Impact of immobilization technology in industrial and pharmaceutical applications

Abstract

The current demands of the world’s biotechnological industries are enhancement in enzyme productivity and development of novel techniques for increasing their shelf life. Compared to free enzymes in solution, immobilized enzymes are more robust and more resistant to environmental changes. More importantly, the heterogeneity of the immobilized enzyme systems allows an easy recovery of both enzymes and products, multiple reuse of enzymes, continuous operation of enzymatic processes, rapid termination of reactions, and greater variety of bioreactor designs. This review summarizes immobilization definition, different immobilization methods, advantages and disadvantages of each method. In addition, it covers some food industries, protein purification, human nutrition, biodiesel production, and textile industry. In these industries, the use of enzymes has become an inevitable processing strategy when a perfect end product is desired. It also can be used in many other important industries including health care and pharmaceuticals applications. One of the best uses of enzymes in the modern life is their application in diagnose and treatment of many disease especially when used in drug delivery system or when used in nanoform.

Introduction

Enzymes increase the rate of chemical reactions without changing or being consumed permanently due to interactions. They also increase reaction rates without changing the balance between reactants and products (Cooper 2000). Reaction rates are accelerated more than a million times; so, reactions that take years to complete in the absence of stimulation can occur within parts of a second in the presence of the appropriate enzyme. Enzymes utilization increased rapidly all over the world; the worldwide figure of industrial enzymes is about €1.6 billion. The liquied enzymes can not be easily separated from the reaction and lost after the first use that means additional cost (Wahba and Hassan 2017).

On the other hand, if the enzyme form changed from liquid form to another solid form, it can be used many times, that means saving money. This can be done using immobilization technology. As shown in Fig. 1, it is difficult to separate the enzyme (in liquid form) from the product, while the immobilized form of enzyme would empower the reusability as it can be separated easily from the product; therefore, the cost of enzymes and product will be reduced significantly (Elnashar and Hassan 2014).

Fig. 1.

a, b Schematic diagram of free and immobilized enzyme reactions. a Reaction of free enzyme with substrate and formation of product, which has to be separated via dialysis; b reaction of immobilized enzyme with substrate and formation of product, which can be separated via filtration

To make the use of enzyme in industrial and biotechnological processes more convenient, different methods have been applied to reduce cost (Hassan et al. 2016). Immobilization is one of these methods. The term immobilization can be defined as “enzyme physically localized or confined in a defined region of space with retention of the catalytic activities, that can be reused repeatedly and continuously”. Thus, the main aim of immobilization is associating the enzyme with an insoluble matrix so that it will be separated easily and reused under stabilized conditions (Tosa et al. 1966).

So, the immobilized enzyme is more convenient than the free one. It offers the possibility of repeating flow processing; also, the easy recovery and low-cost operation can be done in industrial processing (Magdy et al. 2014).

The immobilization technology is a common technique that used mainly to reduce lost of enzymes during the process by reusing enzymes many times that means economic benefits (Eman et al. 2017). In this technology, the enzyme is linked in/on a polymeric matrix in the form of beads, film or membrane, in such a manner that the enzyme cannot be lost into reaction solution (Mohy Eldin et al. 2012; Hassan et al. 2019). Immobilized enzyme technology also facilitates the downstream processing as it can be separated easily from the reaction by filtration. On the other hand, much more efforts and money are needed to separate the soluble enzyme from the reaction process. Also, immobilization makes the enzyme more stable than the soluble one (Ghada et al. 2016). Although the immobilization technology of enzymes has the advantage of reusability and reducing the operation cost, it also has some disadvantages, as shown in Table 1.

Table 1.

Technological properties of immobilized enzyme system

Advantages	Disadvantages
Catalyst reuses Easier reactor operation Easier product separation Wider choice of reactor Stable and more efficient Products free enzyme Suitable for medical and industrial use	Loss or reduction in activity Diffusional limitation Additional cost

Over the previous decade, enzyme immobilization has been developed rapidly. Comparing it nowadays with the previous years; we searched in science direct database for studies dealing with enzyme immobilization. This search includes English-language peer-reviewed publications related to any enzyme immobilization work between 1997 and 2018. As shown in Fig. 2a, the outcome of publications is tending to increase by time. It was only 7256 publication articles in 1997 and become 22,159 in 2018, indicating the increased interest in enzyme immobilization in recent years. This number of publications is divided into research articles, review article, etc. as shown in Fig. 2b.

