Encapsulation of enzymes in mesoporous host materials via the nonsurfactant-templated sol–gel process

doi:10.1016/S0167-577X(99)00287-6

Materials Letters

Volume 44, Issue 1, May 2000, Pages 6-11

https://doi.org/10.1016/S0167-577X(99)00287-6 Get rights and content

Abstract

A new, simple, one-step method for enzyme immobilization is demonstrated with encapsulating alkaline phosphatase in mesoporous sol–gel silica. Since the pore size (e.g., ∼30 Å) of the mesoporous silica is much greater than that (e.g., ∼15 Å) in traditional microporous sol–gel silica, enzymatic activity is increased by a factor of 2 to 10 because of easy diffusion of the substrate molecules. Compared to macroporous materials, the channel is narrow enough to prevent the enzyme from leaking out of its cage.

Introduction

Immobilization of enzymes has been a subject of extensive research efforts because of its immense technological potentials [1], [2], [3]. Among the popular methods is formation of chemical bonding between enzymes and a solid support, which often alters the enzymatic activities. A variety of enzymes and other bioactive substances have also been entrapped in inorganic oxides such as silica for biocatalysis and biosensor applications through conventional sol–gel process [4], [5], [6], [7], [8], [9], [10]. However, because of the microporous nature (i.e., typical pore diameter ≤15 Å and pore volume ≤0.25 cm³ g⁻¹) of conventional sol–gel materials, the catalytic activities of enzymes are hindered by low diffusion rates of substrate molecules and poor accessibility of enzymes inside the materials [4], [5]. Recently, we have developed a nonsurfactant-templating sol–gel route to mesoporous materials [11], [12], [13], in which nonsurfactant organic molecules such as d-glucose, d-maltose, dibenzoyl tartaric acid, etc., were employed as templates or pore-forming agents. Upon removal of the templates by simple extraction, mesoporous silica materials [11], [12] and alumina [13] materials were obtained with large surface areas and pore volumes as well as narrow pore size distributions.

Here we present a new, one-step method for enzyme immobilization by direct encapsulation of enzyme molecules in mesoporous host materials through the nonsurfactant-templating sol–gel route. Since the pore diameter and volume (e.g., ∼30 Å and ∼0.6 cm³ g⁻¹, respectively) in the mesoporous materials are greater than those in conventional sol–gel materials, significantly increased enzymatic activities by a factor of 2 to 10 are achieved. On the other hand, the interconnecting pore size is small enough to prevent the enzyme from leaking out of its cage. This new method is demonstrated in this report by encapsulating alkaline phosphatase (ALP) in mesoporous silica via d-glucose-templated sol–gel reactions of tetraalkyl orthosilicate.

Section snippets

Experimental

The encapsulation of ALP was achieved by hydrolysis and polycondensation, i.e., sol–gel reactions [18], [19], [20], [21], of tetramethyl orthosilicate (TMOS, Aldrich) and tetraethyl orthosilicate (TEOS, Aldrich) in the presence of d-glucose as the nonsurfactant template and ALP at room temperature. The TMOS protocol [6] was modified by introducing d-glucose to the system [11]. As a typical procedure, to a mixture of 5.0 ml of TMOS and 6.0 ml of methanol in a 25-ml beaker, 0.10 ml of 1.0 mmol l⁻¹

Results and discussion

All the sol–gel silica samples were characterized with TGA, IR and N₂ adsorption–desorption isotherms. The removal of the glucose template by water extraction was confirmed by TGA and IR measurements, which showed little weight loss at 750°C and diminished glucose CH absorption band at ∼2940 cm⁻¹, respectively. Brunauer–Emmett–Teller (BET) surface area and pore parameters were determined by N₂ adsorption–desorption isotherms [23] and are summarized in Table 1. Fig. 1 shows N₂

Summary

We have described a simple, one-step method for immobilization of enzymes by encapsulating enzymes such as ALP in mesoporous silica host materials via the sol–gel reactions of tetraalkyl orthosilicate in the presence of a nonsurfactant template such as d-glucose. The enzymatic activities are significantly higher than those observed for encapsulated ALP in conventional microporous sol–gel materials. It is noted that most of surfactant-templated routes to mesoporous materials involve reagents or

Acknowledgements

This work was supported in part by NIH (No. DE09848) and by Drexel University through a Research Achievement Award to Y.W. We thank Dr. A.W. Addison for discussion.

References (25)

S. Braun et al.
Mater. Lett.
(1990)
C.G. Kauffmann et al.
J. Biotechnol.
(1996)
E.E. Kim et al.
J. Mol. Biol.
(1991)
P. Gemeiner (Ed.), Enzyme Engineering: Immobilized Biosystems, translation: J.L. Francis (Ed.), Ellis Horwood, West...
D. Avnir et al.
Chem. Mater.
(1994)
B.C. Dave et al.
Anal. Chem.
(1994)
S. Shtelzer et al.
Biotechnol. Appl. Biochem.
(1996)
M. Reetz et al.
Angew. Chem. Int. Ed. Engl.
(1995)

U. Narang et al.

Chem. Mater.

