, Volume 20, Issue 5–6, pp 801–821 | Cite as

Enzyme immobilization by adsorption: a review

  • Teofil Jesionowski
  • Jakub Zdarta
  • Barbara Krajewska
Open Access


Endowed with unparalleled high catalytic activity and selectivity, enzymes offer enormous potential as catalysts in practical applications. These applications, however, are seriously hampered by enzymes’ low thermal and chemical stabilities. One way to improve these stabilities is the enzyme immobilization. Among various tested methods of this process that make use of different enzyme-carrier interactions, immobilization by adsorption on solid carriers has appeared most common. According to these findings, in this review we present a comparative analysis of the literature reports on the recent trends in the immobilization of the enzymes by adsorption. This thorough study was prepared in order to provide a deeper understanding of the process. Both carriers, carrier modifiers and procedures developed for effective adsorption of the enzymes are discussed. The review may thus be helpful in choosing the right adsorption scheme for a given enzyme to achieve the improvement of its stability and activity for a specific application.


Enzymes immobilization Adsorption Carriers Surface modifying agents Applications 

1 General overview

Low thermal stability, narrow pH range, effective activity in water environment and the loss of catalytic activity after one cycle have been the greatest obstacles in the use of the enzymes in the multiple practical processes (Liese and Hilterhaus 2013; Gray et al. 2013; DiCosimo et al. 2013). However, the enormous catalytic potential offered by the enzymes for innumerable transformations, has stimulated intense studies aimed at the improvement of their properties (Mateo et al. 2007; Brady and Jordon 2009; Fernandez-Lafuente 2009; Garcia-Galan et al. 2011; Cowan and Fernandez-Lafuente 2011; Rodrigues et al. 2013). Among several methods of this improvement that have been proposed, an immobilization of the enzymes is apparently most widely applied (Zhao 2010; Rodrigues et al. 2011; Hanefeld et al. 2013). The term first appeared in the literature at the beginning of twentieth century and referred to the enzymes bound directly to the carriers. At present this term has been extended to include both direct immobilizations on the carriers and the immobilizations supported with the intermediate agents (Cao et al. 2003; Hanefeld et al. 2009).

An immobilization of the enzymes on the solid carriers can be achieved using a broad variety of chemical and physical methods (Cao 2005; Sheldon 2007; Sheldon and van Pelt 2013). Among many methods proposed for the protein immobilization, the most important and useful is the immobilization by adsorption. Adsorption makes use of the physical interactions generated between the carrier and enzyme that include van der Waals forces, ionic interactions and hydrogen bonding. The binding are rather weak and, what is important, typically are does not change the native structure of the enzyme. This prevents the active sites of the enzyme from disturbing and allows the enzyme to retain its activity (Hernandez and Fernandez-Lafuente 2011; Hwang and Gu 2013). Notably, any carrier can be applied for enzyme adsorption, but not every enzyme can be immobilized on all carriers. The reason is that for the successful adsorption of the enzyme to occur, some certain conditions must be met, among which an enzyme-carrier affinity is most important. This is assured by the presence of the specific active groups on the carrier, which enable the generation of the enzyme–carrier interactions. However, if absent, the interactions can be tuned by applying intermediate agents (carrier modifiers) (Fig. 1).
Fig. 1

Enzyme immobilization by adsorption

A wide range of available compounds can be successfully used as the enzyme carriers. The criteria of the choice suitable for a given enzyme and its application include: the cost, availability, stability (or reactivity if necessary) in specific conditions, and the type of reactor. The physicochemical parameters of the carrier that should be taken into an account are: the surface area, particle size, pore structure and type of functional groups present on the surface. A general classification of typical carriers used for the enzyme adsorption is presented in Fig. 2.
Fig. 2

Carriers used for enzyme immobilization by adsorption

In general, the carriers used for the enzyme immobilization by adsorption can be divided into both organic and inorganic origin. The most common inorganic carriers are silicas, titania and hydroxyapatite. The organic carriers by contrast, include compounds of natural origin, such as chitin, chitosan, cellulose and alginate, also the synthetic compounds, mainly polymers. The advantage of these matrices is that they can be readily chemically modified to match conditions for a given enzyme and its application.

Another important advantage of the enzyme immobilization is that the immobilized enzymes may show properties that can be exploited in the reactions performed in non-aqueous environments. Typically, the native enzymes are catalytically active in the aqueous media and they lose the activity in organic solvents. However, when immobilized, the enzymes may have their catalytic properties altered in a manner permitting them to preserve their activities in conditions other than aqueous. This is important for two reasons. One is that such enzymes can be used for the transformations of hydrophobic substrates that can only be performed in organic solvents (Carrea and Riva 2000; Klibanov 2001; Iyer and Ananthanarayan 2008). The other one is that the immobilized enzymes may exhibit catalytic properties in organic solvents different from those in aqueous environments. This can be exploited in guiding the reactions toward the desired products. Excellent examples of such properties are lipases and esterases; in aqueous environments those enzymes catalyze the hydrolysis of esters to alcohols, while in organic solvents they catalyze transesterifications of the same substrates (Klibanov 2001). Additionally, along with the use of the specific organic solvents, the following properties can be achieved: chemo-, regio- and enantioselectivity of the enzymes after immobilization may be customized for a specific purpose (Carrea and Riva 2000; Klibanov 2001), the reactions can be reversed, their yield may be increased, and also the homogeneous product may be obtained rather than a mixture of isomers or enantiomers. Importantly, it should not be overlooked that the water, present in an organic solvent, even in trace amounts, may significantly alter the parameters of the preparation obtained and may even affect the course of the entire process. Interestingly, organic solvents also affect the stabilities of the immobilized enzymes. This is because the enzymes desorb from the carriers to the organic solvents less readily than to the aqueous solutions.

Given the growing importance of immobilized enzymes as well as the complexity of their preparation, this review presents a thorough study on literature dealing with the immobilization of the enzymes by adsorption. Carriers utilized for the immobilization with and without the intermediate agents are reviewed, methods of adsorption on different types of the carriers are compared, and examples of the immobilized enzymes employed as catalysts in practical applications both in aqueous and non-aqueous (organic solvents, surfactants, ionic liquids) media are discussed. The data taken from the literature are presented as a summary in Table 1 and discussed individually in the subsequent sections: Carriers in Sect. 2, Surface modifiers in Sect. 3, and Properties of immobilized enzymes in Sect. 4.
Table 1

Immobilization of enzymes by adsorption



Carrier modifier

Research techniques

Examined properties and applications


Alanine racemase from Geobacillus stearothermophilus

Folded-sheet mesoporous silica

Pore structure characterization

Catalytic properties; chemical, thermal and operational stability. Activity assay based on racemization of l-alanine to d-alanine

Nara et al. (2010)

α-Amylase from Bacillus subtilis

Mesoporous silica SBA-15

XRD, SEM, HR-TEM, pore structure characterization

Optimization of immobilization conditions: effects of pore size, pH and time of immobilization

Ajitha et al. (2010)

α-Amylase from Bacillus species

Mesoporous silica thin film

TEM, FE-SEM, XRS, XRD, EEP, spectrophotometric measurements

Activity and stability versus pH and temperature. Activity assay based on hydrolysis of starch

Bellino et al. (2010)

Amylase from Aspergillus carbonarius

Silica gel


Bradford method

Optimization of immobilization conditions: glutaraldehyde concentration, pH and temperature. Thermal and chemical stability

Nwagu et al. (2011)

Carbonic anhydrase from bovine

Mesoporous silica SBA-15


XRD, FE-SEM, FTIR, 29Si CP MAS NMR, Bradford method, pore structure

Activity; thermal, chemical and storage stability, reuse. Application in hydration and sequestration of CO2

Vinoba et al. (2012)

Carbonyl reductase from Georichum candidum

Silica gel


Comparison of immobilization methods. Stability. Adsorption efficiency. Activity assay based on conversion of 1-acetonaphthone to (S)(–)-1-(1′-naphthyl) ethanol

Bhattacharyya et al. (2010)

Carboxymethyl cellulase from Trichoderma reesei

Large pore silica FDU-12

3-Aminopropyltriethoxysilane; 3-Mercaptopropyl-, Phenyl- and Vinyltrimethoxysilanes

XPS, SAXS, TEM, 13C CP MAS NMR, zeta potential, pore structure, spectrophotometry

Carrier and modifier characteristics. Activity and stability. Amount of adsorbed enzyme versus modifying agent. Application in bioadsorption, biomolecule separation and in pharmaceutical industry

Hartono et al. (2010)

Cellulase from Trichoderma viride

Silica grafted with polyamidoamine dendrymers


Activity; thermal and storage stability, Optimization of immobilization parameters. Application in hydrolysis of carboxymethylcellulose

Wang et al. (2013)

Cellulase from Trichoderma reesei

Mesoporous silica

XRD, SEM, TEM, UV–Vis, 29Si CP MAS NMR, 13C CP MAS NMR, pore structure characterization

Activity and stability. Application in hydrolysis of cellulose to glucose in water. Elaboration of universal immobilization method

Chang et al. (2011)

Chloroperoxidase from Caldariomyces fumago

Mesoporous silica SBA-15

FTIR, XRD, SEM, TEM, fluorescent spectroscopy, pore structure

Activity assay based on oxidation of 4,6-dimethyl dibenzthiophene. Carrier characteristics. Kinetic parameters; catalytic activity, storage and thermal stability

Montiel et al. (2007)

Chlorophyllase from Phaedactylum tricornutum

Silica gel, cellulose


HPLC, spectrophotometric measurements

Kinetic parameters; thermal stability, reuse. Influence of organic solvent and inhibitory agents. Activity assay based on reaction of chlorophyll

Karboune et al. (2005)


Aptamer-silica beads


HPLC, spectrophotometric measurements

Applications in digestion of proteins. Catalytic activity and product stability

Xiao et al. (2012)

Feruloyl esterase (used as Depol 740L)

Mesoporous silica SBA-15

HPLC, Bradford method

Activity assay based on transestrification of methyl hydroxycinnamate with butanol to butyl hydroxycinnamate. Thermal and chemical stability

Thorn et al. (2011)

Glucose oxidase from Aspergillus niger

Rod-like and vesicle-like mesoporous silica


HR-TEM, FTIR, FE-SEM, ampero- and voltometric measurements, pore structure

Applications in electrodes as sensors of glucose detection. Catalytic properties and stability versus various immobilization methods

Zhou et al. (2011)

Glucose oxidase from Aspergillus niger


Second generation dendronized polymer, avidin–biotin system

spectrophotometric measurements

Application in reaction of β-d-glucose to glucono-δ-lactone and H2O2

Fornera and Bauer (2012)

Peroxidase from horseradish

Utilization in oxidation of o-phenylenediamine to 2,3-diaminophenazine

Glucose oxidase from Aspergillus niger

Silica gel 100

Acrylonitrile copolymers

-vinyl pyridine

Lowry method, spectrophoto-metric measurements

Activity assay based on amount of H2O2 formed in hydrolysis of β-d-glucose. Elaboration of the effective immobilization method. Immobilization efficiency and catalytic activity versus pH, temperature and storage time

Godjevargova et al. (2006)

-vinyl imidazole

-N,N-dimethyl aminoethyl methacrylate

Laccase from Aspergillus



1H NMR, 13C NMR, spectrophotometric measurements

Effect of the ionic liquid. Optimum pH and enzyme concentration. Kinetic parameters