Fig. 2.

a Annual number of enzyme immobilization articles indexed in Science Direct data base 1997–2018. b Types of enzyme immobilization articles indexed in Science Direct data base 1997–2018

Choice of support and principal method

Immobilization of enzymes process has three major components: enzyme, the support or matrix, and mode of interaction between enzyme and this matrix.

The support or matrix chosen is essential as it can enhance the operational stability of the immobilization system. Although there is no universal matrix, there are many features that should be found in any material consider as immobilization matrix or support. Matrix properties are of paramount importance in determining the efficacy of the enzyme immobilization system (Brodelius and Mosbach 1987; Jakub et al. 2018).

The choice of the optimal support material can affect the process, where the properties of both the enzyme and the supporting materials determine the properties of the enzyme-supported reaction. Thus, the interaction between the enzyme and matrix leads to an immobilized enzyme with specific mechanical, chemical, biochemical and kinetic properties (Sheldon 2007).

The ideal properties of the support material include inertness towards enzymes, physical resistance to compression, ease of derivatization, high surface area, hydrophilicity, resistance to microbial attack, biocompatibility, resistance to pressure and availability at low cost (Buchholz and Klein 1987; Li et al. 2004).

Therefore, we must first determine the support material based on the final application of immobilized enzyme, and then the immobilization method. Some points that must be considered are listed in Table 2.

Table 2.

Fundamental considerations in selecting a support and methods of immobilization

Property	Points of consideration
Physical	Strength, available surface area, non-compression of particles, form (beads/film/fibers), pore size, degree of porosity, permeability, density, pressure drop and flow rate
Chemical	Hydrophilicity, availability of functional groups for modification, inertness toward enzyme/cell, and reusability of support
Stability	Residual enzyme activity, regeneration of enzyme activity, cell productivity, maintenance of cell availability, and mechanical stability
Resistance	Microbial attack, disruption by chemicals, temperature, pH, organic solvent, proteases, and cell defence mechanism (proteins/cells)
Safety	Biocompatibility, toxicity, health and safety for process workers and end-product users, specification of immobilized preparation (FDA approval) for food, and medical applications
Economic	Cost and availability of support, its mechanicals, reagents, special equipment, technical skill required, Industrial-scale chemical preparation, environmental impact, feasibility for scale-up, effective working life, continuous processing, reusable support, and zero contamination (enzyme/cell-free product)
Reaction	Enzyme/cell loading and catalytic productivity, Flow rate, side reactions, reaction kinetics, multiple enzyme and/or cell systems, diffusion limitations on mass transfer of cofactors, substrates and products

Principal methods for immobilization of enzyme

Choosing the appropriate immobilization method is an essential part of the immobilization process. It plays a key role in determining the activity and properties of the enzyme in a particular reaction (Chiou and Wu 2004). Immobilization methods can be divided into two general categories: chemical and physical methods. Physical methods are characterized by weak monovalent interactions such as hydrogen bonds, van der Waals forces, hydrophobic interactions, affinity binding, ionic binding of the enzyme with the supporting material, or mechanical containment of the enzyme within the support (Costa et al. 2005).

While in the chemical methods, the formation of covalent bonds achieved through ether, thio-ether ether, amide or carbamate bonds (Brena and Batista-Viera 2006) between the enzyme and the supporting material is involved. There are five principal methods available for enzymes immobilization: adsorption, covalent binding, entrapment, encapsulation, and cross-linking (Wahba and Hassan 2015).

Adsorption

Physical adsorption method is one of the simplest immobilization methods; in this method, the enzyme is adsorbed on the surface of the matrix that may be an organic or inorganic matrix (Woodward 1985; Sugahara and Varéa 2014). This adsorption method is based on weak bonds such as van der Waal’s force, electrostatic and hydrophobic interactions, hydrogen bond or ionic bond between enzyme and solid matrix (Flickinger and Drew 1999; Jegannathan et al. 2008). Table 3 shows some advantages and disadvantages of the physical adsorption method.

Table 3.

