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Covalent Enzyme Immobilization
Abstract
IntroductionThe Past The Early Days (1916–1940s)The Underdeveloped Phase (1950s)The Developing Phase (1960s)The Developed Phase (1970s)The Post-developed Phase (1980s)Rational Design of Immobilized Enzymes (1990s–date)Immobilized Enzymes: Implications from the Past Methods of ImmobilizationDiversity versus VersatilityComplimentary versus AlternativeModification versus Immobilization Enhanced StabilityEnhanced ActivityImproved SelectivityProspective and Future Development The Room for Further DevelopmentAn Integration ApproachReferences The Early Days (1916–1940s)The Underdeveloped Phase (1950s)The Developing Phase (1960s)The Developed Phase (1970s)The Post-developed Phase (1980s)Rational Design of Immobilized Enzymes (1990s–date) Methods of ImmobilizationDiversity versus VersatilityComplimentary versus AlternativeModification versus Immobilization Enhanced StabilityEnhanced ActivityImproved Selectivity Enhanced StabilityEnhanced ActivityImproved Selectivity The Room for Further DevelopmentAn Integration Approach
... Weitere Verfahren sind z. B. Einschlussverfahren in Silika-Gelmatrices [Smirnova 2002] oder die Anbindung an Membranoberflächen [Cao 2005] , wobei jedes Verfahren seine Vor-und Eine weitere Methode zur Modifizierung der Oberfläche stellt die Layer-by- Layer (LbL) Technik dar (Abbildung 1–2). Diese findet bei der Immobilisierung von Enzymen bisher allerdings keine Anwendung. ...
... Die Wahl des Trägermaterials erfolgt im Idealfall nach den Anforderungen des späteren Prozesses. Die grundlegenden Anforderungen an das Trägermaterial sind neben der großen Oberfläche , ein Porendurchmesser von 50-100 nm (ab etwa 100 nm kann die Diffusionslimitierung vernachlässigt werden [Cao 2005] ), ein geringes Quellvermögen und mechanische ), 100 µL 1 mM NADH, 8830 µL puffergesättigtem t-Butylmethylether, Substrat: Acetophenon 60 mM, 10 % V V -1 Isopropanol, t = 30 °C, V = 10 mL, 250 rpm, n = 2 (+/-0,11), Aktivität Immobilisat: 9,2 U g . Dafür muss die Katalysatordichte allerdings hoch genug gewählt werden, um ökonomische Umsätze realisieren zu können. ...
... Neben der Wiederverwendbarkeit und einer hohen Aktivität und Stabilität der Immobilisate, kann durch die Immobilisierung die pH-und die Temperaturstabilität beeinflusst werden [Cao 2005] . Diese Versuche sind im Folgenden dargestellt. ...
Alcohol dehydrogenases have been immobilized adsorptivly and covalently on surface modified particles (modified through the layer-by-layer technique,e.g. polyethylene imine). The immobilized enzymes have been subsequently applied in a plug-flow reactor to produce short chain alcohols (2,5-hexanediol). The reaction product was isolated through adsorption on a solid phase of alumina oxide and alumina silicate and was obtained in high purity.
... The utilization of nanofibers as a support for enzymes immobilization was first reported by Jia et al. in 2002 [2]. Thus immobilized, the enzymes offer such benefits vis-à-vis their use in soluble forms as controllability of the reaction, reusability, and often both enhanced stability and activity [3][4][5][6]. Electrospun nanofibers have been proven to provide excellent support for enzyme immobilization [7] because of their high specific surface area-to-volume ratios, mass transfer resistance, and effective loading [8,9]. Moreover, because they can be manufactured in various formats and from different synthetic and natural polymers, they afford great flexibility in surface functional- ity [9]. ...
... As has been described in numerous reviews, the methods applied for enzyme immobilization on nanofibers do not differ from standard bioconjugation techniques [33] and include adsorption, covalent immobilization , enzyme entrapment and encapsulation [5,34,35]. From this list, covalent bonding is often preferred because it provides stable linkage between enzymes and nanofibers. Glutaraldehyde crosslinking [32, 36] and carbodiimide chemistry [37,38] are two covalent techniques most commonly applied for biofunctionalization of nanofibers and typically result in high loading yields. ...
... It also testifies about the quality of the binding of the enzyme to the carrier. Covalent enzyme immobilization typically improves the stability of enzymes [5], as has been reported several times already when nanofibers have been used as support [41] . These parameters are particularly important in case of enzymes with the ability of autolysis, as trypsin . ...
The electrospun chitosan nanofibers provide excellent material for immobilized proteolytic enzymes, and are biocompatible, nontoxic and hydrophilic matrices with large specific area. This paper deals with an application of electrospun chitosan nanofibers and optimizing conditions for their biofunctionalization by model proteolytic enzyme trypsin. Nanofibers from chitosan were prepared using NanospiderTM technology and covalent immobilization of trypsin followed. Three immobilization techniques preserving biocompatibility and utilizing amine and/or hydroxyl groups of chitosan were optimized and compared to simple adsorption to achieve maximum proteolytic activity per cm2 of the functionalized chitosan nanofibers (Tryp-NF). Significant differences were observed. Trypsin immobilized by the carbodiimide one-step protocol demonstrated the highest activity of the three procedures, ranging from 132 to 210 IU/cm2 (i.e., 548–874 IU/mg of nanofibers), depending on the initial amount of trypsin used. Long-term storage stability together with high reusability of Tryp-NF confirmed advantages of the immobilized enzyme. Tryp-NF showed no cytotoxicity toward growth of HeLa cells. The in vivo tests for irritation and skin sensitization demonstrated no undesirable skin reactions.
... Depending on the pH of the solution and the isoelectric point of the protein, its conformation and charge can vary. The protein can be negatively (for instance carboxylate) or positively charged (for instance protonated amino groups), therefore, an ion exchanger can act as carrier via ionic and strongly polar interactions [20,34]. Techniques of layer-by-layer deposition and electrochemical doping method based on electrostatic adsorption have been employed to develop enzymatic biosensors [19] . ...
... Despite of its simplicity and other advantages such as improved protein conformation stability, and retention of the intrinsic electronic and optical properties of the carbon nanoparticles, physical binding may be insufficient for the stable immobilization under harsh industrial conditions. Durability and leaching are always a concern for the application of adsorption methods [34,44] . Additionally , there is the risk of protein partial unfolding, since hydrophobic regions normally buried within the protein's tertiary structure may be exposed upon hydrophobic interaction with the carbon nanoparticle. ...
... Most commonly, covalent binding of an enzyme or a protein to a nanoparticle carrier is based on chemical reactions between the side group of amino acid residues located on the protein surface and functionalized group available on the particle surface. Often, carriers are activated before binding to enzymes [34], usually by directly introducing electrophilic functionalities on the support surface [34,46]. Various functional groups can be easily incorporated on the surface of carbon nanotubes by chemical modification, such as carboxylation, acylation, amidation, esterification, PEGylation, or via polymers wrapping, some of which are non-covalent while the rest are covalent functionalization. ...
... The adsorption of lipase in mesoporous microspheres was still inclined to be monolayer adsorption because the mesopores restricted the distribution of lipase molecules (the pore size is 14.7 nm; the molecular size of CALB is 6.92ˆ592ˆ92ˆ5.05ˆ805ˆ05ˆ8.67 nm 3 [28]). Since the monolayer adsorption of lipase was dominant in mesoporous microspheres, the enzyme activity and the specific activity were obviously lower than those in the gigaporous ones. ...
... Figure 6showed the adsorption parameters of lipase on PST chips. [28]). Since the monolayer adsorption of lipase was dominant in mesoporous microspheres, the enzyme activity and the specific activity were obviously lower than those in the gigaporous ones. ...
Compared with the one immobilized in the conventional mesoporous microspheres, the enzyme immobilized in gigaporous microspheres showed much higher activity and better stability. To gain a deeper understanding, we herein selected lipase as a prototype to comparatively analyze the adsorption behavior of lipase at interfaces in gigaporous and mesoporous polystyrene microspheres at very low lipase concentration, and further compared with the adsorption on a completely flat surface (a chip). Owing to the limited space of narrow pores, lipase molecules were inclined to be adsorbed as a monolayer in mesoporous microspheres. During this process, the interaction between lipase molecules and the interface was stronger, which could result in the structural change of lipase molecular and compromised specific activity. In addition to monolayer adsorption, more multilayer adsorption of enzyme molecules also occurred in gigaporous microspheres. Besides the adsorption state, the pore curvature also affected the lipase adsorption. Due to the multilayer adsorption, the excellent mass transfer properties for the substrate and the product in the large pores, and the small pore curvature, lipase immobilized in gigaporous microspheres showed better behaviors.
... On the other hand, the activity of the laccase may be reduced (Barrios-Estrada et al., 2018); this can come from reducing the free movement of the enzyme, but also its active site may be blocked by the immobilisation agent. Regarding the nature of the bond formed between the enzyme and its carrier, five types of immobilisation techniques can be distinguished: encapsulation (Datta et al., 2013), adsorption (Jesionowski et al., 2014), cross-linking (Yamaguchi et al., 2018), affinity interaction (Andreescu et al., 2006), and covalent bond (Cao, 2006). Each technique has its advantages and disadvantages, and considering these, one needs to find the most suitable method for the given process. ...
Micropollutants are persistent and hazardous materials in low concentrations (ng L⁻¹–μg L⁻¹), including substances such as pharmaceuticals, personal care products and industrial chemicals. The advancement of analytical chemistry has allowed for the detection of micropollutants; however, an efficient and economical treatment solution is yet to be installed. Fungal laccase has been a successful biocatalyst of these compounds. However, large-scale application of free enzyme is currently not feasible for removing water-borne micropollutants, partly due to relatively rapid loss in enzyme stability. In this paper, three types of cyclodextrin, α, β and γCD, were chosen to immobilise the laccase under various conditions with the aim to improve the stability of the enzyme. Laccase activity was chosen as a response parameter, and laccase-cyclodextrin binding was evaluated by Fourier-transform infrared spectroscopy (FTIR). Results showed an optimum using α-cyclodextrin immobilisation. At that level, α-cyclodextrin increased the half-life of laccase and slightly improved its activity in all tested pH by physically bonding to laccase. By protecting the enzyme structure, activity was maintained under a range of circumstances (acidic conditions, from 10 to 50 °C). Under room temperature and at pH 5, α-cyclodextrin-laccase nanocomposite had a better removal efficiency of diclofenac compared to free laccase of the same concentration.