Tavares et al. (2013)

Laccase from Trametes versicolor

Mesoporous silica SBA-15

XRD, TG/DSC, pore structure characterization

Activity assay based on oxidation of phenol and 4-aminoantipyrine. Use in biodegradation of naphthalene. Adsorption efficiency. Activity versus catalytic cycles

Bautista et al. (2010)

Lipase from Candida rugosa

Vesicular silica

FTIR, FE-SEM, TEM, pore structure, Bradford method

Improvement of thermal and chemical stability. Catalytic activity, thermal and chemical stability

Wu et al. (2012)

Lipase from Candida rugosa

Mesoporous silica MSU-H


FTIR, XRD, TG/DTA, pore structure, Bradford method

Application in estrification of linoleic acid with ethanol in organic solvent. Catalytic activity versus different immobilization methods and reuse

Yu et al. (2013)

Lipase from Candida rugosa

Silanized silica

n-Octyltriethoxysilane, 3-Mercaptopropyl-triethoxysilane

Catalytic activity. Activity assay based on hydrolysis of p-nitrophenyl palmitate to p-nitrophenol. Application in estrification of phytosterols from oleic or linoleic acid

Zheng et al. (2012)

Lipase from: Candida rugosa and antarctica, Thermomyces lanuginosus and Mucor javanicus

Silica sol–gel

Multi-walled carbon naotubes

Catalytic activity and amount of enzyme adsorbed versus surface modifier. Activity assay based on hydrolysis of p-nitrophenyl butyrate in DMF. Application in estrification and hydrolysis in organic solvents

Lee et al. (2010)

Lipase from Mucor miehei and Rhizopus oryzae

Mesoporous silica

SEM, TEM, UV–Vis, SAXS, pore structure

Activity assay based on hydrolysis of acetate 4-nitrophenolate to 4-nitrophenol. Effect of pH and type of enzyme on amount of enzyme adsorbed

Gustafsson et al. (2012)

Lipase from porcine pancreas

Mesoporous silica SBA-15

Ionic liquid

SAXRD, FTIR, SEM, TG, 13C CP MAS NMR, pore structure characterization

Activity assay based on hydrolysis of triacetin. Elaboration of fast, universal and effective immobilization. Activity and stability versus temperature, storage time, pH and reuse

Yang et al. (2013)

Lipase from Pseudomonas fluorescens

Mesoporous silica SBA-15

XRD, TEM, HPLC, pore structure, Bradford method

Utilization in biodiesel production. Carrier characteristics. Catalytic activity decrease after reuse

Salis et al. (2010)

Lipase B from Candida antarctica

Fumed silica


Activity assay based on separation of (R,S)-1-phenyl-ethanol and vinyl acetate to R-1-phenylethyl acetate and (S)-1-phenylethanol. Activity and stability vs solvent, water content, temperature and immobilization time. Utilization in enantioselective reactions in hexane

Kramer et al. (2010)

P450 BM-3 monooxygenase from heme domain

Mesoporous silica SBA-15 and MCM-41

XRD, pore structure characterization, spectrophotometric measurements

Carrier characterization. Optimization of immobilization. Activity and adsorption versus carrier. Activity assay based on reaction of p-nitrophenoxy-dodecanoic acid to p-nitrophenolate and on conversion of n-octane

Weber et al. (2010)

Peroxidase from horseradish

Mesoporous silica composite with polypirol

SEM, XRD, TG, pore structure, spectrophotometric measurements

Elaboration of universal enzyme immobilization method. Catalytic activity veruss storage time

Kwon et al. (2012)

Superoxide dismutase from bovine erythrocytes

Mesoporous silica nanoparticles


FTIR, XRD, TG, EA, TEM, pore structure, zeta potential

Immobilization efficiency versus surface modifiers and enzyme concentration. Thermal and chemical stability. Influence of denaturating agents. Industrial uses

Falahti et al. (2012)

Superoxide dismutase from bovine erythrocytes

Mesoporous silica nanoparticles


XRD, FTIR, CD, DSC, TG, pore structure, spectrophotom

Catalytic activity. Elution rate from carrier. Elaboration and optimization of immobilization method

Falahati et al. (2011)

Endo-glucanase, Exo-glucanase, β-glucosidase

Gold nanoparticles; gold-doped magnetic silica nanoparticles



Chemical, thermal and storage stability. Applications in hydrolytic degradation of cellulose

Cho et al. (2012)

Glucose oxidase from Aspergillus niger

Gold nanotubes


DSM, XPS, amperometric measurements

Utilization as an enzymatic biosensor to glucose detection in physiological fluids

Delvaux and Demoustier-Champagne (2003)

Peroxidase from horseradish

Titania sol–gel film

SEM, voltometric measurements

Application in biosensors to H2O2 detection. Influence of pH on catalytic activity. Storage time stability

Yu and Ju (2002)

α-Amylase from Bacillus subtilis


XRD, IR, pore structure characterization

Application and activity assay based on starch hydrolysis. Kinetic parameters. Activity and stability vs buffer, pH, temperature and immobilization time

Reshmi et al. (2007)

Lipase from Candida rugosa

Zirconium dioxide nanoparticles

Eruic acid, Tween 85


Effect of surface modifier, reuse and storage time on catalytic activity and stability. Use in resolution of (R,S)-ibuprofen and (R,S)-1-phenylethanol

Chen et al. (2008)


Layered γ-zirconium phosphate


Optimization of pH and temperature conditions. Chemical and thermal stability. Activity assay based on hydrolysis of N-benzoyl-p-nitroanilide

Geng et al. (2003)

α-Amylase from Bacillus subtilis


XRD, IR, pore structure, spectrophotometric measurements

Activity and stability versus pH and buffer. Kinetic parameters. Application in starch hydrolysis to low molecular weight compounds

Reshmi et al. (2006)

Carbonic anhydrase

Mesoporous aluminosilicates

XRD, FTIR, GC, SEM, TEM, TCD, pore structure characterization

Kinetic parameters; Activity and stability versus pH and temperature. Optimization of immobilization. Activity assay based on reaction of p-nitrophenyl acetate. Application in carbonation process in production of CaCO3 from CO2

Wanjari et al. (2012)

Dextransucrase from Leuconostoc mesenteroides


Spectrophotometric measurements

Activity assay based on hydrolysis of sucrose. Amount of adsorbed enzyme on various carriers. Effects of pH, temperature, inhibitors. Activity and kinetic parameters versus storage time and reuse

Gupta and Prabhu (1995)

Calcium alginate

Alumina gel

Calcium phosphate gel

Endodextranase from Chaetomium erraticum


Bradford method

Kinetic parameters, adsorption efficiency. Optimization of pH of immobilization process. Application in synthesis of isomaltose using dextransucrase

Erhardt and Jordening (2007)


Streamline DEAE


Fructosyl transferase from Steptococus mutans


Influence of carrier structure on adsorption efficiency. Activity assay based on sucrose conversion to fructanes insoluble in ethanol

Bronshyteyn and Steinberg (2002)

Lyase hydroperoxide from Amaranthus tricolor

Ceramic hydroxyapatite

Spectrophotometric measurements

Activity versus temperature and pH. Kinetic parameters; thermal and chemical stability. Use in food industry

Liu et al. (2013)

Urease from Canavalia ensiformis


Kinetic parameters; thermal, chemical and storage stability

Marzadori et al. (1998)

α-Amylase, Urease

Halloysite nanotubes

TEM, XRD, FTIR, pore structure characterization

Activity assay based on starch hydrolysis. Thermal and storage stability, reuse and catalytic activity

Zhai et al. (2010)

β-Galactosidase from Aspergillus oryzae

Cordierite, Acicular mullite

3-Aminopropyltriethoxysilane; Glutaraldehyde

SEM, FTIR, TG/DTA, spectrophotometric measurements

Activity assay based on hydrolysis of o-nitrophenyl-β-galactopyranoside. Optimization of immobilization. Catalytic activity, reuse and adsorption efficiency

de Lathouder et al. (2008)

Lipase from Candida rugosa



Bradford method, pore structure characterization

Carrier adsorption capacity, immobilization efficiency. Activity vs reuse and temperature. Utilization in esterification of fatty acids and sugars (lactose esters)

Zaidan et al. (2012)

Glucose oxidase

Pt nanoparticles/graphene sheets/chitosan film

TEM, amperometric and voltometric measurements

Application in electrode as a sensor for detection of low levels of glucose

Wu et al. (2009)

Hexokinase from bakers yeast



DLS, TEM, zeta potential, pore structure, spectrophotom

Activity assay based on reduction of NADP+ to NADPH. Carrier characteristic. Activity on storage

Castro et al. (2007)

Laccase from Tramates versicolor

Chitosan membrane with epichlorohydrin

Itaconic acid, itaconic acid and Cu(II)


Effects of pH and temperature on catalytic efficiency. Use in bioremediation of hazardous materials

Bayramoglu et al. (2012)

Lipase from Candida rugosa

Chitosan beads

TLC, Bradford metod

Activity assay based on transestrification of cooking oil. Application in transestrification

Nasratun et al. (2010)

Cells from Erwinia sp.D12

Calcium alginate; gelatin transglutaminase

spectrophotometric measurements

Application in sucrose conversion to isomaltulose–sucrose replacement in food industry

Kawaguti et al. (2011)

β-Galactosidase from Kluyveromyces lactis

Cellulose acetate membranes

Oxygen plasma

HPLC, Bradford metod

Kinetic parameters. Optimization of temperature and pH. Activity and stability on reuse. Activity assay based on conversion of lactose to galactooligosaccharides. Application in food industry

Gulec (2013)

Laccase from Cerrena unicolor, Tyrosinase

Cellulose acetate disc membranes, poly(amide) disc membranes

Plasma polymerization, glutaraldehyde


Activity assay based on oxidation of 2,2′-anizo-bis-(3-ethylbenzothiazoline-6-sulfonate) and L-3-(3,4-dihydroxyphenyl)alanine. Kinetic parameters; catalytic activity, thermal, chemical stability and immobilization efficiency

Labus et al. (2012)

Allyl alcohol


Acrylic acid

Lipase from Candida rugosa

Ultrathin film of cellulose acetate and propionate, and of acetate butyrate

AFM, contact angle, spectrophotometric measurements

Activity assay based on hydrolysis of p-nitrophenyldodecanoate. Catalytic properties. Influence of reuse on stability and catalytic activity

Kosaka et al. (2007)

Amyloglucosidase from Rhizopus


Spectrophotometric measurements

Optimization of immobilization. Carrier characteristics. Activity. Storage, thermal and chemical stability, reuse. Activity assay based on starch hydrolysis. Utilized for starch hydrolysis

Ashly and Mohanan (2010)

Carbonic anhydrase from bovine

Poly(acrylic acid-co-acrylamide)/ hydrotalcite nanocomposite hydrogels

Cryo-SEM, FTIR, TEM, fluorescence microscopy

Removal of CO2 from gases. Effect of water content on carrier structure. Catalytic activity. Activity versus temperature and organic solvent. Amount of enzyme adsorbed versus unmodified and modified carrier

Zhang et al. (2009)