Advantage and disadvantage of adsorption method

Advantages	Disadvantages
Little or no damage to enzyme/cells Easy, cheap, and fast No changes happened to carrier or enzyme/cells Reversible	Leakage of enzyme/cells from the support Separation of product is not easy Nonspecific binding

Physical adsorption of the enzyme can be done by soaking the solid matrix in a solution of the enzyme at specific conditions of temperature, pH, ionic strength, etc., for a specific time. Then, the solid matrix containing enzyme was washed to remove unbounded enzymes (Fig. 3).

Fig. 3.

Immobilization methods

Covalent binding

The covalent binding method is the most widely used method for enzyme immobilization (Porath and Axen 1976). This method depends on the formation of a stable covalent bond between functional groups found in enzyme and the functional groups found on the surface of the support material such as amino group, carboxylic group, hydroxyl group and sulfhydryl group (Fig. 3). In some cases, the support materials are inert and need activation before the formation of the covalent bond with the enzyme (Amal et al. 2016; Marcela et al. 2008).

The main disadvantage of this method is that the reaction may be carried out through an active site of the enzyme (amino group). This means that the enzyme is inactivated. So, it is imperative to choose the suitable method of immobilization for every specific reaction (Srere and Uyeda 1976).

Entrapment

Entrapment is one of the easiest methods of enzyme immobilization. In this method, the enzyme is physically restricted inside the network of support material (Fig. 3). Enzyme entrapment can improve the stability of the enzyme with no loss of activity as there is no chemical interaction between enzyme and support material. The critical parameter in this method is the support material choice and its pore size, which affect the reaction between restricted enzyme and substrate. The difference between entrapment method and other methods such as adsorption and covalent binding is that the enzyme is free in movement in solution; however, it is restricted inside the support material (Mohy Eldin et al. 2005; Driscoll 1979; Brodeliu 1985).

Microencapsulation

Microencapsulation method is a type of entrapment method (Fig. 3); it can be done by wrapping enzymes or cells inside the semi-permeable membrane. It has the same advantage of entrapment method as the enzymes or cells are free in movement but limited in a specific area. While the disadvantage of this mehod is that there are many enzymes and cells are big enough so it cannot out or inter the capsule. The most crucial factor is the pore size found in the capsule (Kierstan and Coughlan 1991).

There are three main methods for microencapsulation: formation of special membrane reactors, preparation of emulsions, and formation of microcapsule from this emulsion. This method has recently been used for microencapsulation of enzymes and mammalian cells (Tian et al. 2009).

Cross-linking

The cross-linking method is another irreversible immobilization method. This method relies only on the enzyme; so it is a support material-free method. In this method; the enzyme molecules (or cells) cross-linked with each other to prepare a large three-dimensional complex molecule using poly-functional reagent (Fig. 3). There are several reagents such as glutaraldehyde, hexamethylene diisocyanate, diazobenzidine and toluene diisothiocyanate which can be used to create bonds between amino groups of enzymes in cross-linking formation. This cross-linking can be done chemically or physically (Grobillot et al. 1994). The advantage of this method is that there is no loss in enzyme activity. Also, the enzyme contacts with the substrate directly; while the disadvantage is that some cross-linking reagents may be harmful to the enzyme or cell and it may be denatured the enzyme by the cross-linking reagent.

Glutaraldehyde is the most extensive cross-linking reagent as it is economical and easily obtainable. It aggregates the enzyme molecules by forming Schiff’s base (Migneault et al. 2004). The links formed between enzymes molecules are irreversible. The most crucial factor is the temperature and pH of the enzyme solution. This cross-linking method is widely used in industrial enzymes immobilization such as penicillin acylase and glucose isomerase. This method is cost-effective and straightforward (Sheldon 2007).

Choice of immobilization method

It is very important to choose the suitable method of immobilization to avoid deactivation of the active site found on the surface of the enzyme. So, knowing every enzyme active sites will help in choosing the appropriate method that prevents reaction with it. Also, these active sites can be protected during immobilization technology and in some cases, this is done using the enzyme–substrate or specific inhibitor. Choice of a suitable method of immobilization is based on many factors such as stability, enzyme catalytic activity and also cost factor (Ciaron 2003).

So, the enzyme or cells can be immobilized using different methods, while every method has its own advantage and disadvantage, as shown in Table 4.

Table 4.