Graphical abstract
... Therefore, immobilization approaches such as entrapment or encapsulation can be more preferred in some cases. However, a covalent bond is usually rigid enough to minimize the leaking of the enzyme from carrier support (Cao, 2006). On the other hand, chemical immobilization by covalent bonding on carrier substrates and self-immobilization may alter the structure of the enzyme (Velasco-Lozano et al., 2016). ...
The demand for large-scale production of pure and functional proteins via cost-effective and simple methods is highly emerging at present. In biotechnology, proteins are synthesized in heterologous systems, since natural sources do not always allow satisfactory yields and purity of desired proteins. Heterologous systems are focused to produce a high amount of an expressed protein, often leading to translation mechanism overload. As a result, insoluble aggregates of the protein called inclusion bodies (IBs) are formed. Since the beginning of recombinant protein production, the formation of IBs has been seen as an obstacle and great afford has been made to prevent their presence. On the contrary, many independent studies in the recent decade challenged this wide-accepted opinion and proved the huge potential of IBs. This review focuses on the benefits of tailored-made production of IBs and their emerging use as self-immobilized catalysts used in the synthesis of several industrially interesting products, as well as on their utilization in different areas.
... As for enzyme immobilization through purely ionic forces between the enzyme and support, it is based on the protein ligand interaction principles used in chromatography, namely the reversible immobilization of enzymes which was first used in ion exchangers (Tosa et al., 1966). Depending on the pH of the solution and the isoelectric point, the surface of the enzyme may bear charges (Cao, 2005) and its charge distribution can be readily calculated and displayed using current available modelling systems (Hanefield, 2008). Any ion exchanger can act as carrier in immobilization via ionic and strongly polar interactions. ...
... The above mentioned problems may be solved by combination of techniques to overcome such disadvantages. It may be required to apply modified methods and develop a useful and successful technique [13]. In covalent binding, at first, support must be activated with coupling agents such as glutaraldehyde (which has two types of aldehyde functional groups can bind with amine groups). ...
Porcine pancreas lipase was immobilized on mesoporous chitosan beads. Glutaraldehyde as coupling agent was used through several immobilization techniques. With the aid of FESEM, BET and BJH analysis, the effect of glutaraldehyde on porosity of chitosan was evaluated. It was observed that the total surface area and pore volume of the carrier were significantly improved by addition of glutaraldehyde as cross-linking agent. The surface area exposure and pore volume were substantially increased (both by 4.4 folds). In addition, distribution of enzyme on the carrier was illustrated by fluorescence image. The characteristics of the immobilized lipases such as immobilization efficiency, enzyme activity, pH stability, thermal stability, reusability, storage stability and enzyme leakage were evaluated. In kinetic studies of enzyme, Michaelis–Menten kinetic coefficients of the hydrolytic activity for the immobilized lipase were defined using Lineweaver–Burk plot. The low value of ionization constant, Km (∼0.008 mM) and high value of specific rate, Vmax(∼200 μM/ml.min) indicate strong affinity and high activity of enzyme. The obtained results demonstrate that use of glutaraldehyde has an excellent impact on expansion of the porosity of chitosan and enzyme distribution. The immobilization efficiency increased by1.6 folds and enzyme leakage was minimized (17% reduced to zero); while, glutaraldehyde has improved pH stability (in acidic range), thermal stability, reusability and storage stability of immobilized lipase.
... Blocking free aldehyde groups by ethanolamine, amino acids or proteins prevents potential nonspecific binding of other molecules. Proper washing of activated carrier to remove free glutaraldehyde and blocking free aldehyde groups by ethanolamine after binding of enzyme was performed in our experiments in order to prepare biocompatible biocatalyst [20]. Immobilization usually leads to the increase of enzyme stability and higher temperature resistance. ...
... Nevertheless, all these applications, which involve severe industrial conditions, are often restricted by the deficiency of long-time operational stability, lack of durability, and the difficulty of recovering and recycling used enzymes [16]. These disadvantages can be overcome by enzyme immobilization [18]. ...
In this article, we report results of experiments on covalent immobilization of Candidia rugosa lipase enzyme on modified multiwall carbon nanotubes (MW-CNTs) for oily wastewater treatment application. MWCNTs were produced using chemical vapor deposition (CVD) and surface-modified by nitric acid and organic cross-linkers. Successful attachment and high enzyme loading up to 30 wt % was confirmed via FTIR and TGA analysis. Enzymatic activity and loading, which are dependent on the oxidized MWCNT surfaces, cross-linker types and concentrations, resulted with high thermal and operational stability in the microenvironment conditions. This demonstrates the potential for improved resistance to the severe conditions in industrial applications. Furthermore, the CNTs-immobilized enzyme yielded a catalytic activity about 93 times higher than those immobilized on other reported support materials. Up to 98% biological activity retention was also achieved, marking a significant improvement over literature-reported activities (1-20%). Titrimetric analysis of hydrolyzed samples using MWCNT-Lipase (after 1 hr reaction time at 37°C) resulted in an enzymatic activity increase of about five times over those from lyophilized lipase.
... However, at the time the work of Quiocho and Richards was published, the efforts in immobilization research were focused towards immobilization by adsorption, entrapment and chemical bonding to supports, due to the low activity and stability retention that CLE exhibited. These structures began to be studied more intensively in the 1990's [11,27]. The CLE are formed with proteins in solution that are linked together by a cross-linking agent having sizes from 1 to 100 μm. ...
Structural and functional catalytic characteristics of cross-linked enzyme aggregates (CLEA) are reviewed. Firstly, advantages of enzyme immobilization and existing types of immobilization are described. Then, a wide description of the factors that modify CLEA activity, selectivity and stability is presented. Nowadays CLEA offers an economic, simple and easy tool to reuse biocatalysts, improving their catalytic properties and stability. This immobilization methodology has been widely and satisfactorily tested with a great variety of enzymes and has demonstrated its potential as a future tool to optimize biocatalytic processes.
... Entrapment is defined as the integration of an enzyme within the lattice of a polymer matrix or a membrane, whilst retaining the protein structure of the enzyme [10]. In addition to the immobilisation of enzymes, membranes can also eliminate potential interfering species that may be present in complex media such as serum and food. ...
The development of biosensors for the determination of glutamate has been of great research interest for the past 25 years due to its importance in biomedical and food studies. This review focusses on the various strategies used to fabricate glutamate biosensors as well as their performance characteristics. A brief comparison of the enzyme immobilisation method employed and the performance characteristics of a range of glutamate biosensors are described in tabular form and then described in detail throughout the review: some selected examples have been included to demonstrate the various applications of these biosensors to real samples.
... Nevertheless, all these applications, which involve severe industrial conditions, are often restricted by the deficiency of long-time operational stability, lack of durability, and the difficulty of recovering and recycling used enzymes [16]. These disadvantages can be overcome by enzyme immobilization [18]. ...
We report here on the results of a study of immobilization of Lipase enzyme from Candida Rugosa on Multiwall Carbon Nanotubes (MWCNT) locally produced in a Chemical Vapor Deposition (CVD) reactor. Covalent bonds was formed between the enzyme and MWCNTs using organic cross linkers (NHS and EDC). FTIR results confirmed the covalent binding of the amide functional group of the lipase molecules onto the surface of MWCNT. The produced carbon nanotubes proved to be a good matrix for the lipase loading and immobilization. TGA results and calculations confirmed that 30% wt% of the enzyme was successfully attached to the functionalized MWCNTs. The nanocomposite material revealed high thermal stability and good hydrophobic characteristics which is considered an important property for commercial applications. Findings revealed higher than generally reported biological activity of the protein was successfully retained. Results confirmed that Immobilized lipase on MWCNTs present an excellent opportunity for treatment of oily wastewater. Teste with synthetic water/oil emulsion resulted with oil degradation reached up to 98%.
... Likewise, Dodor et al. (2004) observed that the covalent immobilization of laccase from T. versicolor on kaolinite did not displace the activity-pH profile of the enzyme. On the contrary, other authors reported a shift in the optimal pH for the enzyme after immobilization, due to a change in the microenvironment of the enzyme after the immobilization, which depends on the physical and chemical properties of the carrier (Cao 2005). For instance, the use of a charged support, which can repel or attract the substrate, product, cofactor , and H + depending on the type and quantity of theTable 1 Comparison of different laccase-fsNP conjugates produced with different starting amount of laccase (n=3). ...
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In this study, we proposed integrated tools to evaluate the wind power potential, economic viability, and prioritize 15 proposed sites for the installation of wind farms. Initially, we used modified Weibull distribution model coupled with power law to assess the wind power potential. Secondly, we employed value cost method to estimate per unit cost ($/kWh) of proposed sites. Lastly, we used Fuzzy Technique for Order of Preference by Similarity to Ideal Solution (F-TOPSIS) to rank the best alternatives. The results indicate that Pakistan has enormous wind power potential that cost varies from 0.06 $/kWh to 0.58 $/kWh; thus, sites S12, S13, S14, and S15 are considered as the most economic viable locations for the installation of wind power project, while remaining sites are considered to be less important, due to other complexities. The further analysis using Fuzzy-TOPSIS method reveals that site S13 is the most optimal location followed by S12, S14, and S14 for the development of wind power project. We proposed that government should formulate wind power policy for the implementation of wind power projects in order to meet energy demand of the country.
... Enzymes can be adsorbed onto an inert solid support, entrapped in porous polymeric matrices or gels or encapsulated in beads. Besides that, enzymes can be covalently bonded to solid support via chemical bonding methods [5, 6]. In recent years, polyvinyl alcohol (PVA) which is a cheap and nontoxic synthetic polymer has been widely used for enzymes immobilization due to its advantages compared to alginate beads. ...
Hydrolysis of waste cooking oil (WCO) using immobilized Candida rugosa lipase (CRL) was studied. PVA-Alginate-Sulfate beads were used to immobilize CRL. During the transesterification process, three parameters were considered: pH, temperature and enzyme concentration. The degree of hydrolysis as well as the rate of the hydrolysis were also determined. The morphology of the beads was analyzed using Fourier Emission Scanning Electron Microscopy (FESEM). It was found that the operating conditions, pH = 7.00, temperature = 50°C, and bead loading of 8 g, were most favourable for the hydrolysis of WCO by immobilized CRL to yield maximum fatty acid production and hydrolysis conversion. It was also found that the rate of hydrolysis by immobilized CRL is higher than that of free enzyme which is 96.50% and 68.75%, respectively.
