of lactose
Enzyme Microb. Technol.
(1985) - et al.
Studies on modelling and simulation of lactose hydrolysis by free and immobilized β-galactosidase from Aspergillus niger
Chem. Eng. J.
(1993) - et al.
Use of immobilized lactase in milk systems
Adv. Biochem. Eng. Biotechnol.
(1980) - Nijpels, H. H. Lactases and their applications. In: Enzymes and Food Processing (Birch, G. G., Blakebrough, N., and...
- et al.
Hydrolysis of lactoseA literature review
Proc. Biochem.
(1985)
Beta-galactosidase technologySolution to the lactose problem
CRC Critical Reviews in Food Technology
Enzymatic whey hydrolysis in the pilot plant “Lactohyd”
Biotechnology
Effects of temperature on lactose hydrolysis by immobilized β-galactosidase in plug-flow reactor
Biotechnol. Bioeng.
Hydrolysis of lactose in skim milk by immobilized β-galactosidase (B. circulans)
Biotechnol. Bioeng.
Preparation and use of porous sheets with enzyme action
Nature
β-galactosidase
Hydrolysis of β-galactosides using polymer-entrapped lactase
Biotechnol. Bioeng.
Reduction of lactose in milk by entrapped β-galactosidaseResults of long-term experiments on pilot plant
Milchwissenshaft
Cited by (106)
Industrial production of carbohydrate-degrading enzymes
2023, Valorization of Biomass to Bioproducts: Biochemicals and BiomaterialsEnzyme-catalyzed cascade reactions on multienzyme proteinosomes
2022, Journal of Colloid and Interface ScienceCitation Excerpt :Native enzymes were employed in the synthesis of multienzyme proteinosomes and the enzyme-catalyzed cascade reactions on the surfaces were investigated. As shown in Fig. 6a, in the cascade reactions lactose is hydrolyzed to glucose and galactose by β-gal, GOx catalyzes the oxidation of glucose to gluconolactone and H2O2, and H2O2 acts as a cosubstrate for the HRP catalyzed oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) [62]. To perform the cascade reactions, lactose and TMB were added to the proteinosome solution, and the reactions were monitored by measuring the absorption of the oxidized TMB at 652 nm.
Added value of ionic liquids in a biocatalytic process: An automatic approach
2021, Process Biochemistry