Advantages and disadvantages of immobilization methods

Characteristic	Immobilization method
	Physical adsorption	Covalent binding	Entrapping method	Encapsulation	Cross-linking method
Preparation	Easy	Difficult	Difficult	Easy	Difficult
Enzyme activity	Low	High	High	High	Moderate
Substrate specificity	Unchangeable	Changeable	Unchangeable	Unchangeable	Changeable
Binding force	Weak	Strong	Strong	Moderate	Strong
Regeneration	Possible	Impossible	Impossible	Possible	Impossible
General applicability	Low	Moderate	High	Moderate	Low
Cost of immobilization	Low	High	Low	Low	Moderate

Applications of immobilized enzymes and cells

Enzymes are the key players that are widely used in various industrial and pharmaceutical processes, as well as their involvement in food production and exploration of knowledge in microbiology, biochemistry and other similar disciplines. Contentious efforts are being made for the improvement of enzyme activity, reproducibility, efficiency and also stability during industrial processes (Wang et al. 2010). Enantioselective and regioselective compounds have been produced for pharmaceutical applications by immobilization technology (Ren et al. 2006; Lee et al. 2009).

Industrial applications

Many efforts have been made by researchers to reduce the cost of products by reusing the enzymes in industrial applications. They find that this can be achieved using an immobilization technology that facilitates the reuse of enzymes, while maintaining their stability and effectiveness. In view of the number of researches published in science direct database on the use of immobilization technology in industry between 1997 and 2018, we found that it is constantly increasing, demonstrating the importance of this technology in the industry (Fig. 4a, b).

Fig. 4.

a Annual number of enzyme immobilization in industry articles indexed in Science Direct data base 1997–2018. b Types of enzyme immobilization in industry articles indexed in Science Direct data base 1997–2018

Food industry application

The purified enzymes can be used in the food industry (Table 5). It may be denatured during the purification process. But the enzymes that have been immobilized are stable and can be easily separated. Immobilized enzymes can be used in the production of syrups. Immobilized β-galactosidase has been used in the decomposition of lactose in whey to produce glucose from waste materials and can also be used to produce lactose-free milk (Wahba and Hassan 2015). Also, it can be used in the production of baker’s yeast. Essential oils such as cumin and fennel can be encapsulated inside the polymeric matrix and used as preservative materials that provide health and nutritional benefits. Fennel and cumin essential oils can be used in food products such as bread, beverages, pastries, pickles and cheese (Hussein et al. 2016; Walaa et al. 2018).

Table 5.

Immobilized enzymes used in food industry

Enzyme	Food substrate	Reference
β-Galactosidase and amyloglucosidase	Lactose whey, whey permeates, skimmed milk	Osma et al. (2010)
Pectinase	Pectin	Purnachandra and Saritha (2015)
Laccase	Fruit juice and beer processing	Esmail et al. (2016)
Trypsin	β-Lactoglobulin	Kristal et al. (2018)
Tyrosinase	Phenolic in red wine	Rajendra et al. (2016)
Lipases	Triacylglycerols	Abol Fotouh et al. (2016)
Keratinase	Feather hydrolysates	Cai et al. (2008)

Dairy industry

Immobilization technology of cells in the dairy industry has been widely examined. It modifies the cell physiology and consequence of cell immobilization technology on lactose and also on citrate metabolism. This technology can be used in the production of starter for the dairy industry, including acidification of raw milk prior to ultra filtration, the production of yogurt, cream fermentation and cheese processing. Also, lipase can be used in the conjugation of linoleic acid in dairy food (Denkova et al. 2004).

The increased interest in the development of dairy processes to hydrolyze lactose found in dairy products is because of the problem of lactose intolerance that found in many countries worldwide. In this method, lactose can be converted to glucose and galactose using enzyme β-galactosidase immobilized on a polymeric material (Fig. 5). These monosaccharide sugars are sweeter, soluble and digestible than lactose. Thus, it can be used by people suffering from lactose intolerance problem (Elnashar and Hassan 2014).

Fig. 5.