... An ample variety of versatile methodologies for protein immobilization has piled up in the literature which has provided a wide range of options for this purpose [1][2][3]. However, the choice of an optimal immobilization strategy depends on the final application of the immobilized proteins. ...
... Despite their large-scale commercial availability, however, these biocatalysts still have certain drawbacks, chief among which is their extremely limited reusability [2]. Enzyme immobilization is a technology aimed at enhancing the stability of enzyme-related processes [3], with a view to enabling continuous processing through the reuse of enzymes [4]. Immobilized enzymes can be defined as " enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, which can be used repeatedly and continuously " [5] . ...
Industrial applications require enzymes highly stable and economically viable in terms of reusa-bility. Enzyme immobilization is an exciting alternative to improve the stability of enzymatic processes. The immobilization of two commercial enzymes is reported here (cellulase and xylanase) using three chemical methods (adsorption, reticulation, and crosslinking-adsorption) and two polymeric supports (alginate-chitin and chitosan-chitin). The optimal pH for binding was 4.5 for cellulase and 5.0 for xylanase, and the optimal enzyme concentrations were 170 µg/mL and 127.5 µg/mL respectively, being the chitosan and the ideal support. In some cases, a low concentration of crosslinking agent (glutaraldehyde) improved stability of the immobilization process. Biotechnological characterization showed that the reusability of enzymes was the most striking finding, particularly of immobilized cellulase using glutaraldehyde, which after 19 cycles retained 64% activity. These results confirm the economic and biotechnical advantages of enzyme immobilization for a range of industrial applications.
... Though enzyme immobilization has several advantages, the selection of support material and method of immobilization are important in order to obtain higher performance of enzymatic reaction. Encapsulation is one of the immobilization techniques which can be defined as the process by which the enzyme is enclosed physically or chemically within semi-permeable membrane [8]. The alginate bead is commonly used in the encapsulation process because it is easy for formulation, mild gelation conditions, non-toxic, biocompatibility, low cost and resistance to microbial attack. ...
Multi-enzymes (alpha-amylase, glucoamylase and cellulase) were successfully encapsulated in calcium alginate-clay beads to hydrolyze cassava roots for glucose production. Under the optimal conditions (2% clay solution and 0.2 M CaCl2), the loading efficiency and the immobilization yield of enzymes were calculated to be 97.07% and 52.14%, respectively. The calcium- alginate-clay beads have been analyzed using Fourier Transform Infrared Spectroscopy (FTIR), Field Emission Scanning Electron Microscopy (FESEM) and Energy Dispersive X-Ray (EDX). FTIR results showed that asymmetric and symmetric COO- and OH- group shifted to 1621, 1419 and 3324 cm-1 and proved strong interaction between alginate and clay of the beads. Observation under FESEM showed that the beads have a rough surface and the enzymes were successfully encapsulated in the beads. The presence of the clay elements in the beads was confirmed by EDX analysis where the beads consist of 13.97% C, 43.69% O, 28.96% Ca, 6.46% Al and 6.91% Si. The beads were later used for the saccharification of cassava slurry into glucose. Results showed that the process was successful and the encapsulated enzymes in calcium alginate-clay beads have enhanced the enzymes reusability in the process compared to pure alginate beads where it retained 51.77% of its activity after seven hydrolysis cycles.
... Several non-covalent interactions are involved in this immobilization , including non-specific physical adsorption, bio-specific adsorption, affinity adsorption, electrostatic interaction (also [5,7,12]). ionic binding), and hydrophobic interaction [13]. Compared with other immobilization techniques, adsorption immobilization is advantageous in the following aspects [12]: (1) mild conditions and easy operation; (2) relatively low cost of carrier materials and immobilization procedure; (3) no requirement of chemical additives during adsorption; (4) easy regeneration of carriers for recycling; and (5) high lipase activity recovery. ...
... As for enzyme immobilization through purely ionic forces between the enzyme and support , it is based on the proteinÀligand interaction principles used in chromatography, namely the reversible immobilization of enzymes which was first used in ion exchangers.[9] Depending on the pH of the solution and the isoelectric point, the surface of the enzyme may bear charges [56] and its charge distribution can be readily calculated and displayed using current available modelling systems.[57] Any ion exchanger can act as carrier in immobilization via ionic and strongly polar interactions. ...
The current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes. Employment of enzyme as biocatalysts offers the benefits of mild reaction conditions, biodegradability and catalytic efficiency. The harsh conditions of industrial processes, however, increase propensity of enzyme destabilization, shortening their industrial lifespan. Consequently, the technology of enzyme immobilization provides an effective means to circumvent these concerns by enhancing enzyme catalytic properties and also simplify downstream processing and improve operational stability. There are several techniques used to immobilize the enzymes onto supports which range from reversible physical adsorption and ionic linkages, to the irreversible stable covalent bonds. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. Hence, it is mandatory to obtain information about the structure of the enzyme protein following interaction with the support surface as well as interactions of the enzymes with other proteins. Characterization technologies at the nanoscale level to study enzymes immobilized on surfaces are crucial to obtain valuable qualitative and quantitative information, including morphological visualization of the immobilized enzymes. These technologies are pertinent to assess efficacy of an immobilization technique and development of future enzyme immobilization strategies.
Repeated and unregulated use of pesticides in agricultural lands wreaks havoc on the environment, threatening and weakening the biodiversity and ecosystem. They are a growing concern for global stability and sustainability of the environment. The detection of pesticides in the soil at lower concentrations remains a challenge today. In the current decade, Nanotechnology is the emerging technology that help in promoting a sustainable and reliable approach in the agricultural sector. The pesticide residue analysis is an urgent call to safeguard soil quality, reduce health issues, and protect the ecosystem from the excessive use of pesticides. The use of sensor technology for pesticide detection is considered a promising alternative strategy compared to traditional chromatographic detection techniques. Nanosensors possess excellent sensitivity, can detect even lower concentration, and are more stable and reliable for smart agriculture. Developments of nanosensors and nanobiosensors continue to serve a crucial role in the up-gradation of the agriculture sector. In this chapter, we have covered various types of nanosensors and nanobiosensors used for pesticide residue analysis in soil.
The widespread application of soluble enzymes in industrial processes is considered restrict due to instability of enzymes outside optimum operating conditions. For instance, enzyme immobilization can overcome this issue. In fact, chitosan–based nanofibers have outstanding properties, which can improve the efficiency in enzyme immobilization and the stability of enzymes over a wide range of operating conditions. These properties include biodegradability, antimicrobial activity, non–toxicity, presence of functional groups (amino and hydroxyl), large surface area to volume ratio, enhanced porosity and mechanical properties, easy separations and reusability. Therefore, the present review explores the advantages and drawbacks concerning the different methods of enzyme immobilization, including adsorption, cross–linking and entrapment. All these strategies have questions that still need to be addressed, such as elucidation of adsorption mechanism (physisorption or chemisorption); effect of cross–linking reaction on intramolecular and intermolecular interactions and the effect of internal and external diffusional limitations on entrapment of enzymes. Moreover, the current review discusses the challenges and prospects regarding the application of chitosan–based nanofibers in enzyme immobilization, towards maximizing catalytic activity and lifetime.
The oxidation activity of laccase on a broad range of substrates has attracted great interest in the development of technologies for industrial and environmental applications. In this work, a crude laccase from Marasmiellus palmivorus was immobilized onto chitosan-coated magnetic particles. The support was developed and characterized by X-ray diffraction, vibrating sample magnetometer (VSM), specific surface area, and pore size by BET/BJH method, and Fourier Transform Infrared Spectroscopy (FTIR). Magnetic particles retained approximately 30% of magnetization (19.5 emu/g) after enzyme immobilization. The highest activity of the immobilized crude laccase was 139.84 U.g dry support⁻¹ (48.74% efficiency and 99% yield) when 25 mg protein. g dry support⁻¹ were applied in the support previously activated with 90 mM glutaraldehyde. Enzyme immobilization was fast, reaching maximum recovered activity (36%) after 5 min of contact with the support. The optimum pH of the soluble and immobilized crude laccase was 5.5 and the optimum temperature varied between 40 and 50 °C for the soluble form and between 25 and 35 °C for the immobilized form. Immobilization increased the thermal stability of the enzyme by a factor of 1.33. Decolorization reaction of methyl orange dye using ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) as a mediator allowed 30 cycles of reuse with a percentage of decolorization above 60%.
Textiles represent promising support materials for enzymes. The goal of the present work was to investigate the immobilization of commercial peroxidase on a polyester needle felt and the repeated use in the gentle degradation of norbixin in whey from dairy cheese as a practical application. High enzyme loads were obtained by a 2-step immobilization procedure. First, the number of functional groups on the textile surface was increased by a modification with amino-functional polyvinylamine. Second, the enzyme was immobilized by using 2 types of crosslinking agents. Due to the iron content of peroxidase, inductively coupled plasma–optical emission spectrometry was used for the quantitative determination of the enzyme load on the textile. The enzyme activity was evaluated using common 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) assay for peroxidases. By the variation of enzyme input and crosslinker concentration, a maximal enzyme load of 80 mg/g of textile was achieved, and a maximum specific activity of 57 U/g of textile. For the visualization of the enzyme on the fiber surface, fluorescence microscopy as well as scanning probe microscopy were used. The immobilized peroxidase showed significant activity, even after 50 reuse cycles. In addition, the potential of the new support and enzyme combination in commercial whey bleaching was demonstrated successfully on a 10-L scale.
Bacillus amyloliquefaciens cyclodextrin glucosyltransferase (CGTase) was immobilized on 35 supports by different methods of immobilization. The immobilized enzymes were prepared by physical adsorption on chitin, ionic binding onto Amberalite IRA-45, covalent binding on Duolite XAD761, and entrapment in polyacrylamide had the highest recovered activity. The immobilized preparations retained 12.08–43.5% of the original specific activity exhibited by the free enzyme. Compared to the free enzyme, the immobilized preparations exhibited higher optimum temperature, lower activation energy, lower deactivation constant rate, higher half-life (T1/2) values, and resistance to chemical denaturation. The values of thermodynamic parameters for irreversible inactivation indicated that immobilization significantly decreased entropy (ΔS*) and enthalpy of deactivation (ΔH*). The immobilized enzyme displayed higher Km and lower Vmax values. After using for 10 cycles, the retained catalytic activity were 30.0, 35.0, 69, and 54% of the initial values of the immobilized enzyme on chitin, Amberlite IRA-45, Duolite XAD761, and polyacrylamide respectively.
