β-Galactosidase entrapment in silica gel matrices for a more effective treatment of lactose intolerance
Graphical abstract
Highlights
► A new pharmaceutical formulation for the treatment of lactose intolerance. ► A reliable and reproducible procedure for the immobilization of β-galactosidase. ► A one-pot entrapment of β-galactosidase in a silica network was optimized. ► The entrapment in a silica matrix can prolong the therapeutic action of lactase. ► The sol–gel technique preserves the stability of the enzyme.
Introduction
Lactose intolerance is a very common disorder due to the inability to digest lactose into its constituents, glucose and galactose, because of low levels of lactase.
Lactase, an enzyme of the β-galactosidase (EC 3.2.1.23) family, is produced on the brush border of the small intestine and is responsible for the hydrolysis of lactose into its constituents [1]. Enzyme levels reach their maximum shortly after birth, but they decline with aging: it is estimated that 75% of adults worldwide shows some decrease in lactase activity during adulthood [2]. Lactase deficiency can result in lactose maldigestion, but only in presence of clinical symptoms such as abdominal bloating and pain, flatulence, diarrhea, nausea and borborygmi lactose intolerance occurs [3]. The diagnosis or even the suggestion of lactose intolerance leads many people to avoid milk and/or to consume food prepared with digestive aids. The treatment of lactose intolerance includes four general principles: (i) reduction or restriction of dietary lactose, (ii) substitution with alternate nutrient sources to avoid reductions in energy and protein intake, (iii) regulation of calcium and vitamin D intake and (iv) use of exogenous β-galactosidase [4], [5], [6]. In particular, the intake of digestive supplements is a successful way to alleviate the symptoms of lactose intolerance; actually a number of lactase preparations is commercially available. These supplements are formulated in tablet or capsule form to be taken just before or with meal [7] and the proper dosage should be suited as a function of the seriousness of the symptoms; the final formulation of these preparations consists of a capsule which delivers the enzyme in the small intestine, where it can carry out its therapeutic action.
Enzyme therapy is promising in the treatment of several diseases, in particular of in-born enzyme deficiencies, but many limitations exist for the clinical use of native enzymes because, in addition to the cost, they are unstable and have a short lifetime in the circulation [8]. The polymeric structure of enzymes, in fact, is stabilized by a large number of low-energy bonds, so deactivation is relatively easy. The inactivation phenomena of an enzyme can be of inter- or intramolecular nature: intermolecular phenomena include autolysis and aggregation, while the intramolecular phenomena are due to interactions of the enzyme with poisons such as irreversible inhibitors, or to extremes values of pH or temperature [9].
In the small intestine lactase is exposed to a deactivating environment because of the presence of inhibitors (glucose and galactose) and proteases. Moreover, when taken orally, β-galactosidase has to cross the acid pH of the stomach which can compromise the structural integrity of the enzyme and, as a consequence, its activity in the hydrolysis of lactose: if lactose passes indigested from the small intestine into the colon it can cause physiological effects that result in the clinical manifestations typical of lactose intolerance.
The major lack of the commercial lactase supplements is then the rapid inactivation of the enzyme, which shortens the activity of β-galactosidase thus forcing to several assumptions during the day.
Immobilization can make enzymes more stable and impart a longer circulation lifetime in the organism [8]. It is well known that the immobilization of enzymes (physical adsorption, covalent bonding, gel entrapping, etc.) on proper matrices can prevent their chemical and biological degradation and enhance their stability [10], [11], [12]; it is then potentially possible to prevent the denaturation of β-galactosidase in the intestine and prolong its therapeutic action in the course of time by its entrapment inside a matrix. The entrapment of β-galactosidase within a porous matrix allows to avoid a direct contact of the enzyme with the surrounding medium, thus increasing its stability, but, at the same time, it enables the reagents to reach the catalytic site.
A large number of different supports has been used for lactase immobilization [13], [14], [15], [16], but in the case of a pharmaceutical application the support must possess proper features, biocompatibility at first. In the last years, several ceramic and inorganic oxides have attracted the attention of researchers for their potential application in the biomedical field [17]; in this context silica has gained increasing importance and its use for the entrapment of enzymes, antibody, cells and for the design of controlled drug delivery systems has been investigated [18], [19], [20], [21], [22], [23], [24]. In fact, silica matrices show high biocompatibility–biodegradability, resistance to microbial attack and exhibit higher mechanical strength, enhanced thermal stability and negligible swelling in organic solvents compared to most organic polymers [25], [26], [27], [28]. In addition, silica possesses physico-chemical and textural properties (hydrophilicity/hydrophobicity, surface area, pore volume, etc.) that can be modulated ad hoc according to the final application and the nature of the guest molecule.