Laccase from Trametes versicolor

Cationic resin Amberlite IR-120H


SEM, spectrophotometric measurements

Kinetic parameters; catalytic activity, thermal, chemical and storage stability. Optimization of immobilization method. Activity assay based on oxidation of ABTS

Spinelli et al. (2012)

Lacitase ultra

Macroporous resin


Catalytic activity. Optimization of immobilization conditions. Stabilities and reuse. Application in production of diacylglycerols by glycerolysis of soybean oil

Liu et al. (2012a, b)

Commercial lipase

Celite 545



Effect of metal ions and enzyme concentration on catalytic properties. Storage stability. Activity assay based on estrification of ferulic acid with ethanol to ethyl ferulate in DMSO

Kumar and Kanwar (2011)

Lipase B from Candida antarctica

Ion exchange resin Lewatit

HPLC–MS, SEM, pore structure characterization

Effect of the matrix on immobilization. Catalytic activity. Utilization in vitamin E (tocopherol) transestrification with vinyl acetate to 2-methyl-2-butyl

Torres et al. (2008)

Polymer (Purasorb)

Polypropylene (Accurel EP100)

Lipase from Candida rugosa

Poly(N-methylol acrylamide)


Bradford method

Optimization of immobilization. Effect of temperature, pH, storage and reuse on activity and stability. Activity assay based on butyl butyrate synthesis (in organic solvents) or hydrolysis of olive oil (in aqueous solvent)

Santos et al. (2007)

Lipase from Penicillium camembertii (Lipase G)

MANAE-agarose, Epoxy-SiO2-PVA


Immobilization efficiency, catalytic activity, thermal and chemical stability. Optimization of immobilization conditions

Mendes et al. (2012)

Lipase from P.antarctica, T. lanuginosus 1, T. lanuginosus 2, P. fluorescens, and G. thermocatenulatus

Small/large polyhydroxybutyrate beads

GC, Bradford method

Effects from carriers and lipases on catalytic activity. Activity assay based on hydrolysis of olive oil, estrification of butyric acid with butanol and transestrification of babassu oil. Application in biodiesel production

Mendes et al. (2011)

Lipase from Pseudomonas cepacia

Polyacrylonitrile fibers

Bradford method

Utilization in biodiesel production. Stability in reactor. Influence of amount of adsorbed enzyme, temperature, immobilization time and water content on catalytic activity

Sakai et al. (2010)

Lipase from porcine pancreas

Cross-linked polivinyl alcohol


Influence of water content, substrate concentration and temperature on activity. Storage stability. Activity assay based on hydrolysis of tributyrin to fatty acids

Ozturk and Kilinc (2010)

Lipase „powder” 20 AK from Pseudomonas fluorescens


Spectrophotometric measurements

Catalytic activity and storage stability. Utilization in enantioselective transestrification acylation of β-hydroxy esters with various aryl groups and enantioselective transestrification of 1-phenyl methane

Brem et al. (2011)

Lipase (Pf2001) from Pyrococcus furiosus

Hydrophobicity carriers

Spectrophotometric measurements

Thermal, chemical and storage stability on various carriers. Optimization of enzyme immobilization process. Activity assay based on gum arabic reaction

Branco et al. (2010)

Lipase from Rhizopus delemar, Patalase 20000L from Mucor miehei

Accurel MP1000


Catalytic activity, storage stability. Utilization in acidolysis of tuna oil and caprylic acid to triacyloglycerols

Hita et al. (2009)

Nattokinase from Bacillus subtilis

Polyhydroxybutyrate nanoparticles


Catalytic activity, thermal and chemical stability. Optimization of immobilization and elaboration of universal immobilization method

Deepak et al. (2009)

β-Xylosidase from Aspergillus niger USP-67


MS, Bradford method

Influence of glucose and xylose on catalytic activity. Amount of enzyme adsorbed vs carrier. Thermal and chemical stability. Utilization in hydrolysis of short xylooligomers. Activity assay based on hydrolysis of p-nitrophenyl-β-d-xylopyranoside

Benassi et al. (2013)






Cells from Pleurotus ostreatus

Pumice particles


Amount of enzyme adsorbed versus used carrier. Utilization in the biodegradation of fluorene

Akdogan and Pazarlioglu (2011)

Amberlite XAD-2000

Polystyrene foam


Amberlite XAD-7

Cells from Rhodococcus equi A4



Application in biotransformation of nitrile derivatives

Kubac et al. (2006)

Cycloisomalto-oligosaccharide glucanotransferase

Porous hollow-fiber membranes

Glicid methacrylate, diethyloamine

Amount of enzyme adsorbed versus catalytic activity. Application in dextran production from cycloisomaltooligosaccharides

Kawakita et al. (2002)

Lipase from Thermomyces lanuginosus

Cotton flannel cloth


Influence of numbers of adsorbed enzyme layers on catalytic activity. Activity assay based on estrification of olive oil with poly(vinyl) alcohol

Karimpil et al. (2012)


Nylon membranes

Poly(styrene sulfonate)


Application in protein digestion

Xu et al. (2010)


Magnetic poly(acrylamide-allylglycydyl ether) cryogels


Influence of pH, temperature and ionic strength on activity. Kinetic parameters and storage stability. Optimization of immobilization process

Tuzmen et al. (2012)

Glycolate oxidase from Medicago falcata

Magnetic nanoparticles


Kinetic parameters; catalytic activity, storage, thermal and chemical stability reuse. Utilization in catalytic oxidization of glycolic acid to glyoxylic acid

Zhu et al. (2009)

Laccase from Trametes versicolor

Magnetic mesoporous silica spheres

SAXS, VSM WAXS, SEM, TEM, UV–Vis, pore structure, zeta potential, spectrophoto-metric measurements,

Catalytic activity, amount of enzyme adsorbed. Activity assay based on hydrolysis of 2,2′-azinobis(3-ethylbenzthiazolin-6-sulfonate). Activity versus temperature, pH, reuse

Zhu et al. (2007)

Lipase from Burkholderia

Magnetic nanoparticles Fe3O4–SiO2

[3-(Trimethoxysilyl) propyl] octadecyl dimethyl ammonium chloride

FTIR, SEM, XRD, pore characterization, Bradford method

Carrier characteristics. Kinetic parameters. Application in transesterification of olive oil with methanol in biodiesel production

Tran et al. (2012)

Lipase from Brukholderia

Hydrophobic magnetic particles

Pore structure characterization

Activity and stability. Influence of water and methanol content on transestrification. Biodiesel production

Liu et al. (2012a, b)

Lipase from Candida rugosa

Magnetic chitosan microspheres



Carrier characteristics. Activity. Optimization. Activity assay based on transestrification of soya oil with methanol. Application in biodiesel production

Xie and Wang (2012)

Chloroperoxidase from Caldariomyces fumago

Agarose gel


spectrophotometric measurements

Catalytic activity. Chemical and storage stability. Activity assay based on reaction of benzyl-N-(2-hydroxyethyl)-carbamate ethnolamine to benzyl-N-(2-hydroxyethyl)-carbamate glycine

Pesic et al. (2012)

α-Chymotrypsin from bovine pancreas

Reverse micellar from different substrates


Spectrophotometric measurements, Raman spectroscopy

Activity assay based on reaction of N-glutaryl-l-phenylalanine-p-nitroanilide.

Thudi et al. (2012)

Yeast alcohol dehydrogenase from Saccharomyces cerevisiae

Activity assay based on reaction of but-2-one with NADH as cofactor

Glucose dehydrogenase from Gluconobacter cerinus cerevisiae

Activity assay based on glucose and NADP concentration

Lipase from Thermomyces lanuginosus

Olive pomace powder

SEM, spectrophotometric measurements, Bradford method

Optimization of immobilization. Catalytic activity, thermal and chemical stability. Activity in reuse. Activity assay based on hydrolysis of p-nitrophenyl palmitate.

Yucel (2012)

Polyphenol oxidase from Solanum tuberosum

Mesoporous activated carbon matrices MAC 200 and MAC 400

FTIR, SEM, spectrophoto-metric measurements

Kinetic parameters; catalytic activity, pH, temperature and enzyme concentration. Activity assay based on dopachrome formation from l-DOPA

Kennedy et al. (2007)

Xylanase from Neocallimastix patriciarium

Artificial oil bodies

Activity assay based on hydrolysis of oat spelt xylan. Catalytic activity, thermal and chemical stability. Activity in reuse

Hung et al. (2008)

2 Carriers used for immobilization of enzymes by adsorption

2.1 Inorganic carriers

Among many inorganic carriers used for immobilization of enzymes by adsorption, silicas are apparently those carriers, which have drawn most attention (Erhardt and Jordening 2007; Magner 2013; Hartmann and Kostrov 2013). Silicas of different dispersive-morphological parameters and porous structures have been proposed. A representative silica used for the enzyme immobilization on a large scale is mesoporous silica SBA-15 (Santa Barbara Amorphous) with hexagonal array of pores (Salis et al. 2010; Thorn et al. 2011). It is characterized by small pores, from 5 to about 30 nm in diameter and a hexagonal array of pores (Grudzień et al., 2006; 2007; Hartmann and Kostrov 2013). Large volume of mesopores, close to 1.0 cm3/g and micropores of about 0.8 cm3/g, and also a very well developed surface area from 500 to 1400 m2/g [Hartmann and Kostrov 2013] make this silica an excellent support for the enzyme immobilization. Another mesoporous silica MSU-H (Yu and Fang 2013) has the specific surface area reaching 750 m2/g, pore radius from 7 to 10 nm and pore volume from 0.9 to 1.0 cm3/g. By contrast, in mesoporous silica MCM-41 (Mobil Composition of Matter), with hexagonally ordered mesopores (Choma et al. 2004; Weber et al. 2010), the pore size is from 2 to 8 nm, which is controlled by adjusting the synthesis conditions and/or by applying surfactants with different chain lengths (pore sizes 2–5 nm) or expanders (pore sizes up to 8 nm) in their preparation. The pore volume in this silica is close to 1.0 cm3/g and its surface area exceeds 1,200 m2/g (Magner 2013), which are features that classify this material as a carrier for the enzyme immobilization. FDU-12 (Fudan University Material) (Hartono et al. 2010) is another mesoporous silica material with face-centered cubic structures of spherical mesopores and surface area of about 700 m2/g, pore size from 10 to 15 nm and pore volume from 0.6 to 0.7 cm3/g. This silica and other silica matrices (Falahti et al. 2012) differing in pore size and structure, are also excellent carriers for enzyme immobilization. Likewise, highly ordered mesoporous silicas with 2D and 3D structures and mesopores from 2 to about 30 nm, obtained by a surfactant and block copolymer templating, can be readily applied. The material of 3D structure is a better adsorbent and permits immobilization of a greater amount of the enzyme. The small particle of mesoporous silica (Chang et al. 2011) was reported to have a surface area of 820 m2/g and a pore diameter varying from 2 to 5 nm, while the diameter of particles was close to 150 nm. This small particle mesoporous silica was compared with the large particle mesoporous silica (Chang et al. 2011), which surface area was near 260 m2/g, pore diameter varied from 20 to 40 nm and particle size reached 600 nm. It was found that the larger surface area and smaller particle diameter favoured an immobilization of a greater amount of the enzymes. Cubic Ia3d mesoporous silica nanoparticles (Falahati et al. 2011) have the surface area of over 820 m2/g, pore diameters of about 7 nm and pore volume greater than 1.5 cm3/g. To increase the surface area of the carrier available to the enzyme, the folded sheet mesoporous silica was proposed (Nara et al. 2010). Such a configuration permits an immobilization of the greater amounts of the enzymes at only insubstantial growth of cost of the carrier production. Silicas of smaller surface areas obtained mainly in the processes of hydrolysis and condensation of tetraalkoxysilanes (Grabicka and Jaroniec 2010; Fornera and Bauer 2012; Zheng et al. 2012) were also used for the enzyme immobilization.