The immobilized lactase converts lactose into glucose and galactose as the milk flows through

Protein purification

Purification of proteins is an essential goal in industrial enzymes. It can be done to increase the specific activity of the enzyme and to obtain the enzyme in its pure form. Purification by affinity is the most used technique as it can reduce the time and steps of chromatography. Affinity ligands immobilization to an insoluble matrix can be a powerful tool to isolate a certain substance (protein) from a complex mixture (Silva et al. 2011). Immobilization of carbohydrate-binding proteins, such as lactose, mannose and melibiose, as well as immobilized metal ions is an example of affinity ligands technique. For example, lactase can be purified from a mixture of proteins using immobilized lactose on a solid matrix (Hermanson et al. 1992).

Human nutrition

Nutrients are defined as the food components that have health benefits more than that of traditional nutritional value. New biotechnological tools such as immobilization technology have also been used to isolate and integrate these nutrients into normal food. The synthesis of nutrients has been reported to be successful using immobilized lipases, such as those produced from Lactobacillus ruteri and Candida antartica (Nedovic et al. 2003).

Biodiesel production

Biodiesel is monocrystalline esters of long-chain fatty acids. It is produced through a chemical combination of alcohol with triglycerides in the presence of a catalyst. This catalyst is the lipase enzyme. This enzyme can catalyze the esterification reaction with fewer requirements of energy and mild reaction conditions. Also, it produces fewer by-products or wastes (Fukuda et al. 2001).

Due to the high cost of lipase production, the immobilized lipase is more convenient as it can be reused and more stable (Fig. 6). This enzyme can be immobilized by different methods, and the adsorption method is a widely used technique in recent years (Jegannathan et al. 2010).

Fig. 6.

Enzymatic biodiesel production by immobilized lipase

Textile industry

It is a traditional industry in many countries that has a large proportion of the economy. Microbial enzymes are of great interest in this industry. These enzymes such as amylase, cellulase, laccase, cutinase, pectinase, etc. are used in various textile applications such bio-polishing, as scouring, denim finish and wool processing design (Hassan et al. 2013). The widest enzyme used in this industry from the earlier period until now is cellulase enzymes. Because this industry requires high temperature degrees and also high pH values, the free enzymes are not suitable at those extreme conditions. Thus, immobilized enzymes can react in extreme conditions and also maintain its stability for many cycles. Nanoparticles of polymethyl methacrylate are synthesized and covalently bonded with cellulase enzyme (Chen et al. 2007).

Wastewater treatment

Heavy metals pollution is one of the most critical environmental problems of concern worldwide. Industrial wastewater may contain heavy metals such as Cr, Pb, Cd, Zn, Ni, As, Ag, Cu and Hg (Abdel Hameed and Ebrahim 2007). The most extensive techniques used to eliminate the matrix interference are precipitation, ion-exchange separation, solvent extraction and solid-phase extraction. Biosorbents such as immobilized algae were used to remove heavy metals pollution from wastewater (Shareef 2009). These biosorbents are defined as the selective isolation of metal soluble species that result in the immobilization of metals by bacterial cells such as cyanobacteria. On the other hand, bio-polymeric materials such as carrageenan, alginate can be used in water softening using immobilization technology (Fig. 7); in this method, the metal ions are immobilized on the surface of activated bio-polymeric materials and removed from the water (Ali et al. 2017).

Fig. 7.

Wastewater treatment diagram using immobilization technology

Decolourization of dyes

It is known that large quantities of dangerous compounds are released at different stages of textile processing, including dyes, chromium, alkalis, oils, waxes and phenol (Asamudo et al. 2005). Thus, the conversion of such potential toxic compounds is necessary before disposal. Most of the physico-chemical methods currently used have technical and economic limitations. They are expensive and produce large amounts of sludge and are not suitable for some soluble dyes. Biomass-based materials can be used for the direct enzyme immobilization. An enhancement instability and also lack of chemical modifications are usually achieved (Akhtar et al. 2005). For this purpose, immobilized peroxidase on alginate–starch beads is used to remove dye from textile (Matto and Husain 2009).

Pharmaceutical applications

Manufacturing or processing of enzymes to be used as a drug is an essential secondary aspect of the pharmaceutical industry today. Attempts were made to utilize the benefits of enzymes as medicines in almost all scientific centers of pharmacy in the world. In view of the number of articles published in science direct database that is dealing with immobilization technology in pharmaceutical applications between 1997 and 2018, we found that it is continuously increasing, thus proving the importance of this technology in the pharmaceutical industry Fig. 8a, b. Immobilization technology of enzymes has been used in medicine since 1990 (Tischer and Wedekind 1992). Immobilized enzymes can be used to diagnose or treat the disease in the medical field as well as artificial organs. Cells can also be used in the coating of artificial materials to improve biocompatibility. Enzymes can be encapsulated through the electroporation and used in the medical field. This method is the easiest way of enzyme immobilization in this field, and it is a reversible method for which the enzyme can be regenerated (Lizano et al. 1998).