The choice of the proper immobilization technique is fundamental to preserve to a high degree the structural integrity of the enzyme, which is related to its catalytic activity; an effective technique for this purpose is the sol–gel process [29], [30]. The sol–gel method is particularly attractive for the entrapment of biological molecules. It is characterized by a number of unique features including: (i) mild operative conditions (in particular low temperature); (ii) high versatility; (iii) encapsulation of a guest molecule in the inorganic matrix by a one-step approach [28], [29], [30], [31]. It is now well established that a wide variety of enzymes and other proteins retains its characteristic reactivity and chemical function when confined within the pores of the sol–gel derived matrix, which isolates the biomolecules protecting them from self-aggregation [32]. It is important to highlight that protein molecules encapsulated in a sol–gel matrix are not covalently bound to the support, but they are physically entrapped in the gel network that has grown around them [33]. This can prevent the enzyme denaturation (chemical modification of the protein) that frequently occurs in the presence of covalent linkages between the matrix and the biological molecule and guarantees a sufficient mobility to the enzyme, which experiences conformational changes when it binds the reagent molecule.
Another important parameter in the design of a pharmaceutical system for the treatment of lactose intolerance is the choice of the proper source of the enzyme.
β-Galactosidase, in fact, can be obtained from a wide variety of sources, such as microorganisms, plants and animals, but its properties differs markedly according to the source. In particular it is well known that the optimum pH of enzymes obtained from fungi is 3.5 to 4.5, while β-galactosidases from yeasts have their optimum pH between 6.5 and 7.0 [34]. The commercially available lactase supplements contain a β-galactosidase obtained from GRAS yeasts or fungi but, considering that in the small intestine the pH is close to neutrality, β-galactosidase from yeasts should be more suitable for this application.
The aim of this work has been the optimization of a reliable procedure for the synthesis of a stable β-galactosidase/silica gel composite by using a sol–gel approach. In particular we have investigated the effect of several operative parameters (pH, aging time, enzyme amount, etc.) on the final features (structural and physico-chemical) of the system and subsequently on its stability. This is a preliminary study directed to the realization of a new pharmaceutical formulation for the treatment of lactose intolerance. The main aim is the design of a stable system able to explain its therapeutic action for a long period of time, thus avoiding the frequent administrations required by the traditional commercial preparations.
Section snippets
Materials
Lactozym 3000L®, a liquid preparation of β-galactosidase from Kluyveromyces lactis (specific activity: 4097 U/mL), Tetraethoxysilane (TEOS) (98%, Aldrich), H2O milliQ, Potassium Phosphate monobasic (99%, Aldrich), Potassium Phosphate dibasic (≥98%, Aldrich), o-nitrophenyl-β-d-galactopyranoside (99%, o-NPG, Aldrich), o-nitrophenol (99%, o-NP, Aldrich). All reagents have been used as received.
Synthesis
Enzyme/silica composites were synthesized by a one-step sol–gel process. In a typical experiment, the
Synthetic procedure
It is well known that the sol–gel process is a suitable technique for the immobilization of a wide series of molecules in a porous matrix; we have already verified the effectiveness of the process in the encapsulation of enzymes [19] and in the design of controlled drug delivery systems by the entrapment of several drugs in silica gel matrices [23], [24], [37]. However, in the design of the process parameters it is fundamental to consider the nature of the guest molecule and the final
Conclusions
The obtained results can be summarized in the following points:
- 1.
The optimized sol–gel approach is an effective technique for the immobilization of β-galactosidase in a silica matrix. After 21 days since the gel preparation it is possible to obtain monolithic and homogeneous β-galactosidase/silica gel systems, which are stable in terms of textural properties, enzyme content and catalytic activity.
- 2.
The silica matrix protects the enzyme molecules from the external environment, preserving their
Acknowledgement
The authors want to thank Mrs. Tania Fantinel for the excellent technical assistance.
References (40)
- et al.
Best Pract. Res. Clin. Gastroenterol.
(2006) - et al.
Food Chem. Toxicol.
(2000) Adv. Drug Deliv. Rev.
(1987)- et al.
Enzyme Microb. Technol.
(2006) - et al.
J. Mol. Catal. B: Enzym.
(1998) - et al.
Catal. Today
(2003) - et al.
J. Non-Cryst. Solids
(1999) - et al.
Micropor. Mesopor. Mater.
(2005) - et al.
Micropor. Mesopor. Mater.
(2010) - et al.
J. Control. Release
(2005)