Other silica carriers widely used for the enzyme adsorption are silica gels (Bhattacharyya et al. 2010; Lee et al. 2010). They have very well developed porous structures and surface areas, as well as high mechanical strength and thermal stability. The size of silica gel particles varies from 70 to 150 μm depending on the type and the pore size reaches 250 nm.

Other types of silicas used for the enzyme immobilization are vesicular silica and fumed silica. Vesicular silica (Zhou et al. 2011; Wu et al. 2012) has pores of a diameter ranging from 15 to 20 nm, a pore volume from 0.6 to 1.4 cm3/g and surface area reaching 360 m2/g, while fumed silica (Kramer et al. 2010) has particles of a diameter from 7 to 50 nm and a surface area of 255 m2/g. These silicas have well-developed surface areas, small particles and high mechanical strength, which make them attractive alternatives to the other silicas described.

In order to enhance the affinity of the enzymes to silicas, the modifications of the matrices with polymers were proposed. The modifications consist in mixing silicas with polymers or coating them with polymers (Kwon et al. 2012). One group of polymers are polyamidoamine dendrimers (Wang et al. 2013). These are highly branched complex compounds, which due to the presence of amino groups in their structure, facilitate the development of the enzyme-carrier bonds, thereby giving rise to a more effective immobilization. For instance, it was observed that with an increasing content of the dendrimer on the silica surface, the amount of immobilized enzyme increased from 32 mg/g to almost 87 mg/g after a full modification of the silica. Polyamidoamine dendrimers were reported to enhance the affinity to cellulases, a group of enzymes catalysing the decomposition of cellulose by cleaving β-1,4-glycoside bonds. Importantly, the enzymes immobilized on polyamidoamine dendrimers modified-silicas retained over 80 % of their activity after three full catalytic cycles. A similar effect was reported for a mesoporous silica-polypyrrole composite. The effect was ascribed to the presence of hydroxyl groups in polypyrrole. Another group of polymers applied for silica modifications are aptamers (Xiao et al. 2012). These are short oligonucleotide chains (DNA or RNA fragments) able to form specific bonds with the carrier and the biocatalyst.

The noble metal applied as a carrier for the enzyme immobilization is gold. The preparations made on the basis of gold are used mainly in the electrodes mounted in biosensors (Delvaux and Demoustier-Champagne 2003), but they can also be employed in biodegradation of cellulose (Cho et al. 2012). Gold is hardly soluble but easily malleable so its form can be well managed. For the enzyme adsorption, it is used in the form of nanoparticles, gold-doped magnetic silica nanoparticles (Cho et al. 2012), and nanotubes (Delvaux and Demoustier-Champagne 2003).

Another inorganic carrier employed for the enzyme immobilization is a titania sol–gel film (Yu and Ju 2002). Titanium dioxide is a white solid of high melting point and good adsorption parameters.

Zirconia, a white crystalline solid with a high melting point and high chemical resistance, is also an attractive for the enzyme immobilization, where it is used in the form of nanoparticles (Chen et al. 2008), layered γ-zirconium phosphate (Geng et al. 2003) and as pure zirconium (Reshmi et al. 2007).

In addition to the above-mentioned materials, also alumina gel (Gupta and Prabhu 1995) and aluminium (Reshmi et al. 2006) were tested as the enzyme carriers. Aluminium is a common, malleable and plastic metal. Its derivatives are mesoporous aluminosilicates (Wanjari et al. 2012), well characterized (Jaroniec and Fulvio 2013), which are a class of compounds made of aluminium, silicon and oxygen. They can be of either natural (zeolites) or synthetic origin.

Some enzymes were also reported to be immobilized on cordierite and mullite (de Lathouder et al. 2008). The former is a rare mineral belonging to the group of silicates, whereas the latter, a mineral related to aluminosilicates in structure and composition and it was used in the immobilization in the form of acicular mullite.

Other minerals reported as the enzyme carriers are halloysite (Zhai et al. 2010) and mica (Zaidan et al. 2012). Mica is a multi-element mineral of a complex chemical composition that includes mainly aluminium, silicon, calcium, sodium and potassium, and in smaller amounts, lithium, magnesium, iron and manganese. The benefits offered by mica are its high thermal and chemical resistance.

Hydroxyapatite is another mineral used as a carrier for the immobilization of enzymes by adsorption (Fargues et al. 1998; Marzadori et al. 1998; Bronshyteyn and Steinberg 2002). Built of calcium, phosphorus, oxygen and hydrogen, hydroxyapatite is easily available; it occurs in nature and can also be chemically synthesized. Being a component of bones, hydroxyapatite shows high biocompatibility. It also displays high resistance to a wide range of reaction conditions. Its important advantage is the ability to bind practically all enzymes, where it is typically used as a powdered solid or as ceramic hydroxyapatite (Liu et al. 2013).

Also bentonite was reported to be an enzyme carrier with high protein adsorption capacity (Erhardt and Jordening 2007). Bentonite does not dissolve in water but readily swells, which is why bentonite-supported enzymes can be used in water environments.

Useful as a carrier in the enzyme immobilization also appeared to be a mesoporous activated carbon (Kennedy et al. 2007) of different pore sizes, such as MAC 200 and MAC 4000.

2.2 Organic carriers

Of particular interest among organic carriers for the enzyme adsorption is chitosan (Krajewska 2004; Nasratun et al. 2010; Bayramoglu et al. 2012). Chitosan is a polyaminosaccharide obtained from chitin by deacetylation. Chitosan is a nontoxic, biocompatible and gel-forming cationic compound that can readily be prepared in different geometrical configurations, such as membranes, beads, nanoparticles, fibers, hollow fibers or sponges (Krajewska 2005). It can also be applied in the microcrystalline form (Castro et al. 2007). The special advantage of chitosan is that when dissolved in acidic solutions, it bears multiple positive charges on –NH3 + groups along its linear chains. This feature allows it to readily develop electrostatic interactions with molecules containing negatively charged groups (Alatorre-Meda et al. 2009; Krajewska et al. 2011, 2013a, b). Another adavantage of chitosan is that it can easily be chemically modified, which is possible due to the presence of modifiable functional groups (–NH2 and –OH) on chitosan chains (Honarkar and Barikani 2009).

A common organic compound used as an enzyme carrier is calcium alginate [Gupta and Prabhu 1995]. Alginate is an anionic polysaccharide that offers attractive gel-forming, concentrating and stabilizing properties. Commercial varieties of alginate are extracted from seaweeds, including the kelp Macrocystis pyrifera, Ascophyllum nodosum, and various types of algae from Phaeophyceae family. In addition to its pure form, it can also be used admixtured, e.g. with gelatin and transglutaminase (Kawaguti et al. 2011). It easily forms spherical particles with a well-developed surface area that endow it with good adsorption properties.

Alternatively used, organic carrier is cellulose. This is a polysaccharide of natural origin, made of glucose molecules. On the industrial scale, cellulose is obtained from wood. It is frequently used in the form of colourless cellulose acetate. It is a thermoplastic but hardly combustible polymer, insoluble in water. Different structures made of cellulose acetate are utilized, e.g. cellulose acetate membranes (Gulec 2013), cellulose acetate disc membranes (Labus et al. 2012) or ultrathin film of cellulose admixtured with acetate propionate and acetate butyrate (Kosaka et al. 2007).

Agarose gel (Pesic et al. 2012), a polysaccharide polymer, typically applied for separation of nucleic acids, is also used for enzyme immobilization, which is due to its morphological structure and beneficial adsorption properties.

In addition to natural polymers, synthetic polymers form a large and varied group of the enzyme carriers (Kumar and Kanwar 2011; Brem et al. 2011). Effectively, any polymerization can be designed to prepare a polymer with the customized properties. These properties can also be adapted by preparing the polymer composites. The synthetic polymers most commonly used as enzyme carriers include: poly(vinyl alcohol) (PVA) (Mendes et al. 2012) (commercial product LentiKats) (Kubac et al. 2006); cross-linked poly(vinyl alcohol) (Ozturk and Kilinc 2010); poly(N-methylolacrylamide) (Santos et al. 2007); polypropylene (commercial products Accurel EP100 (Torres et al. 2008) and Accurel MP1000 (Hita et al. 2009); polystyrene in the form of foam (Akdogan and Pazarlioglu 2011), in which immobilization is facilitated by a large number of pores; and poly(acrylic acid-co-acrylamide)/hydrotalcite nanocomposite hydrogels (Zhang et al. 2009). An interesting enzyme carrier is the biodegradable and thermo-shrinkable hydroxybutyrate. It is used in the form of poly(hydroxybutyrate) nanoparticles (Deepak et al. 2009) and small or large poly(hydroxybutyrate) beads (Mendes et al. 2011). Another carrier, poly(o-toluidine) built of particles of o-toluidine isomer has active –NH2 groups (Ashly and Mohanan 2010). Furthermore, poly(acrylonitrile) (PAN), a polymer widely used in the production of synthetic fibres, as an enzyme carrier is used in the form of electrospun fibres (Sakai et al. 2010). PAN mats are about 25 μm thick and they are made to have a radius of about 400 nm. The material is very simple and its production is inexpensive. The stiff and elastic PAN carriers show high porosity and ability to interact with other materials, including enzymes and can be used in the various types of the reactors.

The carriers for the enzyme adsorption, if prepared in the form of membranes, e.g. as porous hollow fibre membranes (Kawakita et al. 2002), cotton flannel cloth (Karimpil et al. 2012) and nylon membranes (Xu et al. 2010), are special as they serve both as an enzyme support and at the same time as a separation phase, which, for instance, can separate the reagents of different molar masses.

An interesting option seems to be the carriers containing magnetic particles in their structure (Zhang et al. 2008; Zhu et al. 2009; Tran et al. 2012). Such magnetic matrices provide a good control of the process, as upon application of the magnetic field, the immobilized enzyme can be isolated and the catalyzed reaction terminated. Examples of such magnetic carriers include: a mixture of silicas with iron(II) and iron(III) oxides (Zhu et al. 2007); chitosan microspheres (Xie and Wang 2012); magnetic poly(acrylamide-allylglycydyl ether) cryogels (Tuzmen et al. 2012); and hydrophobic magnetic particles (Liu et al. 2012a, b).

Commercially available, ion-exchange resins, such as Lewatit (Wu et al. 2009), Amberlite IR-120H (Spinelli et al. 2012) or Amberlite XAD-2000 and Amberlite XAD-7 (Akdogan and Pazarlioglu 2011), typically used in the form of gels, are characterized with a highly developed porous structures and the presence of multiple active groups. These characteristics allow them to act as good enzyme supports.