Efficient immobilization of enzymes on support surfaces requires an exact match between the surface chemistry and the specific enzyme. A successful match would normally be identified through time consuming screening of conventional resins in multiple experiments testing individual immobilization strategies. In this study we present a versatile strategy that largely expands the number of possible surface functionalities for enzyme immobilization in a single, generic platform. The combination of many individual surface chemistries and thus immobilization methods in one modular system permits faster and more efficient screening, which we believe will result in a higher chance of discovery of optimal surface/enzyme interactions.
The proposed system consists of a thiol‐functional microplate prepared through fast photochemical curing of an off‐stoichiometric thiol‐ene (OSTE) mixture. Surface functionalization by thiol‐ene chemistry (TEC) resulted in the formation of a functional monolayer in each well, whereas, polymer surface grafts were introduced through surface chain transfer free radical polymerization (SCT‐FRP). Enzyme immobilization on the modified surfaces was evaluated by using a rhodamine labeled horseradish peroxidase (Rho‐HRP) as a model enzyme, and the amount of immobilized enzyme was qualitatively assessed by fluorescence intensity (FI) measurements. Subsequently, Rho‐HRP activity was measured directly on the surface. The broad range of utilized surface chemistries permits direct correlation of enzymatic activity to the surface functionality and improves the determination of promising enzyme‐surface candidates. The results underline the high potential of this system as a screening platform for synergistic immobilization of enzymes onto thiol‐ene polymer surfaces. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1267–1277, 2017
Nanoporous gold (NPG) electrodes were prepared by dealloying sputtered gold:silver alloys. Electrodes of different thickness and pore sizes areas were prepared by varying the temperature and duration of the dealloying procedure and then used as supports for FAD-dependent glucose dehydrogenase (Glomorella cingulata) and bilirubin oxidase (Myrothecium verrucaria). Glucose dehydrogenase was immobilised by drop casting a solution of the enzyme with an osmium redox polymer together with a cross-linked polymer while bilirubin oxidase was covalently attached via carbodimide coupling to a diazonium modified NPG electrode. The stability of the bilirubin oxidase modified NPG electrode was significantly improved in comparison to that of a planar gold electrode. Enzyme fuel cells were prepared; the optimal response was obtained with a 500 nm thick BOx modified NPG cathode and a 300 nm GDH modified anode that generated power densities of 17.5 and 7.0 µW cm-2 in phosphate buffered saline and artificial serum, respectively.
Solid-binding peptides (SBP’s) have the ability to bind with high affinity and selectivity to the surfaces of a diverse range of solid materials. They offer a simple and versatile method for the directed immobilisation and orientation of proteins and enzymes onto solid supports without impeding their catalytic functionality. This chapter describes SBPs and their potential applications in the isolation, purification and reuse of thermostable enzymes using readily-available and inexpensive silica-based matrices. We suggest some prospects for the introduction of thermostable enzymes immobilised onto solid matrices using SBPs into several of areas of applied biotechnology. In particular, we outline conceptual applications for immobilised enzymes in cell-free synthetic biology, biofuels production and in gas phase biocatalysis for the capture of carbon dioxide.
This study aimed to prepare robust immobilized formate dehydrogenase (FDH) preparations which can be used as effective biocatalysts along with functional oxidoreductases, in which in situ regeneration of NADH is required. For this purpose, Candida methylica FDH was covalently immobilized onto Immobead 150 support (FDHI150), Immobead 150 support modified with ethylenediamine and then activated with glutaraldehyde (FDHIGLU), and Immobead 150 support functionalized with aldehyde groups (FDHIALD). The highest immobilization yield and activity yield were obtained as 90% and 132%, respectively when Immobead 150 functionalized with aldehyde groups was used as support. The half-life times (t(1/2)) of free FDH, FDHI150, FDHIGLU and FDHIALD were calculated as 10.6, 28.9, 22.4 and 38.5 h, respectively at 35 degrees C. FDHI150, FDHIGLU and FDHIALD retained 69, 38 and 51% of their initial activities, respectively after 10 reuses. The results show that the FDHI150, FDHIGLU and FDHIALD offer feasible potentials for in situ regeneration of NADH.
Microencapsulation as a major technique of enzyme immobilization is reviewed. Fabrication of enzyme-containing microcapsules is broadly categorized according to whether a semipermeable membrane coating or a porous polymeric network structure is primarily responsible for enclosure of enzymes within the internal microcapsule phase. Microcapsules are mostly spherical with diameters usually in the micro- to millimeter range and exhibit core-shell structures of variable complexity. Liposomes and vesicles formed from amphiphilic (co)-polymers (polymersomes) are widely used to prepare membrane-coated microcapsules. Hydrogels, sol–gels and other organic–inorganic hybrid materials, or layer-by-layer structures made through controlled assembly of polyelectrolytes are used to prepare microcapsules composed of internal polymeric networks. Method combination where polymeric network microcapsules are coated with a suitable membrane to generate tailored core-shell structures is also used for enzyme encapsulation. Advances in the material sciences contribute to the development of microcapsules with improved properties such as enhanced morphological stability, reduced enzyme leakage, and designed physicochemical permeability and enzyme biocompatibility. Soluble enzymes, but also enzyme aggregates and other insoluble enzyme preparations, are encapsulated. Multienzyme microcapsules are interesting small-scale bioreactors for confined and even compartmentalized cascade biotransformation. Techniques of microcapsule preparation are described, and applications of microencapsulated enzymes in biotechnology and biomedicine are discussed.
Keywords:
immobilization;
microencapsulation;
microcapsule;
membrane coating;
biocatalysis
Since immobilization of lipases enhances their productivity, stability and selectivity, a series of surface modified silica gel supports was developed and used for hydrophobic adsorption of Lipase AK from Pseudomonas fluorescens and Lipase PS from Burkholderia cepacia. © 2014, Universitatea Babes-Bolyai, Catedra de Filosofie Sistematica. All rights reserved.
The lipase enzyme converts long chain acyltriglycerides into di- and monoglycerides, glycerol and fatty acids. The catalytic site in lipase is situated deep inside the molecule. It is connected through a tunnel to the surface of the molecule. In the unbound state under aqueous conditions, the tunnel remains closed. The tunnel can be opened when the enzyme is exposed to a lipid bilayer or a detergent or many hydrophobic/hydrophilic surfaces.
In the present study, the lipase was subjected to three-phase partitioning (TPP) which consisted of mixing in tert-butanol and ammonium sulphate to the solution of lipase in the aqueous buffer. The enzyme formed an interfacial precipitate between the tert-butanol rich and water rich phases. The stability of the enzyme subjected to TPP was found to be higher (T m of 80 °C) than the untreated enzyme (T m of 77 °C). The activity of the enzyme subjected to TPP (3.3 U/mg) was nearly half of that of the untreated one (5.8 U/mg). However, the activity of the treated enzyme was higher (17.8 U/mg) than the untreated one (8.6 U/mg) when a detergent was incorporated in the assay buffer.
The structure determination showed that the substrate binding site in the treated enzyme was more tightly closed than that of the untreated protein.
Graphical abstract
The Van Der Waals distances between atoms of residues in the active site of Thermomyces lanuginosus lipase change after the enzyme is subjected to three phase partitioning (TPP). (a) native lipase, (b) TPP treated lipase.
Glycosides have attracted great interest due to the growing evidence of their beneficial effect on human health. The discovery of new drugs from plants leads to the isolation of many substances that are used clinically and then served as prototypes for the synthesis of new therapies. Its estimated that around 40% of the available medicines in current therapeutics have been developed from natural sources: 25% from plants, 13% from microorganisms, and 3% from animals. (De)glycosylation increases biologic activity. The scarcity of these compounds makes them very expensive to isolate. Glycosides can be hydrolyzed by β-glycosidase. Several in vitro studies (preclinical trials) have demonstrated multiple beneficial effects of glycosides in prevention or in therapeutics of different pathologies.
Present study aimed to immobilize the bacterial tannase on suitable carrier and its subsequent applicability in fruit juice clarification. Tannase from Bacillus subtilis PAB2 was immobilized on chitin-alginate bead by adsorption-entrapment technique. Maximum immobilization (82 %) with an activity yield of 67 % was achieved in presence of 1.5 % (w/v) sodium alginate, 1 M CaCl2 and 1 % (w/v) flake chitin, incubation at 4 °C temperature, and adsorption period of 90 min. Thermal and pH stabilities of immobilized tannase increased in respect to free tannase. The storage stability and reusability of the immobilized tannase were also improved significantly, retaining 83 % activity after storing 90 days at 4 °C and it showed 79 % residual activity after 10 times repeated use. Then, immobilized tannase was applied for clarification of jamun and cashew apple juice to investigate its debittering property. Enzymatic treatment removed 60 and 51 % tannin from jamun and cashew apple juice, respectively, after incubation at 40 °C for 120 min. Other qualitative indicator viz., total titratable acidity, color, and viscosity also reduced, whereas, flavonoid (460.49 μg/ml; 994.44 μg/ml), glucose (6.95 mg/ml; 9.19 mg/ml), and antioxidant activity (69.28 %; 78.44 %) were significantly increased in clarified jamun and cashew apple juice, respectively.
Immobilization of enzymes onto different carriers increases enzyme's stability and reusability within biotechnological and pharmaceutical applications. However, some immobilization techniques are associated with loss of enzymatic specificity and/or activity. Possible reasons for this loss are mass transport limitations or structural changes. For this reason an immobilization method must be selected depending on immobilisate's demands. In this work different immobilization media were compared towards the synthetic and hydrolytic activities of immobilized trypsin as model enzyme on magnetic micro-particles.
Porcine trypsin immobilization was carried out in organic and aqueous media with magnetic microparticles. The immobilization conditions in organic solvent were optimized for a peptide synthesis reaction. The highest carrier activity was achieved at 1 % of water (v/v) in dioxane. The resulting immobilizate could be used over ten cycles with activity retention of 90 % in peptide synthesis reaction in 80 % (v/v) ethanol and in hydrolysis reaction with activity retention of 87 % in buffered aqueous solution. Further, the optimized method was applied in peptide synthesis and hydrolysis reactions in comparison to an aqueous immobilization method varying the protein input. The dioxane immobilization method showed a higher activity coupling yield by factor 2 in peptide synthesis with a maximum activity coupling yield of 19.2 % compared to aqueous immobilization. The hydrolysis activity coupling yield displayed a maximum value of 20.4 % in dioxane immobilization method while the aqueous method achieved a maximum value of 38.5 %. Comparing the specific activity yields of the tested immobilization methods revealed maximum values of 5.2 % and 100 % in peptide synthesis and 33.3 % and 87.5 % in hydrolysis reaction for the dioxane and aqueous method, respectively.