(1994)

Y. Wei et al.

Adv. Mater.

(1998)

Cited by (109)

Recent progresses in the adsorption of organic, inorganic, and gas compounds by MCM-41-based mesoporous materials
2020, Microporous and Mesoporous Materials
Adsorbent mesoporous materials are highly efficient for the remediation of different compounds in environmental applications such as organic, inorganic, and gas molecules. These compounds are removed by the traditional adsorption process in batch assessments. In this review, we report the recent progress on the MCM-41-based mesoporous material syntheses and its strategic use as an adsorbent material for the removal of different pollutants. Also approached the main factors that affect its synthesis and the methods of functionalization most used to prepare the MCM-41-based functionalized mesoporous materials. On the other hand, the factors that affect the adsorption process were also investigated. In addition, it is possible to affirm that adsorption technology has attracted a lot of attention in the removal of environmental pollutants, mainly due to the fact that it is a technique that requires the use of few steps and equipment, easy operation, low cost, does not generate residues or by-products, eco-friendly, and high energy efficiency, which allow its wide application on the remediation processes.
Silica nanowires with tunable hydrophobicity for lipase immobilization and biocatalytic membrane assembly
2018, Journal of Colloid and Interface Science
Citation Excerpt :
These properties benefit mesoporous silicate materials to accommodate various enzymes with high loading [10,23]. However, activity of enzymes immobilized in mesopores could be compromised in reactions when the substrates are difficult to diffuse into the confined space. [26–29]. An alternative strategy to tackle these issues is to immobilize enzyme on low dimensional substrates (e.g., nanowires, nanorods, and nanosheets), which possess high external surface areas for accommodating enzyme with more accessible active sites [30–32].
Silica nanowires (NWs) with tailored hydrophobicity are synthesized by capping different length of alkyl groups on surface via a one-pot anisotropic sol-gel growth approach. Lipase from Burkholderia Cepacia (BCL) is successfully immobilized onto the silica NWs via hydrophobic interaction. The specific activity of the immobilized BCL increases with the increasing length of the capping alkyl groups and surface hydrophobicity of the NWs. BCL immobilized onto the octadecyl groups-capped silica NWs displays the highest specific catalytic activity, which is also notably higher than that of BCL immobilized on octadecyl groups-modified mesoporous silicate. The superior performance can be ascribed to the combination of the interfacial activation of lipases induced by capped-octadecyl groups on the NWs and the improved mass transfer efficiency of reactants around the one-dimensional silica NWs. The BCL-loaded NWs are further used as “building blocks” to assemble filter paper-like biocatalytic membrane via vacuum-assisted filtration method. The free-standing biocatalytic membrane can be operated in a continuous mode to avoid the separation of catalyst from reaction products. This work provides new opportunity in enzyme immobilization and biocatalytic membrane preparation through using discrete silica NWs as supports and building blocks.
Formulation and characterization of chitosan encapsulated phytoconstituents of curcumin and rutin nanoparticles
2017, International Journal of Biological Macromolecules
Citation Excerpt :
The promising attributes of nanoparticles play a significant role when formulated with good drug carriers. Chitosan is a natural polymer prepared by the N-deacetylation of chitin, found in the shells of marine crustaceans, which shows biocompatible, hemocompatible, biodegradable and high drug loading efficiency when used as polymers for the formulation of nanoparticles [3–7]. Curcumin has proven to overcome the multi-drug resistance in cancer therapy with many combinations which include Dasatinib [8], Quercetin [9], Bicalutamide [10], α-Tomatine [11], Berberine [12], Green Tea Catechins [13] etc.
Curcumin and Rutin are natural polyphenolic molecules exhibits several pharmacological actives like antibacterial, anticancer, antioxidant, chemo-preventive and anti-inflammatory properties. However till date, no studies have been reported on their combination efficacy, especially in treating multi-drug resistance of cancers because of their poor solubility and bioavailability. Hence in the present study, an attempt has been made to load both these drugs into a single nanoparticlulate system to enhance their bioavailability and efficacy. This novel formulation was prepared by solvent evaporation technique and was evaluated for particle size and shape using Zeta Sizer, Scanning Electron Microscopy (SEM) and Fourier Transform Infra Red (FT-IR) Spectroscopy. The optimized formulation was further subjected to in vitro and in vivo evaluations. The prepared nanoparticles were in the size range of 25–100 nm and the release profile was found to be Non −Fickian transport. In-vivo pharmacokinetic studies were carried in rabbits and the pharmacokinetic profile was studied. The results indicate that oral bioavailability of Curcumin and Rutin has been increased to 3.06 and 4.24 folds respectively when compared to their pure drugs. This data suggest that the present novel nanoparticles loaded with these combinational drugs may have better therapeutic potential in treating drug resistant cancers.