Beside the materials described above, there are also substances, which although less frequently utilized in enzyme immobilizations, feature good adsorption properties, examples being artificial oil bodies (Hung et al. 2008) and olive pomace powder (Yucel 2012). An interesting approach to the immobilization of enzymes is also the use of reverse micelles (Thudi et al. 2012), in which the hydrophobic part is directed outside the micelle to allow an enzyme attachment.

2.3 Commercial products

An example of commercially available enzyme carriers is Stremaline DEAE (Erhardt and Jordening 2007), which is a composite made of agarose with a quartz core and diethylaminoethyl ligands on the surface. Another example are the epoxy-activated polymer supports, such as Eupergit (Katchalski-Katzir and Kraemer 2000; Erhardt and Jordening 2007) commercialized by Rhon Haas and Sepabeads commercialized by Resindion (Barbosa et al. 2013). Both are available in the form of macroporous beads. Eupergit supports are copolymers, of which a main component is poly(methacryl amide), while Sepabeads are polystyrenic adsorbents. Commercially available are also Celite (Kumar and Kanwar 2011) and Celite 545 silica carriers (Brem et al. 2011). Their main component is diatomaceous earth, which is the sedimentary rock formed as a result of diatoms exoskeletons deterioration. In the naturally occurring form it is admixtured with crystobalite, quartz and alumina. Its particle size varies from a few micrometers to a millimetre, but in the commercial product it has particles of diameters from 10 to 200 μm. Also worthy of note is Sepharose, a crosslinked, beaded-form of agarose, a polysaccharide polymer material extracted from seaweed. The great advantage of Sepharose is that its surface can be chemically modified in order to better adapt it to the functional groups of the protein (Benassi et al. 2013). Chitosan is another biopolymer manufactured for the enzyme immobilization and marketed under the brand name Chitopearl (Fuji Spinning, Tokyo, Japan) (Krajewska 2004). Different Chitopearl beads are produced and they can differ in the type and length of side ligands to be rightly chosen for a particular immobilization. The commercial products utilized in enzyme adsorption also include a group of polyvinyl supports available under the name Lentikats (Kubac et al. 2006), as well as the polymer matrices Accurel EP100 (Torres et al. 2008) and Accurel MP1000 (Hita et al. 2009).

2.4 Summary of data on enzyme carriers

The foregoing presentation of materials considered and studied for immobilizing enzymes by adsorption, shows that their variety is very rich. It includes organic and inorganic, natural and synthetic materials, that may be configured as beads of different sizes, membranes, fibers, hollow fibers, capsules, sponges in order to best match the conditions of a specific biotransformation in a given bioreactor. Importantly, it also shows that there are no universal carriers for all enzymes and their applications. Effectively, the choice of a specific material is determined by many factors and for each enzyme and each process this should be made individually, as it may happen that a drawback of one material in one process can be its advantageous feature in another one. The following general comment on the enzyme carrier materials can, however, be proposed. Silicas are perhaps the most common enzyme carriers. Their features, advantageous for this application result chiefly from the well-developed surface area, high availability and low cost. High thermal stability and chemical resistance shown by silica materials are also characteristic for minerals, such as mica or hydroxyapatite. The carriers based on metals, such as titanium, aluminium or zirconium, also show high mechanical strength; however, they show higher affinity to some groups of enzymes, which restricts their application. The magnetic organic and inorganic carriers, which use permits a good control of the enzymatic process, have become very popular but their widespread use is limited by their high cost. For the same reason, the use of carriers based on gold is limited. The interest in materials of natural origin, such as chitin, chitosan or cellulose, stems from their high biocompatibility and availability, but their application is limited by their selective affinity to certain enzymes, but foremost by their lower durability in the process conditions as compared to inorganic materials. By contrast, synthetic polymer matrices are widely used for enzyme adsorption, as they can be tailored to suite the specific enzyme and the conditions of a specific process. Moreover, their production is relatively facile and rapid, and what is more, they show high thermal and chemical resistance.

3 Surface modifying agents

The prerequisite for the successful immobilization of an enzyme by adsorption on a solid carrier is the existence of specific functional groups on the surface of both the enzyme and the carrier. These give rise to the interactions sufficiently strong for the enzyme-carrier binding (adsorption) to occur (Kosaka et al. 2007; Gustafsson et al. 2012; Wu et al. 2012). When such groups are absent, the carrier is subjected to a chemical modification (Cho et al. 2012; Mendes et al. 2012; Zaidan et al. 2012).

The modifying agent should have at least two reactive groups in its molecule; one should enable it to chemically anchor on the carrier and the other one, to physically interact with the enzyme. Typical compounds meeting this condition are bifunctional carbonyl compounds, among them glutaraldehyde being apparently most common (see Fig. 4) (Delvaux and Demoustier-Champagne 2003; de Lathouder et al. 2008; Thudi et al. 2012). Glutaraldehyde with the formula CH2(CH2CHO)2 contains two reactive aldehyde groups. It is used as a disinfectant and preservative. Having high affinity to bacteria, fungi and proteins, it is a good enzyme immobilizer. Also, beneficially for the immobilization, its five-atom carbon chain serves as a spacer for enzymes, making their active sites easier accessible for the substrates.

Compounds frequently used as the carrier modifiers for adsorption of enzymes are also silanes, such as 3-aminopropyltrimethoxysilane (Mansur et al. 2001; Zhou et al. 2011) and 3-aminopropyltriethoxysilane (Falahati et al. 2011, 2012; Vinoba et al. 2012), mercaptopropyltrimethoxysilane and mercaptopropyltriethoxysilane (Cho et al. 2012). The latter two compounds interact stronger with the carrier surface, which is due to the presence of three methoxy or ethoxy groups in their molecules. In the process of surface functionalization, the groups undergo hydrolysis to hydroxyl groups allowing the formation of hydrogen and covalent bonds with the carrier. On the contrary, the presence of –SH or –NH2 groups compatible with the enzyme functional groups, facilitates generation of carrier-modifier-enzyme interactions. Other trialkylsilanes used as carrier modifiers include n-octyltriethoxysilane (Zheng et al. 2012), phenyltrimethoxysilane, vinyltrimethoxysilane (Hartono et al. 2010) and [3-(trimethoxysilyl)propyl] octadecyl dimethyl ammonium chloride (Tran et al. 2012). The attachment of the most common surface modifying agents to silica particles is shown in Fig. 3.
Fig. 3

Representative silica surface modifying agents used in enzyme immobilization, a silica particle grafted with glutaraldehyde, b silica particle modified with 3-aminopropyltriethoxysilane, c silica particle functionalized with mercaptopropyltriethoxysilane, d silica particle grafted with vinyltrimethoxysilane

Polymers constitute another group of useful compounds for a carrier modification. Their usefulness originates from the fact that they can be chemically prepared of monomers desired for a given process and their chain lengths can be controlled. Polyethyleneimine (Karimpil et al. 2012), polystyrene (Castro et al. 2007) and poly(styrene sulfonate) (Xu et al. 2010) are widely applied. In addition to the branched second generation dendronized polymers (Fornera and Bauer 2012), the use of acrylonitrile copolymers was also reported (Godjevargova et al. 2006). This compound generates interactions with vinyl pyridine, vinyl imidazole and N,N-dimethyl-aminoethyl-methacrylate.

Owing to both acid–base properties and the ability to form hydrogen bonds, amines are also considered as carrier modifiers, the most common among them being diethylamine (Kawakita et al. 2002), diethylaminoethyl (DEAE) (Karboune et al. 2005) and monoaminomethyl-N-aminoethyl as an agarose gel modifier (Pesic et al. 2012)

Also, carboxylic acids have properties classifying them for the use as modifiers. An example of a long-chain carboxylic acid is erucic acid (Chen et al. 2008), while a short-chain carboxylic acid containing two carboxyl groups and an additional reactive carbonyl group is itaconic acid (Bayramoglu et al. 2012), both shown in Fig. 4.
Fig. 4

Representative surface modifying agents used in enzyme immobilization

A new approach to a carrier functionalization is the use of plasma. Oxygen plasma (Gulec 2013) and plasma polymerization: allyl alcohol, allyl amine and acrylic acid were proposed (Labus et al. 2012). The high cost is, however, a serious disadvantage of the method.

4 Immobilized enzymes

The unquestionable advantage of the enzyme immobilization by adsorption process is the versatility. The method can be applied for the enzymes of different types, which catalyse diverse sorts of reactions. Clearly, it is not possible to immobilize any enzyme on any carrier. The range of carriers for a given enzyme is limited by the enzyme-carrier affinity. However, it is possible to propose a carrier that will be optimal for assuring both the desired parameters of the process and the target properties of the immobilized enzyme.

Enzymes most commonly studied in the immobilized form are lipases (Sakai et al. 2010; Adlercreutz 2013; Ansorge-Schumacher and Thum 2013). Lipases catalyse the hydrolysis of esters formed by short- and long-chain alcohols, mono- and multi-hydroxides, and saturated and unsaturated carboxylic acids of short and long chains. The catalysts based on lipases are used in the reactions of esterification or transesterification of different substrates (Brem et al. 2011; Liu et al. 2013; Yu and Fang 2013), and in the process of biodiesel production (Salis et al. 2010; Mendes et al. 2011; Tran et al. 2012). A wide use of this group of proteins and their affinity to many carriers permit their immobilization on many organic and inorganic carriers. Organic carriers seem to be preferred for the immobilization of lipases. They include a wide and highly diverse gamut of polymers, such as cross-linked PVA (Ozturk and Kilinc 2010) and epoxy activated PVA (Mendes et al., 2012), poly(N-methylol acrylamide) (Santos et al. 2007), small and large poly(hydroxybutyrate) beads (Mendes et al. 2011) and polyacrylonitryle electrospun fibres (Sakai et al. 2010), organic matrices of natural origin, including chitosan beads (Nasratun et al. 2010), MANAE-agarose and cellulose ultrathin film (Kosaka et al. 2007), commercial polymer products, e.g. polypropylene membranes Accurel EP100 and Accurel MP1000 (Hita et al. 2009), adsorbent Purasorb (Torres et al. 2008). Lipases were also immobilized on other materials such as buthyl and octadecyl sepabeads (Branco et al. 2010), cotton flannel cloth (Karimpil et al. 2012), olive pomace powder (Yucel 2012) and commercial ion exchange resin Lewatit. By contrast, among inorganic carriers widely used there are different silicas, such as mesoporous silicas (Gustafsson et al. 2012), e.g. SBA-15 (Yang et al. 2013) or MSU-H (Yu and Fang 2013), vesicular silica (Wu et al. 2012), fumed silica (Kramer et al. 2010), silanized silica (Zheng et al. 2012), silica sol–gel film (Lee et al. 2010) and commercial silica-based products Celite (Brem et al. 2011) and Celite 545 (Kumar and Kanwar 2011; see Fig. 5). Other inorganic carriers used for the adsorption of lipases are zirconia nanoparticles (Chen et al. 2008), mica (Zaidan et al. 2012), and magnetic carriers (Liu et al. 2012a, b; Xie and Wang 2012).
Fig. 5

Lipase immobilization onto glutaraldehyde-modified silica surface

An interesting immobilization of lipases was performed in sol–gel derived silica using the multi-walled carbon nanotubes as additives to protect the inactivation of the enzymes during the sol–gel process and to enhance their stability. The immobilized lipases displayed not only higher activities, but also active lifetime as much as five times longer than that of the free enzymes. Similar effects were also observed when a mesoporous silica carrier was modified by carboxyl-functionalized ionic liquid (Yang et al. 2013).