By immobilizing trypsin in dioxane, a beneficial effect on the synthetic trypsin activity resilience compared to aqueous immobilization medium was shown. The results indicate a substantial potential of the micro-aqueous organic protease immobilization method for preservation of enzymatic activity during enzyme coupling step. These results may be of substantial interest for enzymatic peptide synthesis reactions at mild conditions with high selectivity in industrial drug production.
Abstract：In this study, lipase from Candida sp. 99-125 was immobilized on glutaraldehyde-activated APTES-functionalized or epoxy-functionalized porous ceramic monoliths. The activity and stability of the immobilized lipases were investigated. The results indicated that the stabilities of immobilized lipases were improved significantly compared to native lipase. The immobilized lipases were used as catalysts for the production of oil gelling agent by using mannitol and fatty acid vinyl ester as substrates. Take Man-8 as an example, the yield of this oil gelling agent was more than 78% after 48 h reaction that catalyzed by the immobilized lipases. After the 5th batch reaction, more than 50% of yield can be maintained. Furthermore, the results of gelation tests indicated that the oil gelling agent can gel various hydrocarbons and oils. When the oil gelling agent was used to recover diesel from a biphasic mixture, more than 90% of the initial diesel can be recovered.
This chapter demonstrates with a wide range of examples the high potential of continuous-flow techniques for stereoselective biotransformations catalyzed by enzymes, especially hydrolases. Benefits of the various reactor setups are discussed, for example, the continuous flow packed bed reactors can enhance the productivity of enzyme-catalyzed processes or miniaturized continuous flow systems allow fast high-throughput screenings. Continuous flow microreactor systems enabled integration of enzyme-catalysis with chemical steps (e.g., in dynamic kinetic resolutions) or with continuous extraction or performing multienzyme-catalyzed multistep biotransformations. Thus, the potential of continuous-flow microreactor systems with immobilized biocatalysts is demonstrated as process development tool for stereoselective biotransformations.
A new organic-inorganic hybrid zeotype compound with amphiphilic one-dimensional nanopore and aluminosilicate composition was developed. The framework structure is composed of double aluminosilicate layers and 12-ring nanopores; a hydrophilic layer pillared by Q(2) silicon atom species and a lipophilic layer pillared by phenylene groups are alternately stacked, and 12-ring nanopores perpendicularly penetrate the layers. The framework topology looks similar to that of an AFI-type zeolite but possesses a quasi-multidimensional pore structure consisting of a 12-ring channel and intersecting small pores equivalent to 8-rings. The hybrid material with alternately laminated lipophilic and hydrophilic nanospaces can be assumed as a crystallized Langmuir-Blodgett film. It demonstrates microporous adsorption for both hydrophilic and lipophilic adsorptives, and its outer surface tightly adsorbs lysozyme whose molecular size is much larger than its micropore opening. Our results suggest the possibility of designing porous adsorbent with high amphipathicity.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
1. Introduction
2. Screening for Novel Biocatalyst
2.1 Chromogenic and Fluorogenic Assays
2.2 Fermentation Assays
2.3 Metagenomic Assays
3. Development of Biocatalysts
3.1 Enzyme Immobilization
3.1.1 Cross-Linked Enzymes
3.1.2 Nanobiocatalysis
3.2 Hybrid Enzymes
3.3 Multienzyme Reactions
3.4 By Submerged Fermentation
3.5 Pretreatments
4. Raw Materials
5. Reaction Media
In this paper a novel enzymatic glucose biosensor has been reported in which platinum coated alumina membranes (Anodisc™s) have been employed as templates for the growth of polypyrrole (PPy) nanotube arrays using electrochemical polymerization. The PPy nanotube arrays were grown on Anodisc™s of pore diameter 100nm using potentiostatic electropolymerization. In order to optimize the polymerization time, immobilization of glucose oxidase (GOx) was first performed using physical adsorption followed by measuring its biosensing response which was examined amperometrically for increasing concentrations of glucose. In order to further improve the sensing performance of the biosensor fabricated for optimum polymerization duration, enzyme immobilization was carried out using cross-linking with glutaraldehyde and bovine serum albumin (BSA). Approximately six fold enhancement in the sensitivity was observed in the fabricated electrodes. The biosensors also showed a wide range of linear operation (0.2-13mM), limit of detection of 50μM glucose concentration, excellent selectivity for glucose, notable reliability for real sample detection and substantially improved shelf life.
Copyright © 2015 Elsevier B.V. All rights reserved.
Alginate has revolutionized the way in which proteins and enzymes are used in daily life, right from food, textiles, medicines and surgical advancement to environment. Alginate is a biopolymer with unique physical and chemical properties that makes it functionally an ideal material for attachment with proteins. Immobilization of enzymes on alginate is well known to show altered catalytic functions and improved operational stability with no or minimal drawbacks. The GRAS (Generally Recognized as Safe) status of alginate enables it to be readily used as an encapsulation material for food proteins for their better gastrointestinal absorbance, and also in delivery of several proteins and peptides for their therapeutic effects. The exclusive interactions of alginate with proteins as a function of ionic strength, pH and metallic ions make it a support of choice for purification of enzymes and proteins by simple chromatographic separations and affinity precipitation. However biomedical in vivo use of alginate, specifically in tissue engineering, necessitates the use of extra pure form of alginate. This review highlights the impact of interactions between proteins and alginate on the functional properties of the proteins. The review also emphasizes certain core applications areas where alginate is used to a large extent.
Graphene oxide (GO) offers interesting physicochemical and biological properties for biomedicine due to its versatility, biocompatibility, small size, large surface area, and its ability to interact with biological cells and tissues. GO is a two-dimensional material of exceptional strength, unique optical, physical, mechanical, and electronic properties. Ease of functionalization and high antibacterial activity are two major properties identified with GO. Due to its excellent aqueous processability, amphiphilicity, surface functionalization capability, surface enhanced Raman scattering (SERS), and fluorescence quenching ability, GO chemically exfoliated from oxidized graphite is considered a promising material for biological applications. In addition, due to π-π* transitions, a low energy is required for electron movement, a property important in Biosensor and Bioimaging applications of GO. In this article, we present an overview of current advances in GO applications in biomedicine and discuss future perspectives. We conclude that GO is going to play a vital role in Biomedical applications in the near future.
Biocatalysis has emerged as a rich field within Biotechnology, enabling the application of enzymes in a wide range of industries ranging from pharmaceuticals and fine chemicals to food and energy. This striking development of Biocatalysis is due to novel technologies such as bioinformatics, high-throughput screening (HTS), directed evolution, as well as other well-established techniques such as enzyme immobilization and protein engineering or medium engineering. Sustainable manufacturing is a major driver of Biocatalysis, which will provide many real challenges and opportunities for the future. In this article, some of the main methods in enzyme technology have been reviewed, as well as several biotechnological applications of enzymes in industry. Finally, a brief overview of the situation of Biocatalysis in both Spanish academia and industry has also been reported.
Protein nanofibrils self-assembled from crystallin proteins, a waste material from the fishing industry, have been identified as a suitable material to be used as a bionanoscaffold. In this study, a successful method for the functionalization of crystallin protein nanofibrils by immobilizing several enzymes of industrial relevance (glucose oxidase, β-galactosidase, pectinase, α-amylase, and laccase) through a glutaraldehyde cross-linking approach is reported. The extent of functionalization is evaluated by using gel electrophoresis, transmission electron microscopy, and thermostability studies. The functionalized fibrils are investigated further for reusability studies—the use of protein nanofibrils as nanoscaffolds results in a significant increase in enzyme thermostability and reusability relative to the free enzyme in solution under the same conditions. Finally, as an example and proof of concept, the use of the developed functionalization method in a biosensor platform for glucose and lactose detection is shown, by utilizing the crystallin protein nanofibrils as nanoscaffolds.
A simple and inexpensive methodology, based on the use of micro-centrifuge filter tubes, is proposed for establishing the best enzyme immobilization conditions.
The immobilized biocatalyst is located inside the filter holder during the whole protocol, thus facilitating the incubations, filtrations and washings. This procedure minimizes the amount of enzyme and solid carrier needed, and allows exploring different immobilization parameters (pH, buffer concentration, enzyme/carrier ratio, incubation time, etc.) in a fast manner. The handling of immobilized enzymes using micro-centrifuge filter tubes can also be applied to assess the apparent activity of the biocatalysts, as well as their reuse in successive batch reaction cycles. The usefulness of the proposed methodology is shown by the determination of the optimum pH for the immobilization of an inulinase (Fructozyme L) on two anion-exchange polymethacrylate resins (Sepabeads EC-EA and Sepabeads EC-HA).
The micro-scale procedure described here will help to overcome the lack of guidelines that usually govern the selection of an immobilization method, thus favouring the development of stable and robust immobilized enzymes that can withstand harsh operating conditions in industry.
Enzymes require some flexibility for catalysis. Biotechnologists prefer stable enzymes but often this stabilization comes at the cost of reduced efficiency. Enzymes from thermophiles have low flexibility but poor catalytic rates. Enzymes from psychrophiles are less stable but show good catalytic rates at low temperature. In organic solvents enzymes perform poorly as the prior drying makes the enzyme molecules very rigid. Adding water or increasing reaction temperature improves flexibility and catalytic rates. In case of hydrolases, flexibility and enantioselectivity have interdependence. Understanding the complex role of protein flexibility in biocatalysis can help in designing biotechnological processes.
Carboxylated single-walled carbon nanotubes (SwCNTCOOH) were used as a support for the covalent immobilization of phenylalanine ammonia-lyase (PAL) from parsley by two different methods. The nanostructured biocatalysts (SwCNTCOOH-PALI and SwCNTCOOH-PALII) with low diffusional limitation were tested in the batch-mode kinetic resolution of racemic 2-amino-3-(thiophen-2-yl)propanoic acid (1) to yield a mixture of (R)-1 and (E)-3-(thiophen-2-yl)acrylic acid (2) and in ammonia addition to 2 to yield enantiopure (S)-1. SwCNTCOOH-PALII was a stable biocatalyst (>90 % of the original activity remained after six cycles with 1 and after three cycles in 6 M NH3 with 2). The study of ammonia addition to 2 in a continuous-flow microreactor filled with SwCNTCOOH-PALII (2 M NH3, pH 10.0, 15 bar) between 30–80 °C indicated no significant loss of activity over 72 h up to 60 °C. SwCNTCOOH-PALII in the continuous-flow system at 30 °C was more productive (specific reaction rate, rflow=2.39 μmol min−1 g−1) than in the batch reaction (rbatch=1.34 μmol min−1 g−1).