Fig. 8.

a Annual number of enzyme immobilization in pharmaceutical applications articles indexed in Science Direct data base 1997–2018. b Types of enzyme immobilization in pharmaceutical applications articles indexed in Science Direct data base 1997–2018

Immobilized enzymes in clinical medicine

Enzymes can be used as therapeutic agents because they perform complex chemical reactions under physiological conditions (Junyu et al. 2019). Many of these interactions occur in organisms that are healthy and normal but are weak in case of disease or morally deficient patient. An externally applied enzyme can confer on the organism a biological function that is not hereditary in this organism. So, the immobilized enzyme has many advantages rather than the soluble one. Several enzymes have been evaluated with their clinical use (Michael and Robert 1986). For example; the immobilized form of bilirubin oxidase was used to remove bilirubin from the circulatory system. Some applications of enzymes in clinical approaches listed in Table 6.

Table 6.

Some immobilized enzymes with their potential for medical therapy

Enzyme	Reactor (support material)	Disease	Enzyme action	References
α-Galactosidase	Sepharose 4B	Heart and kidney failure	Remove terminal galactose	Garman (2007)
Arginase	External surface of Amicon® fibers in a chamber	Familial hyperargininemia	Remove arginine from blood	Kanalas et al. (1982)
	Polyethylene glycol
Asparaginase	External surface of fibers in Cordis-Dow hollow fiber hemodialyser	Cancer (acute lymphocytic leukemia)	Destroy asparagines, an essential amino acid in tumor growth	Richards and Kilberg (2006)
	Polyacrylamide and polyacryldextran microcapsules
	Artificial cells
	Reed blood cell ghost
	Polyethylene glycol
	Liposomes
	Methacrylate plates
Bilirubin oxidase	Sepharose in a packed bed reactor	Liver disease (failure)	Converts bilirubin to biliverdin	Filip et al. (2017)
Carboxypeptidase G1	Polysulfone hollow fibers	Tumors treated with methotrexate	Inhibits folate metabolism (antagonize methotrexate)	David and John (2004)
Heparinase	Sepharose 4G in a fluidized bed reactor	Extracorporeal circulation	Removing disaccharides to inactivate hebarin	Bhushan et al. (2017)
Liver microsomal enzymes	Large agarose beads in packed bed reactor	Liver failure	Various detoxification steps	Lahtela et al. (1986)
Phenylalanine ammonia lyase	Amicon® hollow fibers	Phenylketonuria (PKU)	Metabolizes phenylalanine	Zhang and Liu (2015)
	Polyethylene glycol
UDP glucuronyl transferase	Agarose beads	Liver failure	Conjugates bilirubin and other toxins to water soluble form	Fujiwara et al. (2016)
Urea cycle enzymes	Blood dialysed through hollow fibers; packed bed sepharose 4B reactor	Liver failure	Metabolize ammonia	Guy Helman et al. (2014)
α-1,4-Glucosidase	Albumin	Type II glycogenosis	Metabolizes glycogen for its removal from cells	Mohy Eldin et al. (2012)
β-Glucuronidase	Reed blood cells ghost	Mucopolysaccharides VII	Metabolizes mucopolysaccharides to prevent its accumulation	Maruti et al. (2008)
Catalase	Collodion microcapsules	Inflammatory diseases mediated by oxygen free radicals	Inhibits preparation of hydroxyl radicals	Chang (2007)
Fibrinolysin	Sephadex	Thromboembolic occlusive vascular disease	Clot dissolution	Wong and Mine (2004)
Glutaminase	Polyethylene glycol	Cancer	Destroys glutamine, an essential amino acid for tumor growth	Erickson and Cerione (2010)
Tyrosinase	Collodion artificial cells	Liver failure	Metabolize tyrosine and free phenol	Arora et al. (2016)
Urokinase	Agarose beads	Thromboembolic occlusive vascular disease	Activate plasminogen in blood to lays clots	Senatore et al. (1986)

Thrombolytic enzymes

Lysing percent of plasma coagulation was used to determine fibrinolysis activity (Mona et al. 2016) Immobilized streptokinase can be used to treat clotting coagulation that requires fibrin therapy and can be a valuable treatment in case of expected stenosis problems (Everse 1981).