In contrast, in the process of the lipase adsorption on zirconia nanoparticles, it was demonstrated that the nanoparticles modified with a carboxylic surfactant of a long alkyl chain significantly enhanced the activity and enantioselectivity of the immobilized lipases in the organic media (Chen et al. 2008). The use of the surfactant in the preparation changed the surface of the nanoparticles from hydrophilic to hydrophobic. It was interpreted that the interaction between the hydrophobic surface of zirconia and lipases induced the conformational rearrangement of lipases into an active, stable form.

Another group of the enzymes of extensive industrial significance, preferably used in the immobilized form, are amylases (Reshmi et al. 2006; Bellino et al. 2010). The catalysts based on amylases are used on the industrial scale for the hydrolysis of starch. In contrast to lipases, amylases are more specific and their immobilization is possible mostly on inorganic matrices, including mesoporous silicas, such as SBA-15 (Ajitha and Suguman 2010) or silica thin film (Wang et al. 2013), and also silica gel (Nwagu et al. 2011), halloysite nanotubes (Zhai et al. 2010) and metals, such as zirconium and aluminium.

Laccase is another enzyme used in the industry in the immobilized form (Bayramoglu et al. 2012; Xie and Wang 2012). Its major task is to oxidize simple phenolic derivatives, as well as other compounds containing aromatic moieties. The enzyme can thus be used in bioremediation (Bautista et al. 2010). Interestingly, laccase shows an affinity to organic carriers, which is considerably higher comparing to the inorganic carriers. In this context it was immobilized on chitosan membrane (Reshmi et al. 2007), cellulose acetate disc membranes (Labus et al. 2012) and commercial cationic resin Amberlite IR-120H (Spinelli et al. 2012). Among inorganic carriers, the enzyme was immobilized on different silicas (Tavares et al. 2013), e.g. on the commercial mesoporous SBA-15 (Bautista et al. 2010), on magnetic mesoporous silica spheres (Zhu et al. 2007) and on silica gels functionalized with different organosilanes (Rekuc et al. 2010).

Another group of the immobilized enzymes utilized on the industrial scale are oxidases (Kennedy et al. 2007; Zhu et al. 2009; Fornera and Bauer 2012). They are widely applied for catalyzing redox reactions that involve molecular oxygen as an electron acceptor. In these reactions oxygen is reduced to water or hydrogen peroxide. Of special significance in this enzyme family is the glucose oxidase. The reason is that this enzyme is applied in glucose biosensors, which are exploited as measuring devices in real time, in situ measurements, for instance in food industry, but foremost in medicine. Notably, the immobilization of the glucose oxidase was shown to enable constructing glucose biosensors with improved durabilities, sensitivities, linear ranges and detection limits (Delvaux and Demoustier-Champagne 2003; Wu et al. 2009; Zhou et al. 2011).

Another enzymes from the oxidases family are peroxidases, which catalyze the reactions of oxidation, commonly with hydrogen peroxide as a substrate, and are also used in the immobilized form, mainly for the treatment of industrial wastewaters (Montiel et al. 2007; Pesic et al. 2012). The immobilized peroxidases are also used in biosensors for detection of H2O2 (Yu and Ju 2002). According to the literature, these enzymes are immobilized mainly on inorganic silica-based carriers, such as mesoporous silica (Fornera and Bauer 2012), commercial SBA-15 (Zhou et al. 2011), rod-like and vesicle-like mesoporous silica (Zhou et al. 2011), silica gel SG/67 and silica gel 100 (Godjevargova et al. 2006), and also on magnetic nanoparticles based on Fe3O4 (Zhu et al. 2009), gold nanotubes (Delvaux and Demoustier-Champagne 2003) and mesoporous activated carbon matrices MAC 200 and MAC 400 (Kennedy et al. 2007). Peroxidases were also immobilized on an inorganic–organic carrier that was made of platinum nanoparticles/graphene sheets/chitosan film (Wu et al. 2009), where the enzyme-carrier interactions were mediated by the chitosan film.

Immobilization by adsorption has also been applied to other enzymes, such as carbonic anhydrases. This group of enzymes catalyzes the reversible interconversion of carbon dioxide and water to bicarbonate and protons. It has been proposed to exploit the reaction in CO2 capture and storage (Fransen et al. 2013). In the process, commonly known as mineral carbonation (Wanjari et al. 2012; Vinoba et al. 2012; Zhang et al. 2009), carbonic anhydrase serves to catalyze the CO2 hydration. If performed in the presence of Ca2+ ions, the reaction is followed by CaCO3 precipitation. This bio-based proposal constitutes a new, eco-friendly approach to capture, store or sequester CO2 done to avoid the growth of its concentration in the atmosphere. To prepare carbonic anhydrases for this process, the enzymes were adsorbed on inorganic carriers, such as mesoporous silica SBA-15 and mesoporous aluminosilicates, where their stabilities were greatly enhanced, as well as on the complex organic system poly(acrylic acid-co-acrylamide)/hydrotalcite nanocomposite hydrogel. An interesting example of enzymes of practical applications are also cellulases (Hartono et al. 2010; Chang et al. 2011), responsible for the hydrolysis of cellulose, for which they were immobilized on mesoporous silicas materials.

Among the noteworthy immobilized enzymes there are also ureases (Krajewska 2009b; Marzadori et al. 1998; Zhai et al. 2010; Krajewska et al. 1990). The enzymes are responsible for the hydrolysis of urea to carbonic acid and ammonia (Krajewska 2009a, Krajewska et al. 2012). The reaction can be exploited in removal of urea from aqueous solutions that is a problem faced in numerous areas, examples being urea-producing industry, agriculture and natural environment, food production and medicine (Krajewska 2009b). In the latter area, an immobilized urease was considered as a part of the wearable/portable artificial kidney, alternative to the classical hemodialytic device. Important are also analytical applications of immobilized ureases in various biosensing systems, mainly biosensors both for the determinations of urea and of pollutants that are urease inhibitors (Krajewska et al. 1997; Krajewska and Zaborska 2007) (spectrometric, potentiometric, conductometric, amperometric, acoustic, thermal) (Krajewska 2009b). Practical, cost-effective and portable analytical devices, especially useful in the in situ and real-time measurements. The biosensors are predicted to become widely accepted for use, once their storage and operational stabilities are improved.

An overview of enzymes immobilized by adsorption is presented in Table 1 along with the carriers, on which they were immobilized, carrier-modifiers, with which carriers had their surfaces modified, techniques that the systems were studied with, and importantly, their properties and possible applications. The overview may thus serve as a guide for making the right choices while preparing enzymes immobilized by adsorption.

5 Conclusions

Enzymes as the effective catalysts have advantageous features, among which the high catalytic efficiency, specificity and mild conditions of operation made them attractive alternatives to the chemical catalysts for a great variety of applications. This has intensified the studies on the immobilization of the enzymes, in order to improve their catalytic properties. From many methods proposed for the enzyme immobilization, the most common is the adsorption on the solid carriers. The most important advantage of this immobilization is that a wide gamut of carriers can be used and that the enzymes of practically each class can be immobilized. Equally important is the fact, that this immobilization leaves the enzyme structure intact, which allows enzymes to retain their activity and also facilitates the transport of the substrates to the enzyme’s active centre. Comparing with the chemical enzyme immobilizations, a disadvantage of enzyme adsorption is a low stability of the immobilized enzymes, which may lead to a fast washing out of the enzyme from the carrier. However, as follows from the presented survey of the literature overview, the adsorption remains the fastest and most universal method of the enzyme immobilization.



This work was supported by research Grant no. 3/32/443–DS-PB/2014 from Poznan University of Technology, Poznań, Poland (TJ, JZ), and by DS WCh/43 from the Faculty of Chemistry of the Jagiellonian University, Kraków, Poland (BK).