In this work we report a fully aqueous and highly efficient method for the encapsulation of enzymes in silica nanocapsules during their formation process. In this approach, enzymes are first enclosed into unilamellar vesicles self-assembled by an amphiphilic precursor polymer – poly(ethylene glycol) substituted hyperbranched polyethoxysiloxane (PEG-PEOS) – in water. After subsequent condensation under basic conditions enzyme-loaded silica nanocapsules are obtained. Due to the significant volume shrinkage during the PEG-PEOS conversion, the encapsulation efficiency is very high, i.e. by adding only 5 wt% PEG-PEOS almost 50% of the enzyme from the solution is encapsulated. As compared with the free enzyme, the protease encapsulated by this means preserves almost 40% of its activity, exhibits significantly enhanced stability against the change of environmental conditions, and can be repeatedly regenerated without a significant activity loss.
The flat sheet support for enzyme immobilization was prepared by UV-initiated photopolymerization of 2-hydroxyethyl-methacrylate (HEMA) in the presence of an initiator (αα′-azoisobutyronitrile; AIBN). An affinity dye, Procion Green H-E4BD, was attached covalently under alkaline conditions and the pHEMA-Procion Green H-E4BD-attached film was used for the immobilization of lysozyme via adsorption. The amount of attached dye on the pHEMA film was 160 μmol m2 and the water content of the dye-attached pHEMA film was 69%. The lysozyme adsorption capacity of the dye-attached pHEMA film was determined under conditions of different pH and with different initial concentrations of enzyme in the medium. The maximum lysozyme adsorption capacity of the dye-attached pHEMA film, under the specified experimental conditions was 3.92 g m−2. Non-specific adsorption of the lyzozyme on the pHEMA film was negligible. Optimum reaction pH was 6.0 for the free and 7.0 for adsorbed enzyme. The free enzyme had an optimum temperature of 35°C, whereas it shifted to 40°C for the immobilized enzyme system. The enzyme could be repeatedly adsorbed and desorbed from the dye-attached pHEMA film without any significant loss in adsorption capacity.
Watermelon α‐galactosidase (EC 3.2.1.22) was immobilized on a natural (chitin) and a synthetic anion‐exchange (Amberlite IRA‐938) support by covalent coupling methods. The procedure entails the activation of supports with 1,1′‐carbonyldiimidazole (CDI), followed by immobilization of the enzyme on to these supports without and with a spacer arm; γ‐aminobutyric acid (GABA). Optimization of activation was performed by changing the CDI concentrations and coupling efficiencies. The comparison of two immobilization techniques for both chitin and Amberlite IRA‐938 was made by comparing different enzyme concentrations against enzyme activity yield. Furthermore, the storage stability of the immobilized enzymes was also investigated and chitin immobilized α‐galactosidase was found to be better. Although the activity yield of immobilized enzymes were the same for both supports, the short storage stability of immobilized enzyme on Amberlite IRA‐938 is currently a drawback to its applications.
The reaction of the nucleophilic pyridine nitogen with strong electrophiles such as cyanogen bromide, fluorodinitrobenzene and p-toluene-sulfonyl chloride to yield glutaconic aldehyde is well known (1,2). This reaction was the base for a quantitative method for the determination of Cyanids (3), and it was recently used as a tool for the following of activation of polysaccharides with cyanogen bromide (4). In the following, we describe that polymers containing pyridine can be also converted to polyaldehydes (glutaconic aldehyde) by the reaction with cyanogen bromide. This polyaldehyde can be used directly for the binding of protein and small ligands.
Reductive alkylation in which Schiff’s bases formed between an aldehyde and an amino group may be reduced with sodium borohydride (NaBH4) or sodium cyanoborohydride is a convenient and gentle method to specifically introduce alkyl groups into proteins at ε-amino groups of lysine (1–4). We have used this reaction to couple enzymes to carbohydrate supports which had been oxidized with sodium metaperiodate (NaI04) including dextran-coated, controlled-pore glass (Dextran-CPG), Glycophase-CPG and Sepharose. These are neutral, hydrophilic supports with a highly porous structure. Glycophase-CPG is porous glass coated with a glyceryl silane (Glass≡Si(CH2)3OCH2CHOHCH2OH).
Lipase from Candida cylindracea was immobilized by adsorption on a hydrophobic zeolite type Y. The maximal amount of bound protein of 8.2 mg g(-1) and an immobilization efficiency of 33% were achieved under optimum conditions. The kinetics of lipase binding to the zeolite were assessed using a general model of topochemical reactions. Based on the values of the parameters of the specific kinetic model, we propose that the adsorption process is controlled by surface kinetics. This was later experimentally confirmed. The activation energy for lipase adsorption on the zeolite is 43 kJ mol(-1).
Partially purified acylase from Xanthomonas compestris (X. compestris) catalyzed the condensation of 7-amino des acetoxy cephalosporanic acid (7-ADCA) and D(-) alpha amino phenyl glycine to form cephalexin. At pH 6.2, 46% of 7-ADCA was converted to cephalexin in 45 minutes. Immobilization of this enzyme, by initial adsorption on bentonite followed by entrapment in alginate beads, helped to stabilize the enzyme and rendered the biocatalyst reusable.
Modified short fibre with unsaturated bonds on the surface was added into the photo-crosslinking polyurethane prepolymer and a kind of fibre-containing photo-crosslinkable polyurethane prepolymer FPCPU was obtained. FPCPU can be crosslinked by polymerization of the double bonds on the ends of the polyurethane prepolymer and on the surface of the fibre at the existence of the photo-sensitive agents and at UV light irradiations. The obtained FPCPU is a kind of hydrophilic gel, possessing a multi-phase composite structure. The added short fibre can reinforce the system well because of crosslinking with the bulk prepolymer and the fibre's retained orientation and crystallinity. Under same conditions, the strength of FPCPU is 2-3 times higher than that of PCPUM which containing no fibre. The obtained new carrier can be used to entrap the Bacillus Substilis living cells producing α-amylase. The enzyme activity produced is usually about 40%-50% higher than that of the untrapped free cells at same conditions.
Immobilized Metal Affinity Chromatography (IMAC) is a purification method wherein the chemistry of protein-metal interactions is the basis for adsorption. A protein's metal affinity is dependent on the frequency and location of electron donating species on its surface, notably the distribution of histidyl residues. Other methods related to IMAC which have also been developed include metal affinity precipitation, and metal-enhanced two-phase aqueous extraction. Also, biospecific affinity chromatography and immobilized enzyme reactors can exploit protein-metal interactions to immobilize a species. Thus IMAC and its related techniques can be useful in a broad range of applications. In this paper various aspects of IMAC will be explored. IMAC may be understood by examining the nature of the stationary phases and the proteins which display affinity towards metals. A description of factors which effect chromatography performance such as choice of metal, eluent, and buffer composition will convey the notion that IMAC may be applied to the purification of a variety of proteins by manipulating column conditions. Finally, the application of genetic engineering in order to modify a protein's metal affinity will be addressed.
This communication deals with the application of photo-crosslinkable resins to inclusion of enzymes, microbial cells and organelles. The method consists of mixing (a) liquid oligomers of suitable photo-crosslinkable resin(s) containing photo-sensitive functional groups, (b) an appropriate initiator, and (c) an enzyme solution or suspension of cells or organelles, followed by illumination with near-ultraviolet light for only a few minutes. This simple and convenient procedure produces tailor-made matrices in which biologically active macromolecules are successfully entrapped.
Cellulase crosslinked by glutaraldehyde was fixed on alumina beads by radiation polymerization of hydrophilic monomers at low temperatures to get immobilized enzyme beads. The immobilized enzyme beads were tested by the enzymatic hydrolysis of the two kinds of cellulosic materials, cellobiose and chaff (insoluble material). The preparation conditions of the immobilized enzyme beads, applied to the pretreatment of cellulosic feed such as chaff are investigated. The enzyme was fixed on the surface of the polymer matrices with porous structure by radiation polymerization using hydrophilic monomers such as hydroxyethyl methacrylate, hydroxypropyl methacrylate etc. Chaff pretreated by electron beam irradiation and subsequent mechanical crushing was hydrolyzed by the immobilized enzyme beads. The hydrolysis conversion yield of cellulose component of chaff was about 50%. The hydrolysis yield varied with the irradiation dose of the pretreatment, the properties of polymer matrix, and the crosslinking parameters of the enzyme.
β-Galactosidase and glucose oxidase have been immobilized on cellulose-polyacrylamide (C-PAM) graft copolymers, using the azide method or through glutaraldehyde. The original (C-PAM) copolymers were prepared via radiation-induced grafting under controlled conditions by both post-irradiation and simultaneous procedures. The optimum conditions for coupling of the copolymers to the enzymes were established as were the levels of activity of the immobilized enzymes. Grafting by the simultaneous route was seen to be more efficient than postirradiation grafting. Indirect evidence of grafting was found from elemental and thermal analysis. Binding of β-galactoside was found to be more successful than that of glucose oxidase. A relationship between the level of immobilized enzyme activity and the extent of grafting in the copolymer was established for the β-galactosidase system.
Two commercially available lipases, Lipase OF (non-specific lipase from Candida rugosa) and Lipolase 100T (1,3-specific lipase from Aspergillus niger), were immobilized on insoluble hydrophobic support HDPE (high density polyethylene) by the physical adsorption method. Hydrolysis performance was enhanced by mixing a non-specific Lipase OF and a 1,3-specific Lipolase 100T at a 2 : 1 ratio. The results also showed that the immobilized lipase maintained its activity at broader temperature (25∼55°C) and pH (4∼8) ranges than soluble lipases. In the presence of organic solvent (isooctane), the immobilized lipase retained most of its activity in upto 12 runs of hydrolysis experiment. However, without organic solvent in the reaction mixture, the immobilized lipase maintained most of its activity even after 20 runs of hydrolysis experiment.
It may seem like an unlikely union, but the marriage between enzyme technology and polymer chemistry could provide fast, environmentally friendly organic syntheses.