Diabetics

Immobilization of glucose oxidase on a polycarbonate membrane modified by the urethane coupling with poly (l-lysine) has been described to be activated using glutaraldehyde (Shin-ichiro et al. 1998). The pH stability and thermal stability of the immobilized enzyme were higher than that of non-immobilized one. A comparison between the enzyme activity and the immobilization method showed that the amount of glucose oxidase adsorbed on the normal polycarbonate membrane was negligible. Whereas, covalent binding with aldehyde groups in the derived membrane string was with no observed leakage. The membrane can be used as a glucose sensor (Maryam et al. 2019; Montaser et al. 2016).

Biomedical analysis

Immobilization technology of enzymes or cells can be used to develop precise and specific analytical techniques to estimate many biochemical compounds (Kuan-Jung et al. 2018). The principle of this assay depends primarily on the reaction of the substrate and the immobilized enzyme. An increase in the product level or decrease in substrate level is the monitor of this assay (Maslova et al. 2018). A list of selected examples of immobilized enzymes used in the assay of some substances is shown in Table 7.

Table 7.

A list of selected examples of immobilized enzymes that can be used in assay of some substances

Immobilized enzyme	Substance assayed	References
Glucose oxidase	Glucose	Ren et al. (2006)
Urease	Urea	Guy Helman et al. (2014)
Cholesterol oxidase	Cholesterol	Rajendra et al. (2016)
Lactate dehydrogenase	Lactate	Makler et al. (1998)
Alcohol oxidase	Alcohol	Edalli et al. (2016)
Hexokinase	ATP	Driscoll (1979)
Galactose oxidase	Galactose	Fujiwara et al. (2016)
Penicillinase	Penicillin	Mohy Eldin et al. (2012)
Ascorbic acid oxidase	Ascorbic acid	Garman (2007)
L-Amino acid oxidase	L-Amino acids	Hassan et al. (2016)
Cephalosporinase	Cephalosporin	Mohy Eldin et al. (2012)

Enzyme-linked immunosorbent assay (ELISA)

This test is used as a tool for the detection of antigens or antibodies in a sample (Farre et al. 2007). This technology measures the links between enzyme and other antibody or antigen. The procedure is based on the immobilization technology where the antigen is immobilized on the solid support (in this case plate well). The color amount is a proportion to the amount of antibody found in the sample that bound to an antigen found in well (Ernesto et al. 2015).

Antibiotic production

One of the most important antibiotic groups, historically and medically, is the β-lactam group, especially penicillin and cephalosporin (Gao et al. 2018). Production of antibiotic is a vital role in the applied microbiology field. Since it is difficult to produce antibiotic with continuous fermentation of free cells, using cell immobilization is preferred in this technology. Thus, a lot of attempts have been done to immobilize various microbial cells in/on different support materials for antibiotic production. Immobilization of Penicillium chrysogenum for the production of penicillin antibiotic is the widely used system in the production of antibiotics (Ogaki et al. 1986). Also, covalently binding penicillin G acylase can be used on different support materials in converting penicillin G into 6-amino penicillinic acid; which is the backbone of other β-lactam antibiotics as shown in Fig. 9.

Fig. 9.

Some semi-synthetic penicillins and naturally produced penicillin G

Drug delivery

Immobilization of drugs into solid carrier has been increased in applications and used as a therapeutic agent in recent years. The carrier itself must be biodegradable and non-toxic material. So, gelatin and human serum albumin were used as a carrier because of their biodegradability and low toxicity (Shweta et al. 2014 and Aldobaev et al. 2018).

Drug delivery system occurs when a polymer is combined with an active agent or a drug and the active agent can release from the polymeric material in a predetermined manner (Fig. 10a, b). The release of that active agent may be constant for a long time, or it may be affected by environmental conditions such as pH or temperature. The main advantage of this system is that it can keep the drug level within the required range for a long time with fewer needs of administrations. So, this system depends on the immobilization of the drug in/on the support materials that must be biodegradable (Saad et al. 2018).