  1. Adlercreutz, P.: Immobilisation and application of lipases in organic media. Chem. Soc. Rev. 42, 6406–6436 (2013)Google Scholar
  2. Ajitha, S., Suguman, S.: Tuning mesoporous molecular sieve SBA-15 for the immobilization of α-amylase. J. Porous Mater. 17, 341–349 (2010)Google Scholar
  3. Akdogan, H.A., Pazarlioglu, N.K.: Fluorene biodegradation by P. osteratus-part II: biodegradation by immobilized cells in a recycled packed bed reactor. Proc. Biochem. 46, 840–846 (2011)Google Scholar
  4. Alatorre-Meda, M., Taboada, P., Sabin, J., Krajewska, B., Varela, L.M., Rodriguez, J.R.: DNA-chitosan complexation: a dynamic light scattering study. Colloid Surf. A 339, 145–152 (2009)Google Scholar
  5. Ansorge-Schumacher, M.B., Thum, O.: Immobilised lipases in the cosmetics industry. Chem. Soc. Rev. 42, 6475–6490 (2013)Google Scholar
  6. Ashly, P.C., Mohanan, P.V.: Preparation and characterization of Rhizopus amyloglucosidase immobilized on poly(o-toluidine). Process Biochem. 45, 1422–1426 (2010)Google Scholar
  7. Barbosa, O., Torres, R., Ortic, C., Berenguer-Murcia, A., Rodrigues, R.C., Fernandez-Lafuente, R.: Heterofunctional supports in enzyme immobilization: from traditional immobilization protocols to opportunities in tuning enzyme properties. Biomacromolecules 14, 2433–2462 (2013)Google Scholar
  8. Bautista, L.F., Morales, G., Sanz, R.: Immobilization strategies for laccase Trametes versicolor on mesostructured silica materials and the application to the degradation of naphthalene. Bioresour. Technol. 101, 8541–8548 (2010)Google Scholar
  9. Bayramoglu, G., Gursel, I., Yilmaz, M., Arica, M.Y.: Immobilization of laccase on itaconic acid grafted and Cu(II) ion chelated chitosan membrane for bioremediation of hazardous materials. J. Chem. Technol. Biotechnol. 87, 530–539 (2012)Google Scholar
  10. Bellino, M.G., Regazzoni, A.E., Soler-Illia, G.J.A.A.: Amylase-functionalized mesoporous silica thin films as robust biocatalyst platforms. Appl. Mat. Interfaces 2, 360–365 (2010)Google Scholar
  11. Benassi, V.M., da Silva, T.M., Pesslea, B.C., Guisan, J.M., Mateo, C., Lima, M.S., Jorge, J.A., de Polizeli, M.L.T.M.: Immobilization and biochemical properties of β-xylosidase activated by glucose/xylose from Aspergillus niger USP-67 with transxylosylation activity. J. Mol. Catal. B 89, 93–101 (2013)Google Scholar
  12. Bhattacharyya, M.S., Singh, A., Banerjee, U.C.: Immobilization of intracellular carbonyl reductase from Geotrichym candidum for the stereoselective reduction of 1-naphthyl ketone. Bioresour. Technol. 101, 1581–1586 (2010)Google Scholar
  13. Brady, D., Jordon, J.: Advances in enzyme immobilisation. Biotechnol. Lett. 31, 1639–1650 (2009)Google Scholar
  14. Branco, R.V., Gutarra, M.L.E., Freire, D.M.G., Almeida, R.V.: Immobilization and characterization of a recombinant thermostable lipase (Pf2001) from Pyrococcus furiosus on suports with different degrees of hydrophobicity. Enzyme Res. 2010, 1–8 (2010)Google Scholar
  15. Brem, J., Turcu, M.C., Paizs, C., Lundell, K., Tosa, M.I., Irimie, F.D., Kanerva, L.T.: Immobilization to improve the propeties of Pseudomonas flourescens lipase for the kinetic resolution of 3-aryl-3-hydroxy esters. Proc. Biochem. 47, 119–126 (2011)Google Scholar
  16. Bronshyteyn, M., Steinberg, D.: Immobilization of fructosyltransferase from Streptococcus mutans on hydroxyapatite surfaces induces the formation of multimeric complexes. Lett. Appl. Microbiol. 34, 205–209 (2002)Google Scholar
  17. Cao, L., van Langen, L., Sheldon, R.A.: Immobilised enzymes: carrier-bound or carrier-free? Curr. Opin. Biotechnol. 14, 387–394 (2003)Google Scholar
  18. Cao, L.: Immobilised enzymes: science or art? Curr. Opin. Biotechnol. 9, 217–226 (2005)Google Scholar
  19. Carrea, G., Riva, S.: Properties and synthetic applications of enzymes in organic solvents. Angew. Chem. Int. Ed. 39, 2226–2254 (2000)Google Scholar
  20. Castro, L.B.R., Silva, F.F., Carmona-Ribeiro, A.M., Kappl, M., Petri, D.F.S.: Immobilization of hexokinase onto chitozan decorated particles. J. Phys. Chem. B 111, 8520–8526 (2007)Google Scholar
  21. Chang, R.H.Y., Jang, J., Wu, K.C.W.: Cellulase immobilized mesoporous silica nanocatalysts for efficient cellulose-to-glucose conversion. Green Chem. 13, 2844–2850 (2011)Google Scholar
  22. Chen, Y.Z., Yang, C.T., Ching, C.B., Xu, R.: Immobilization of lipases on hydrophobilized zirconia nanoparticles: highly enantioselective and reusable biocatalysts. Langmuir 24, 8877–8884 (2008)Google Scholar
  23. Cho, E.J., Jung, S., Kim, H.J., Lee, Y.G., Nam, K.C., Lee, H.J., Bae, H.J.: Co-immobilization of three cellulases on Au-doped magnetic silica nanoparticles for the degradation of cellulose. Chem. Commun. 48, 886–888 (2012)Google Scholar
  24. Choma, J., Kloske, M., Jaroniec, M., Klinik, J.: Benzene adsorption isotherms on MCM-41 and their use for pore size analysis. Adsorption 10, 195–203 (2004)Google Scholar
  25. Cowan, D.A., Fernandez-Lafuente, R.: Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization. Enzyme Microb. Technol. 49, 326–346 (2011)Google Scholar
  26. de Lathouder, K.M., van Benthem, D.T.J., Wallin, S.A., Mateo, C., Fernandez Lafuente, R., Guisan, J.M., Kapteijn, F., Moulijn, J.A.: Poliethyleneimine (PEI) functionalized ceramic monoliths as enzyme carriers: preparation and performance. J. Mol. Catal. B 50, 20–27 (2008)Google Scholar
  27. Deepak, V., Pandian, S.B.R.K., Kalishwaralal, K., Gurunathan, S.: Purification, immobilization and characterization of nattokinase on PHB nanoparticles. Bioresour. Technol. 100, 6644–6646 (2009)Google Scholar
  28. Delvaux, M., Demoustier-Champagne, S.: Immobilisation of glucose oxidase within metallic nanotubes arrays for application to enzyme bionsensors. Biosens. Bioelectron. 18, 943–951 (2003)Google Scholar
  29. DiCosimo, R., McAuliffe, J., Poulose, A.J., Bohlmann, G.: Industrial use of immobilized enzymes. Chem. Soc. Rev. 42, 6437–6474 (2013)Google Scholar
  30. Erhardt, F.A., Jordening, H.J.: Immobilization of dextranase from Chaetomium erraticum. J. Biotechnol. 131, 440–447 (2007)Google Scholar
  31. Falahati, M., Ma’mani, L., Sabuory, A.A., Shafiee, A., Foroumadi, A., Badiei, A.R.: Aminopropyl functionalized cubic Ia3d mesoporous silica nanoparticle as an efficient suport for immobilization of superoxide dismutase. Biochim. Biophys. Acta. 1814, 1195–1202 (2011)Google Scholar
  32. Falahti, M., Saboury, A.A., Ma’mani, L., Shafiee, A., Rafieepour, H.A.: The effect of functionalization of mesoporous silica nanoparticles on the interaction and stability of confined enzyme. Int. J. Biol. Macromol. 50, 1048–1054 (2012)Google Scholar
  33. Fargues, C., Bailly, M., Grevillot, G.: Adsorption of BSA and hemoglobin on hydroxyapatite support: equilibria and multicomponent dynamic. Adsorption 4, 5–16 (1998)Google Scholar
  34. Fernandez-Lafuente, R.: Stabilization of multimeric enzymes: strategies to prevent subunit dissociation. Enzyme Microb. Technol. 45, 405–418 (2009)Google Scholar
  35. Fornera, S., Bauer, T., Dieter Schluter, A., Walde, P.: Simple enzyme immobilization inside glass tubes for enzymatic cascade reactions. J. Mater. Chem. 22, 502–511 (2012)Google Scholar
  36. Fransen, M.C.R., Steunenberg, P., Scott, E.L., Zuilhof, H., Sanders, J.P.M.: Immobilised enzymes in biorenewables production. Chem. Soc. Rev. 42, 6491–6533 (2013)Google Scholar
  37. Garcia-Galan, C., Berenguer-Murcia, A., Fernandez-Lafuente, R., Rodrigues, R.C.: Potential of different enzyme immobilization strategies to improve enzyme performance. Adv. Synth. Catal. 353, 2885–2904 (2011)Google Scholar
  38. Geng, L., Li, N., Xiang, M., Wen, X., Xu, D., Zhao, F., Li, K.: The covalent immobilization of trypsin at the galleries of layered γ-zirconium phosphate. Colloid Surf. B 30, 99–109 (2003)Google Scholar
  39. Godjevargova, T., Nenkova, R., Konsulov, V.: Immobilization of glucose oxidase by acronitrile copolymer coated silica supports. J. Mol. Catal. B 38, 59–64 (2006)Google Scholar
  40. Grabicka, B.E., Jaroniec, M.: Adsorption properties of ordered mesoporous silicas synthesized in the presence of block copolymer Pluronic F127 under microwave irradiation. Adsorption 16, 385–396 (2010)Google Scholar
  41. Gray, C.J., Weissenborn, M.J., Eyers, C.E., Plitsch, S.L.: Enzymatic reactions on immobilised substrates. Chem. Soc. Rev. 42, 6378–6405 (2013)Google Scholar
  42. Grudzień, R.M., Grabicka, B.E., Felix, R., Jaroniec, M.: Polymer-templated organosilicas with hexagonally ordered mesopores: the effect of organosilane addition at different synthesis stages. Adsorption 13, 323–329 (2007)Google Scholar
  43. Grudzień, R.M., Grabicka, B.E., Jaroniec, M.: Adsorption and structural properties of channel-like and cage-like organosilicas. Adsorption 12, 293–308 (2006)Google Scholar
  44. Gulec, H.A.: Immobilization of β-galactosidase from Kluyveromyces lactis onto polymeric membrane surfaces: effect of surface characteristics. Colloid Surf. B 104, 83–90 (2013)Google Scholar
  45. Gupta, A., Prabhu, K.A.: Immobilization and properties of dextransucrase from Leuconostoc mesenteroides culture, LM1. J. Gen. Appl. Microbiol. 41, 399–407 (1995)Google Scholar
  46. Gustafsson, H., Johansson, E.M., Barrabino, A., Oden, M., Holmberg, K.: Immobilization of lipase from Mucor miehei and Rhizopus oryzae onto mesoporus silca-the effect of varied particle size and morphology. Colloid Surf. B 100, 22–30 (2012)Google Scholar
  47. Hanefeld, U., Cao, L., Magner, E.: Enzyme immobilisation: fundamentals and application. Chem. Soc. Rev. 42, 6211–6212 (2013)Google Scholar
  48. Hanefeld, U., Gardosi, L., Magner, E.: Understanding enzyme immobilisation. Chem. Soc. Rev. 38, 453–468 (2009)Google Scholar
  49. Hartmann, M., Kostrov, X.: Immobilization of enzymes on porous silicas-benefits and challenges. Chem. Soc. Rev. 42, 6277–6289 (2013)Google Scholar
  50. Hartono, S.B., Qiao, S.Z., Liu, J., Jack, K., Ladewig, B.P., Hao, Z., Lu, G.Q.M.: Functionalized mesoporous silica with very large pores for cellulase immobilization. J. Phys. Chem. 83, 8353–8362 (2010)Google Scholar
  51. Hernandez, K., Fernandez-Lafuente, R.: Control of protein immobilization: coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performance. Enzyme Microb. Technol. 48, 107–122 (2011)Google Scholar
  52. Hita, E., Robles, A., Camacho, B., Gonzalez, P.A., Esteban, L., Jimenez, M.J., Munio, M.M., Molina, E.: Production of structured triacylglycerols by acidolysis catalyzed by lipases immobilized in a packed bed reactor. Biochem. Eng. J. 46, 257–264 (2009)Google Scholar
  53. Honarkar, H., Barikani, M.: Applications of biopolymers. I. Chitosan. Monatsh Chem. 140, 1403–1420 (2009)Google Scholar