Sephadex G-100 modified with chloracetic acid and CEAH was prepared into cationic and anionic carriers and activated by CNBr. β-Galactosidase I (β-D-galactoside galactohydrolase EC 3.2.1.23) isolated and partially purified from gram chicken bean was immobilized on modified Sephadex G-100 by means of adsorption and crosslinking reaction. Both the anionic and cationic gel carriers have high protein binding capacity and high yield of enzyme activity. Kinetic results showed that the enzyme activity attained its maximum at 57 °C for cationic carriers and 52 °C for anionic carriers. In addition, the operational pH range of the immobilized enzyme was increased. Storage stability of the immobilized enzyme preparations at room temperature was better than that of soluble enzyme. Results of the repeated batch experiments suggested that immobilized enzymes could be reused.
Several enzymes (aldolase, aminoacylase, arginase, carboxypeptidase B, cholinesterase, cyclodextrin glycosyltransferase and glucoamylase) were immobilized by covalent coupling on polyacrylamide bead polymers possessing carboxylic functional groups activated by water-soluble carbodiimides. The factors influencing the immobilization process were studied. The catalytic activities of the immobilized enzymes were influenced advantageously by the structure of carbodiimide used as coupling agent. Immobilized enzymes of highest catalytic activity could be obtained if the carbodiimide was introduced into the reaction mixture in a stoichiometric quantity relative to the carboxylic functional groups located on the support and the support/protein ratio was from 1:0.25 to 1:1. It was found that the hydrogen ion concentration of the coupling reaction mixture has a profound effect on the immobilization of enzymes. In the function of the ionic strength of coupling reaction mixture, the catalytic activity of immobilized enzyme produced showed an apparent optimum. The increasing porosity of support was favourable for the immobilization of enzymes. The insertion of a spacer in the support appeared to be disadvantageous for the enzyme immobilization.
A reaction scheme is proposed to facilitate the simultaneous stereoselective hydrolysis of DL-Phe-Me and separation of L-Phe using aphron-immobilized α-chymotrypsin. While > 90% of the enzyme could be retained on the aphrons the immobilized α-chymotrypsin displayed < 1% specific activity compared to the free enzyme. This was due to the cumulative denaturing effects of the surfactants used for aphron formulation (Softanol 120 and SDS) and exposure to a decanol-water interface. The stereoselectivity of the immobilized enzyme was not significantly altered however.
A raw-starch-digesting amylase, Dabiase K-27, was immobilized covalently on an enteric coating polymer (hydroxypropyl methylcellulose acetate succinate: AS) as a carrier which is autoprecipitating in an insoluble state below pH 4 as well as reversibly soluble-insoluble depending on pH. Dabiase immobilized on AS (D-AS) showed a sharp response of solubility to slight changes of pH without decrease in enzymatic activity. Moreover, D-AS in an insoluble state had good properties of sedimentation and a large portion of D-AS spontaneously precipitated after 10 min at pH 4. D-AS was used successively for repeated ethanol production from raw starch, in which D-AS and flocculating yeast cells were separated simultaneously from a product solution by sedimentation in a reactor with a conical bottom. In the five batches of 10% raw starch, the total amount of ethanol produced from 150 g of raw starch was 61 g, a value of which corresponds to the average ethanol productivity of 0.85 g/l/hr. The repeated ethanol production by a combination of D-AS and flocculating yeast cells is a promising procedure for effectively using the enzyme and recovering the product solution economically in a heterogeneous culture system containing a solid substrate.
Endo-D-galacturonanase of Aspergillus sp. was irreversibly adsorbed on polyethyleneterephthalate in an acetate 0.1 mol l⁻¹ buffer solution of pH 4.2. Immobilization of the enzyme resulted in lowering of its activity, the measure of which depended on the amount of the enzyme fixed on the carrier. The highest relative activity (42.4%) had the preparation containing 5.25 mg of the enzyme per 1 g of the carrier. The velocity and intensity of the sorption of the enzyme depended on the ionic strength of the medium, whilst pH, on the other hand, was of no influence. Endo-D-galacturonanase immobilized in a 0.1 mol l⁻¹ buffer was characteristic a) of its fixation strength in salt solutions of various ionic strength and pH, in a 3 mol l⁻¹ guanidine solution, and also in sodium pectate and pectin solutions, b) of its high stability during a long-lasting storage at 4 °C, c) of its operational stability. The immobilization led to a partial change of the action pattern onto the high-molecular substrate, manifested in lowering the decrease of viscosity to degradation degree ratio.
The pectin esterases from tomatoes and Aspergillus foetidus were immobilized by covalent attachment to CNBr-activated Sepharose 4B and by adsorption to polyethylene terephthalate and the properties of the immobilized enzymes were compared. The relative activity of tomato pectin esterase after the immobilization to both supports was almost 7%, whereas the activity of A. foetidus pectin esterase covalently immobilized on CNBr-activated Sepharose 4B was close to 11.5% and a value of almost 23% was measured with the enzyme immobilized by adsorption to polyethylene terephthalate. The pH-optima of both pectin esterases were unchanged after their immobilization, their temperature stability and temperature optimum of activity, however, significantly increased. The differences in the action of free and immobilized pectin esterases were also observed when the final esterification degree of the substrate was compared: the immobilized enzymes, unlike the free pectin esterases, did not act on pectin showing a higher esterification degree. An increase in K m.app which was 5-fold for the tomato pectin esterase and 4-7-fold for the A. foetidus pectin esterase was observed after the immobilization. The immobilization of both pectin esterases on Enzacryl AA which had been activated by diazotization resulted in complete loss of activity; this indicates the role of the residues of tyrosine (and histidine, resp.) in the catalytic action of these enzymes, which has been observed in earlier experiments.
Exo-D-galacturonanase (E.C. 3.2.1.67) isolated from carrot was irreversibly adsorbed on poly(ethylene terephthalate) in a 0.1 mol l⁻¹ acetate buffer solution of pH 5.1. Activity of the immobilized enzyme depended on the amount of the enzyme bound to the support, on the ionic strength, and, to a very little extent, also on the pH of the reaction medium during immobilization. Activity of the immobilized enzyme dropped; the greatest relative activity (51.8%) had the preparation composed of 9.12 mg of the enzyme per 1g of the support. The pH optimum for catalytical activity of the immobilized enzyme was identical with that of the dissolved enzyme (5.1). Immobilization of the enzyme did not change its thermal optimum, and its thermal stability did not improve, either. The substrate specificity did not alter by immobilization, no differences were found in the mode of action on the polymeric substrate; digalacturonic acid was degraded by the immobilized enzyme like by the dissolved one. Differences in the kinetics of polymeric substrate degradation by the bonded exo-D-galacturonanase were manifested by a lower V' app value and by an increased K'm value. Exo-D-galacturonanase preparation obtained by adsorption on poly(ethylene terephthalate) was extraordinarily stable during storage at 4 °C, and towards action of salts and pH changes; its operation stability was high.
4, 6-Dichloro-s-triazinyl Duolite A7 (CC-A7) as a carrier of immobilized enzymes was studied. The reactivity of the second and the third organic chlorines of s-triazines attached to the carrier toward amino acids or proteins was investigated. The results show that CC-A7 can bind a large quantity of amino acids or proteins and these compounds seem to be bound covalently via the second organic chlorine of s-triazine of the carrier under the usual condition. It is also shown that this carrier can bind a sufficient amount of protein and enzyme activity of aminoacylase and retains a high activity in column operation. These results indicate that 4, 6-dichloro-s-triazinyl Duolite A7 is a convenient and useful carrier for the industrial application of immobilized enzyme.
GlucoamyIase[α-1, 4; 1, 6-glucan-4: 6-glucohydroease, EC 3, 2.1.3] from Rhizopusniveus was entrapped in polyacrylamide gels and adsorbed onto SP-Sephadex C-50 to elucidate the thermostability mechanism of immobilized enzymes. The thermal stability of immobilized glucoamylase entrapped in polyacrylamide gels was enhanced slightly compared with glucoamylase in free solution, and was independent of the acrylamide monomer concentration and N, N'- methylene-bis (acrylamide) content. To explain this phenomenon, the cellular structure of polyacrylamide gel was taken into consideration in addition to interactions between glucoamylase and gel, and a decrease in dielectric constant in the gel [S. Moriyama et al., Agric. Biol. Chem., 41, 1985 (1977)15]. On the other hand, immobilized glucoamylase bound to SP-Sephadex by ionic interaction showed lower stability than free glucoamylase, and much greater stability than glucoamylase in the presence of dextran sulfate, a constituent of SP-Sephadex. Thermal stabilities for the free and immobilized enzymes were also compared at the pH not in the bulk solution, but in the SP-Sephadex.
A polymerizable NAD derivative, N⁶-[N-[N-(2-hydroxy-3-methacrylamidopropyl)carbamoyl-methyl]carbamoylmethyl]-NAD, formate dehydrogenase, and malate dehydrogenase were entrapped all together in polyacrylamide gels. The entrapment was carried out by radical copolymerization, and consequently NAD was bound on the matrix which enclosed the enzymes. These gels had the function of producing L-malate from oxalacetate and formate. The L-malate production was also continuously done in a column reactor for 3 days. Another gel was similarly prepared with N⁶-[N-(6-methacrylamidohexyl)carbamoylmethyl]-NAD, horse liver alcohol dehydrogenase, and diaphorase. This gel was shown to catalyze the formation of resorufin from resazurin and ethanol. This gel was applicable to ethanol analysis using a fluorescence spectrophotometer to determine resorufin. The analyzer was usable for one week. © 1982, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.
Glucose oxidase was immobilized on the surface of a p-benzoquinone-carbon paste electrode by coating the enzyme-loaded surface with a nitrocellulose film. The electrode was able to oxidize glucose electrocatalytically. It showed high current response to glucose, and was stable for more than a week. The electrode can be used as a glucose sensor that is relatively insensitive to variations of oxygen tension in sample solutions.
Horseradish peroxidase (HRP) immobilized on TiO2 coated cellulose microfibers was used to study the catalytic oxidation of pyrogallol to purpurogallin in a flow injection (FI) system. The Michaelis-Menten constants for the enzymatic oxidation were determined for the immobilized enzyme, Km = 47 mM and V = 22 μmol min−1, and compared with those obtained for free enzyme, Km = 11 mM and V = 53 μmol min−1. The activity of the immobilized enzyme is reduced presumably due to its interaction with the oxide surface. However, the level of activity of the immobilized phase does not decrease with time under the conditions used in the FI system after various operation cycles.