Fig. 10.

a Drug release by time. b Mechanism of drug entrance to blood circulation

Enzyme immobilization on nanoparticles

Nanoparticles materials are one of the most efficient support materials used in enzyme immobilization, as it has specific features such as surface area, effective enzyme loading and mass transfer resistance (Gupta et al. 2011; Shivani et al. 2019; Naderi Peikam and Jalali 2018). These nanoparticles materials can be used in immobilization of enzymes that are used in industrial and medicinal applications, Table 8 summarizes some applications of nano-immobilized enzymes. The enzyme shows Brownian movement in aqueous solution when bounded with nanoparticles. Additionally, there is an additional advantage for magnetic nanoparticles as it can be separated easily using a magnetic field (Neda et al. 2019; Masi et al. 2017). The advantage of nanoparticles in immobilization is that it can improve stability and performance and reduce protein unfolding other advantages and disadvantages listed in Table 9. There are different types of nanoparticles (metal oxide, metal, carbon nanotubes, nanorods, nanofibers, porous, magnetic and polymeric nanoparticles) used in immobilization technology (Cipolatti et al. 2014; Almadiy and Nenaah 2018).

Table 8.

Some applications of nanimmobilized enzymes

Enzyme	Nanoparticle	Application	References
β-Glucosidase	Ironoxide nanoparicles	Biofuel production	Gao et al. (2018)
	ZnO nanoparicles	Lactose hydrolysis
Superoxide dismutase	Nano Fe3O4 coated on gold electrode	Biosensors	Gao et al. (2018)
α-Amylase	Silica nanoparicles	Detergent for removal of starch soils	Fu et al. (2013)
	Cellulose coated magnetic nanoparicles	Starch degradation
Lysozyme	Chitosan nanofibers	Antibacterials	Saad et al. (2018)
Cholesterol oxidase	Fe3O4 nanoparicles	Analysis of total cholesterol in serum	Gao et al. (2018)
Laccase	Chitosan-magnetic nanoparicles	Environmental pollutants bioremediation	Saad et al. (2018)
Keratinase	Fe3O4 nanoparicles	Synthesis of keratin	Gao et al. (2018)
Lipase	Fe3O4 nanoparicles	Hydrolysis of pNPP	Gao et al. (2018)
	Polystyrene nanoparicles	Aminolysis, estrification, trans-estrification
Glucose oxidase	Thiolated gold nanoparicles	Estimation of glucose level	Ren et al. (2006)

Table 9.

Advantages and disadvantages of nanoparticles as immobilization support material

Advantages	Disadvantages
Effective enzyme loading	Large scale application
Mass transfer resistance	Cost of preparation process
High mechanical strength	Difficult in separation (except magnetic)
High surface area
Minimum diffusional problems

Biomedical application of nano-immobilization

These nanomaterials supports are studied as drug delivery systems for a large group of substances, for example, drugs used for diabetes, cancer treatment, bones and tendons regeneration (Fu et al. 2013). As the gradual slow release of the drug is an important key that increase efficiency and reduce the side effects of drugs. In this technique, the nanoparticles are associated with the inner cell membrane by endocytosis, then escape from the endosomes, and degrade in the lysosome; the therapeutic agent diffuses into the cytoplasm and transfers to target organelle as shown in Fig. 11 (Faraji and Wipf 2009).

Fig. 11.

Steps detailing the cytosolic delivery of therapeutic agents via nanoparticles carriers

Conclusions

Enzyme immobilization is a technology widely used in various fields and industries such as bioremediation, environmental monitoring, biotransformation, food industry, textile industry, detergent industry, pharmaceutical industry, diagnostics etc. This technology has economic and technical advantages. Vast numbers of enzymes have been used in the immobilized form in various processes. Immobilization of enzymes can lower the cost and also provide operational stability to enzymes. Recently, there are many methods of immobilization that are used. But unfortunately, there is no universal material or method that can be used as support material; i.e., the carrier, and also immobilization method might differ from enzyme to enzyme and also according to application. Future investigations should endeavor at adopting the development of enzyme analgesia and provide new prospects for the pharmaceutical and industrial sector.

Compliance with ethical standards

Conflict of interest

There is no conflict of interest.