Lipase immobilization for catalytic applications obtained using fumed silica deposited with MAPLE technique
2016, Applied Surface Science
Citation Excerpt :
A different approach is enzyme encapsulation in which the enzyme is added in the early stage of the formation of a structure like polymer or sol–gel. This generally improves not only the enzyme activity but also the enzyme stability [14–16]. On the other hand the way nanoparticles aggregate has been studied [17] observing that “two-scale” (nano- and micro-) aggregation occurs, depending on the surface and the concentration of the nanoparticles suspension.
Lipases are enzymes used for catalyzing reactions of acylglycerides in biodiesel production from lipids, where enzyme immobilization on a substrate is required.
Silica nanoparticles in different morphologies and configurations are currently used in conjunction with biological molecules for drug delivery and catalysis applications, but up to date their use for triglycerides has been limited by the large size of long-chain lipid molecules.
Matrix assisted pulsed laser evaporation (MAPLE), a laser deposition technique using a frozen solution/suspension as a target, is widely used for deposition of biomaterials and other delicate molecules. We have carried out a MAPLE deposition starting from a frozen mixture containing fumed silica and lipase in water. Deposition parameters were chosen in order to increase surface roughness and to promote the formation of complex structures. Both the target (a frozen thickened mixture of nanoparticles/catalyst in water) and the deposition configuration (a small target to substrate distance) are unusual and have been adopted in order to increase surface contact of catalyst and to facilitate access to long-chain molecules. The resulting innovative film morphology (fumed silica/lipase cluster level aggregation) and the lipase functionality (for catalytic biodiesel production) have been studied by FESEM, FTIR and transesterification tests.
Immobilization of Candida Antarctica lipase B on epoxy modified silica by sol-gel process
2016, Journal of Molecular Catalysis B: Enzymatic
Citation Excerpt :
For avoiding these defects, various methods for enzyme immobilization have been carried out, such as physical methods(adsorption, entrapment, encapsulation) and chemical methods (covalent bonding and cross-linking). Silica with large surface area and pore volumes exhibits exceptional adsorptive affinity with enzymes and the silica supports have good biocompatibility, high specific surface area and good mechanical properties [4–6]. Some researchers entrapped the enzyme in pure silica matrixes to achieve immobilized enzyme by a sol-gel process [7–11].
An epoxy-functionalized silica support was prepared through 3-glycidoxypropyltrimethoxylsilane(KH560)modified silica sol in the presence of triblock copolymer F127 (F-560-S). The surface chemistry, micromorphology and pore structure of the supports were characterized by TG, FTIR, SEM and N₂ adsorption-desorption, which showed that the BET surface areas and pore volumes of epoxy-activated supports increased with the addition of F127. The Candida Antarctica Lipase B (CALB) was immobilized on the resulting carrier by covalent link. The lipase absorbed amounts were 58 mg/g support and 375 mg/g support on the normal silica (N-S) and modified silica (F-560-S), respectively. The immobilized lipases were examined as biocatalysts for transesterification of 1-phenethanol and vinyl acetate in nonaqueous medium. Solvents and temperature were investigated for influence of morphology and KH560 on the immobilized lipase. The catalytic activity of the CALB immobilized on F-560-S was improved in various solvents, especially in polar solvents. The immobilized CALBs maintained high activity, while the control experiment using free lipase gave very low ester production in the temperature range of 0–60 °C. Furthermore improved thermal stability of CALB immobilized on F-560-S compared to the CALB on N-S was observed. The CALB immobilized on F-560-S exhibited high operational stability in organic media which still retained 88.3% of its original activity for 12 h consecutive 7 runs.
Enzyme Immobilization for Organic Synthesis
2016, Organic Synthesis Using Biocatalysis
Enzyme immobilization has achieved important goals during the recent years trying to overcome the drawbacks of free enzyme catalysis. Up to now many strategies can be used in order to immobilize enzymes, some methods can be very simple such as adsorption or more complex as covalent immobilization. This chapter will cover some aspects of enzyme immobilization and its use on organic synthesis transformations.

View all citing articles on Scopus

View full text

Encapsulation of enzymes in mesoporous host materials via the nonsurfactant-templated sol–gel process

Abstract

Introduction

Section snippets

Experimental

Results and discussion

Summary

Acknowledgements

Mater. Lett.

J. Biotechnol.

J. Mol. Biol.

Chem. Mater.

Anal. Chem.

Biotechnol. Appl. Biochem.

Angew. Chem. Int. Ed. Engl.

Chem. Mater.

Adv. Mater.