The immobilization of endopolygalacturonase (endo-PG) on various supports (cellulose, PVP, nylon-6, Biogel, silica propanediol, silica propylamine, silica and γ-alumina) activated with TiCl3 has been evaluated by measuring the adsorption and activity parameters. The γ-alumina-Ti support shows the greatest adsorption value and the highest half-time of conservation of endo-PG at 25 °C and pH 3.0 (340 h). However, the observed catalytic activities in the 3.0–4.0 pH range, which are found to be lower compared with that of soluble enzyme, reduce the possibility of practical application of the activated γ-alumina support in fruit juice technology. The 30% reduction in activity values as a function of pH found in the γ-alumina-supported endo-PG compared with that of the same samples activated with TiCl3 is attributed to the catalytic action of dispersed titanium salt.
We have studied the influence of several parameters (temperature, NaBH3CN concentration, pH, contact time, ribonuclease A concentration) which control the immobilization of ribonuclease A onto aldehyde Spherosil beads.Response Surface Methodology has been used to examine the interactions between these parameters, and the observed results prove its suitabil- ity for this purpose.We have shown that the pH is an independent variable. The conditions for optimum activity of immobilized ribonuclease A are similar whether using RNA or CMP as substrate.
β-Galactosidase from Aspergillus niger was immobilized effectively on a porous ceramic monolith by adsorption and intermolecular cross-linking. The binding efficiency reached 80% and no enzyme leaching was observed even under vigorous mechanical agitation. Immobilization did not change the pH optimum of lactose hydrolysis. The enzyme decay followed first-order kinetics and the thermal stability of the immobilized lactase was considerably enhanced. At 50 °C and pH 3.6 the half-life of the immobilized lactase was 180 days and that of the free enzyme 24 days. The kinetics of lactose hydrolysis by both free and immobilized lactase were studied in a batch reactor system in the absence of any mass transfer limitations. In both cases the totally competitive galactose inhibition kinetic model predicted the experimental data. Simulation of the performance of a laboratory continuous flow immobilized lactase reactor system showed that experimental results could be predicted by the ideal plug flow model when an apparent effectiveness factor nf=0.65 is used to take into account the external mass transfer limitations.
The partially purified bromoperoxidase of Corallina pilulifera (Corallinaceae, Rhodophyta) was immobilized on the following matrices: Cellulofine (covalent binding), DEAE-Cellulofine (ionic binding), or alkylsilane treated Controlled-Pore Glass (physical adsorption), and entrapped in the soft gels using photo-crosslinkable resin prepolymer (ENT-2000), polyurethane prepolymer (PU-6), or κ-carrageenan. These different forms of immobilized bromoperoxidase were tested for the bromination reactions of monochlorodimedone and uracil. The immobilization techniques using DEAE-Cellulofine and ENT-2000 were found to be suitable for the bromoperoxidase reaction. The immobilized enzyme on DEAE-Cellulofine showed the highest activity and a half-life of 45 days when it was used for the conversion of uracil to 5-bromouracil.
In this report, the authors present initial results and limitations of a polymeric system for the immobilization of enzymes. Enzymes attached to insoluble polymers of natural and synthetic origin are gaining importance in many industrial and biomedical applications. Graft copolymers are used as enzyme supports and in this study a novel polymeric system of alginic acid-polyacrylamide graft copolymer is described which was used for immobilizing enzymes. (Refs. 4).
A bienzymatic sensing layer containing two enzymes able to work sequentially, choline oxidase (ChOD) and phospholipase D (PLaseD), was used to design an electrochemical biosensor for the detection of either a water-soluble (choline) or insoluble (phosphatidylcholine) substrate. A photocrosslinkable polymer, poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ), was used as host-matrix for enzyme immobilization. Controlled amounts of PVA-SbQ and of the two enzymes were directly coated on a platinum disk, then photopolymerized. The compatibility of working conditions for choline and phosphatidylcholine detection in the presence of Triton X-100 and CaCl2 was investigated. The effect of the activity ratio PLaseD / ChOD on the sensor performance was determined. The sensitivities to choline and to phosphatidylcholine were 18 mA.1mol and 0.66 mA.1.mol respectively, the detection limit being 1.5.10 M for choline and 1.5.10 M for phosphatidylcholine. The linear range extended up to ca. 10 M for choline and ca. 2.10 M for phosphatidylcholine and the response time was close to 30 seconds for choline and ca. 2 min for phosphatidylcholine.
Proteolytic enzymes immobilized by adsorption on a solid support are widely used for synthetic reactions in low water systems. However, stability of such derivatives has to be optimized to avoid of reversible and irreversible inactivation of enzyme by organic solvent. Immobilization of enzyme, previously involved in non-covalent complex with polyelectrolyte, could be a possible way to suppress inactivation processes. In the present work, noncovalent complex between a-chymotrypsin and polycation Polybrene was immobilized on Celite. Stability of this biocatalyst against irreversible inactivation by organic solvents as DMSO, DMFA and AcN at 30 °C was studied. It was observed that the complexation with Polybrene could lead to either stabilization or destabilization of enzyme in dependency on type and concentration of organic cosolvent. Maximal protective effect, defined as ratio of half-life times of enzymes, immobilized in/out of complex with Polybrene, was about 50-fold for inactivation by dry DMFA and 30-fold for inactivation by dry AcN and 20% solution of DMSO.
The immobilization of the globular enzyme horse heart cytochrome c (R30A) in mesoporous molecular sieves was studied. Cytochrome c was physically adsorbed into the all silica MCM-41 (32A), aluminosilicate MCM-41 (32A), all silica MCM-48 (34A) and Nb-TMS1 (33A). The physical adsorption of the enzyme showed clear dependences on molecular sieve composition. The cytochrome c cannot be removed from the molecular sieves unless the pH is > 9. Silanation of the molecular sieve pore openings prevented significant leaching even at high pH values. The reduction/oxidation activities of the immobilized cytochrome c are also reported.
An interaction of proteins with acid polysaccharides and other polyelectrolytes results in changes of properties of protein microenvironment under influence of electrostatic field of polyelectrolytes. Alterations of protein properties, such as the kinetic parameters of enzymatic reactions, protein denaturation, the rate of interaction with glutar aldehyde etc. may be completely explained by the influence of polyelectrolytes electrostatic field on distribution of reaction components in ionic atmosphere of electrolytes. The real pK values, of ionogenic groups, protein conformation and other parameters characterizing the state of protein molecules were not changed in any system investigated.
If one of the reaction component is H+ ion (hemoglobin denaturation, enzymatic reactions which depend on the state of protein ionogenic groups etc.) the effect of polyelectrolyte is similar to that of changes of pH. Polyanions' action resembles the effect of decreasing of pH (increases the local concentration of H+ ions); polycations' action is similar to that of elevation of pH.
If one of the reaction components is a compound able to interact electrostatically with polyelectrolyte, the influence of polyelectrolyte can be seen on the reaction steps the rate of which is dependent upon the concentration caused by polyelectrolytes.
If the parameter under investigation is sensitive to the state of protein aggregation, the effect of polyelectrolytes may be based on changes in protein concentration caused by polyelectrolytes.
The conception given above allows to explain some effects described in literature. In particular, it allows to explain the anticoagulation action of heparin and other polyanions.
The relationships observed appear to be common for different protein-polyelectrolyte systems. The magnitude and the direction of the effects are dependent on the value and sign of changes of components (z
i
) and an electrostatic potential (ω) of protein-polyelectrolyte complexex. Provided ω andz
i
are known, a degree of polyelectrolyte effect on any protein properties thez and ω values may be calculated. For from the effect of polyelecrolyte on some protein properties thez and ω values may be calculated. For example, this approach was used to determine the degree of etherification of pectines using their effect on the rate of ATEE hydrolysis catalysed by α-chymotrypsin.
The absence of changes in proteins' molecules themselves allows to conclude that polyelectrolytes do not diminish the biological value of protein if they are used as artificial food.
The purpose of the work presented here was to prepare a support material for enzymes and “affinity ligands” with the following characteristics: low cost, durability, rigidity, and high capacity. Our study encompassed conjugates of porous and nonporous silicas with organic polymers and macroporous ion-exchange resins. Poly-ethyleneimine (PEI), polyacrylic acid (PAA), poly (methyl vinyl ether/maleic anhydride) were attached to porous glass and silica in various combinations. The composite of silica beads with PEI and PAA is a good support for the enzyme trypsin as judged by the activity against N-α-benzoyl-L-arginine ethyl ester.Amberlyst (macroporous, sulfonated polystyrene) was activated by treatment with thionyl chloride; the resulting resin was either used directly or reacted with a diamine. The diamine derivative was used for enzyme coupling or transformed further to the succinyl or p-aminobenzoyl derivative. None of these derivatives were particularly good as supports for the enzyme trypsin. Duolite converted to a PAA, succinyl, or succinimide derivative was a good support. The enzyme-resin adduct has good activity and stability.The resin is quite durable and of low cost. The Duolite-trypsin has good activity against protein. In addition, this derivative was active in 7 M urea. The proteolytic activity was nearly doubled by urea, presumably as a result of substrate (casein) denaturation. The michaelis constants and pH dependences are compared for trypsin conjugates with Duolite A-7, Silica-PEI-PAA, agarose, and porous glass. A cost comparison reveals that the Duolite and silica derivatives are much less expensive than agarose and glass.
A commercial preparation of β glucosidase obtained from Aspergillus niger was immobilized in calcium alginate, after enzyme crosslinking with a bifunctional agent, which increases its molecular size with the formation of enzymatic aggregates. The immobilized enzyme exhibited changes of activity profiles related to temperature and pH, in comparison to the free enzyme. However, there was no shift in optimum pH and temperature upon immobilization. The catalyst showed an improved thermal stability when compared with the free enzyme, yielding a half-life of 23 days, at 50°C. The free enzyme exhibited a 9-day half life, determined at the same temperature.
The adsorption of various enzymes (proteases, lipases and peroxidases) onto the surface of talc (a hydrophobic support) and non- talc materials was investigated. In general, adsorption was favored by the hydrophobicity of the support. We found little evidence for the ionic interactions that characterize adsorption onto mineral supports (clays, porous glasses). Modification of the hydrophobic-hydrophilic balance of the talc support produced new immobilized biocatalysts with high enzyme activity (both lipases and horseradish peroxidase). This represents the first example of this type of talc-protein interaction.