  54. Hung, Y.J., Peng, C.C., Tzen, J.T.C., Chen, M.J., Liu, J.R.: Immobilization of Neocallimastix patriciarum xylanase on artificial oil bodies and statistical optimization of enzyme activity. Bioresour. Technol. 99, 8662–8666 (2008)Google Scholar
  55. Hwang, E.T., Gu, M.B.: Enzyme stabilization by nano/microsized hybrid materials. Eng. Life Sci. 1, 49–61 (2013)Google Scholar
  56. Iyer, P.V., Ananthanarayan, L.: Enzyme stability and stabilization-aqueous and non-aqueous environment. Process Biochem. 43, 1019–1032 (2008)Google Scholar
  57. Jaroniec, M., Fulvio, P.F.: Standard nitrogen adsorption data for α-alumina and their use for characterization of mesoporous alumina-based materials. Adsorption 19, 475–481 (2013)Google Scholar
  58. Karboune, S., Neufeld, R., Kermasha, S.: Immobilization and biocatalysis of chlorophyllase in selected organic solvent systems. J. Biotech. 120, 273–283 (2005)Google Scholar
  59. Karimpil, J.J., Melo, J.S., D’Souza, S.F.: Immobilization of lipase on cotton cloth using the layer-by-layer self-assemble technique. Int. J. Biol. Macromol. 50, 300–302 (2012)Google Scholar
  60. Katchalski-Katzir, E., Kraemer, D.M.: Eupergit® C, a carrier for immobilization of enzymes of industrial potential. J. Mol. Catal. B 10, 157–176 (2000)Google Scholar
  61. Kawaguti, H.Y., Hoffmann Carvalho, P., Figueira, J.A., Sato, H.H.: Immobilization of Erwinia sp. 12 cells in alginate-gelatin matrix and conversion of sucrose into osomaltulose using response surface methodology. Enzyme Res. 1, 1–8 (2011)Google Scholar
  62. Kawakita, H., Sugita, K., Saito, K., Tamada, M., Sugo, T., Kawamoto, H.: Production of cycloisomaltooligosacharides from dextran using enzyme immobilized in multilayers porous membranes. Biotechnol. Prog. 18, 465–469 (2002)Google Scholar
  63. Kennedy, L.J., Selvi, P.K., Padmanabhan, A., Hema, K.N., Sekaran, G.: Immobilization of polyphenol oxidase onto mesoporous activated carbons-isotherm and kinetic studies. Chemosphere 69, 262–270 (2007)Google Scholar
  64. Klibanov, A.M.: Improving enzymes by using them in organic solvents. Nature 409, 241–246 (2001)Google Scholar
  65. Kosaka, P.M., Kawano, Y., El Seound, O.A., Petri, D.F.S.: Catalytic activity of lipase immobilized onto ultrathin films of cellulose esters. Langmuir 23, 12167–12173 (2007)Google Scholar
  66. Krajewska, B., Leszko, M., Zaborska, W.: Urease immobilized on chitosan membrane. Preparation and properties. J. Chem. Tech. Biotechnol. 48, 337–350 (1990)Google Scholar
  67. Krajewska, B., Zaborska, W., Leszko, M.: Inhibition of chitosan-immobilized urease by boric acid as determined by integration methods. J. Mol. Catal. B 3, 231–238 (1997)Google Scholar
  68. Krajewska, B.: Application of chitin- and chitosan-based materials for enzyme immobilizations: a review. Enzyme Microb. Technol. 35, 126–139 (2004)Google Scholar
  69. Krajewska, B.: Membrane-based processes performed with use of chitin/chitosan materials. Sep. Purif. Tech. 41, 305–312 (2005)Google Scholar
  70. Krajewska, B., Zaborska, W.: Double mode of inhibition-inducing interactions of 1,4-naphthoquinone with urease. Arylation vs oxidation of enzyme thiols. Bioorg. Med. Chem. 15, 4144–4151 (2007)Google Scholar
  71. Krajewska, B., Wydro, P., Jańczyk, A.: Probing the modes of antibacterial activity of chitosan. Effects of pH and molecular weight on chitosan interactions with membrane lipids in Langmuir films. Biomacromolecules 12, 4144–4152 (2011)Google Scholar
  72. Krajewska, B., Wydro, P., Kyzioł, A.: Chitosan as a subphase disturbant of membrane lipid monolayers. The effect of temperature at varying pH: I. DPPG. Colloid Surf. A 434, 349–358 (2013a)Google Scholar
  73. Krajewska, B., Kyzioł, A., Wydro, P.: Chitosan as a subphase disturbant of membrane lipid monolayers. The effect of temperature at varying pH: II. DPPC and cholesterol. Colloid Surf. A 434, 359–364 (2013b)Google Scholar
  74. Krajewska, B.: Ureases I. Functional, catalytic and kinetic properties: a review. J. Mol. Cat. B 56, 9–21 (2009a)Google Scholar
  75. Krajewska, B.: Ureases. II. Properties and their customizing by enzyme immobilizations: a review. J. Mol. Cat. B 59, 22–40 (2009b)Google Scholar
  76. Krajewska, B., van Eldik, R., Brindell, M.: Temperature- and pressure-dependent stopped-flow kinetic studies of jack-bean urease. Implications for the catalytic mechanism. J. Biol. Inorg. Chem. 17, 1123–1134 (2012)Google Scholar
  77. Kramer, M., Cruz, J.C., Pfromm, P.H., Rezac, E., Czermak, P.: Enantioselective transestrification by Candida antarctica lipase B immobilized on fumed silica. J. Biotechnol. 150, 80–86 (2010)Google Scholar
  78. Kubac, D., Cejkova, A., Masak, J., Jirku, V., Lemaire, M., Gallienne, E., Bolte, J., Stloukal, R., Martinkova, L.: Biotransformation of nitriles by Rhodococcus equi A4 immobilized in LentiKats. J. Mol. Catal. B 39, 59–61 (2006)Google Scholar
  79. Kumar, A., Kanwar, S.S.: Synthesis of ethyl ferulate in organic medium using celite-immobilized lipase. Bioresour. Technol. 102, 2162–2167 (2011)Google Scholar
  80. Kwon, S.W., Jeong, B.O., Lee, E.H., Kim, Y.S., Jung, Y.: Conducting polimer silica composites for immoilization of enzymes. Bull. Korean Chem. Soc. 33, 1593–1596 (2012)Google Scholar
  81. Labus, K., Gancarz, I., Bryjak, J.: Immobilization of laccase and tyrosinase on untreated and plasma-terated cellulosic and polyamide membranes. Mater. Sci. Eng. C 32, 228–235 (2012)Google Scholar
  82. Lee, S.H., Doan, T.T.N., Won, K., Ha, S.H., Koo, Y.M.: Immobilization of lipase within carbon nanotube-silica composites for non-aqueous reaction systems. J. Mol. Catal. B 62, 169–172 (2010)Google Scholar
  83. Liese, A., Hilterhaus, L.: Evaluation of immobilized enzymes for industrial applications. Chem. Soc. Rev. 42, 6236–6249 (2013)Google Scholar
  84. Liu, C.H., Huang, C.C., Wang, Y.W., Lee, D.J., Chang, J.S.: Biodiesel production by enzymatic transestrification catalyzed Brukholderia lipase immobilized in hydrophobic magnetic particles. Appl. Energy 100, 41–46 (2012a)Google Scholar
  85. Liu, N., Wang, Y., Zhao, Q., Cui, C., Fu, M., Zhao, M.: Immobilisation of lecitase ultra for producion of diacylglycerols by glycerolysis of soybean oil. Food Chem. 134, 301–307 (2012b)Google Scholar
  86. Liu, Q., Kong, X., Zhang, C., Chen, Y., Hua, Y.: Immobilisation of a hydroperoxide lyase and comparative enzymological studies of the immobilised enzyme with membranę-bound enzyme. J. Sci. Food Agric. 93, 1953–1959 (2013)Google Scholar
  87. Magner, E.: Immobilisation of enzymes on mesoporous silicate materials. Chem. Soc. Rev. 42, 6213–6222 (2013)Google Scholar
  88. Mansur, H.S., Orefice, R.L., Lobato, Z.P., Vasconcelos, W.L., Mansur, E.S., Machado, L.J.C.: Adsorption/desorption behavior of bovine serum albumin and porcine insulin on chemically patterned porous gel networks. Adsorption 7, 105–116 (2001)Google Scholar
  89. Marzadori, C., Miletti, S., Gessa, C., Ciurli, S.: Immobilization of jack bean urease on hydroxyapatite: urease immobilization on alkaline soils. Soil Biol. Biochem. 30, 1485–1490 (1998)Google Scholar
  90. Mateo, C., Palomo, J.M., Fernandez-Lafuente, G., Guisan, J.M., Fernandez-Lafuente, R.: Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 40, 1451–1463 (2007)Google Scholar
  91. Mendes, A.A., Freitas, L., de Carvalho, A.K.F., de Oliviera, P.C., de Castro, H.F.: Immobilization of a commercial lipase from Penicillium camembertii (Lipase G) by different strategies. Enzyme Res. 2011, 1–8 (2011)Google Scholar
  92. Mendes, A.A., Oliveira, P.C., Velez, A.M., Giordano, R.C., de Giordano, R.L.C., de Castro, H.F.: Valuation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis. Int. J. Biol. Macromol. 50, 503–511 (2012)Google Scholar
  93. Montiel, C., Terres, E., Dominguez, J.M., Aburto, J.: Immobilization of chloroperoxidase on silica-based materials for 4,6-dimethyl dibenzophene oxidation. J. Mol. Catal. B 48, 90–98 (2007)Google Scholar
  94. Nara, T.Y., Togashi, H., Sekikawa, C., Inoh, K., Hisamatsu, K., Sakaguchi, K., Mizukami, F., Tsunoda, T.: Functional immobilizationof racemase by adsorption on folded-sheet mesoporous silica. J. Mol. Catal. B 64, 107–112 (2010)Google Scholar
  95. Nasratun, M., Hasrul, A.S., Sureena, A., Nurul Aini, M.A., Ruwaida, A.R., Shalyda, M.S., Ideris, A., Rozaimi, A.S., Sharifuddin, J.H., Ahamad Nordin, N.I.A.: Immobilization of lipase from Candida rugosa on chitosan beads for transesterification reaction. J. Appl. Sci. 10, 2701–2709 (2010)Google Scholar
  96. Nwagu, T.N., Okolo, B.N., Aoyagi, H.: Immobilization of raw starch digesting amylase on silica gel: a comparative study. Afr. J. Biotechnol. 10, 15989–15997 (2011)Google Scholar
  97. Ozturk, T.K., Kilinc, A.: Immobilization of lipase in organic solvent in the presence of fatty acid additives. J. Mol. Catal. B 67, 214–218 (2010)Google Scholar
  98. Pesic, M., Lopez, C., Alvaro, G., Lopez-Santin, J.: A novel immobilized chloroperoxidase biocatalyst with improved stability for the oxidation of amino alcohols to amino aldehydes. J. Mol. Catal. B 84, 144–151 (2012)Google Scholar
  99. Rekuć, A., Bryjak, J., Szymańska, K., Jarzębski, A.B.: Very stable silica-gel-bound laccase biocatalysts for the selective oxidation in continuous systems. Bioresour. Technol. 101, 2076–2083 (2010)Google Scholar
  100. Reshmi, R., Sanjay, G., Sugunan, S.: Enhanced activity and stability of α-amylase immobilized on alumina. Catal. Commun. 7, 460–465 (2006)Google Scholar
  101. Reshmi, R., Sanjay, G., Sugunan, S.: Immobilization of α-amylase on zirconia: a heterogeneous biocatalyst for starch hydrolysis. Catal. Commun. 8, 393–399 (2007)Google Scholar
  102. Rodrigues, R.C., Ortiz, C., Berenguer-Murcia, A., Torres, R., Fernandez-Lafuente, R.: Modifying enzyme activity and selectivity by immobilization. Chem. Soc. Rev. 42, 6290–6307 (2013)Google Scholar
  103. Rodrigues, R.C., Berenguer-Murcia, A., Fernandez-Lafuente, R.: Coupling chemical modification and immobilization to improve the catalytic performance of enzymes. Adv. Synth. Catal. 353, 2216–2238 (2011)Google Scholar
  104. Sakai, S., Liu, Y., Yamaguchi, T., Watanabe, R., Kawabe, M., Kawakami, K.: Production of butyl-biodiesel using lipase physically-adsorbed onto electrospun polyacrylonitryle fibers. Bioresour. Technol. 101, 7344–7349 (2010)Google Scholar
  105. Salis, A., Casula, M.F., Bhattacharyya, M.S., Pinna, M., Solinas, V., Monduzzi, M.: Physical and chemical lipase adsorption on SBA-15: effect of different interactions on enzyme loading and catalytic performance. Chem. Cat. Chem. 2, 322–329 (2010)Google Scholar
  106. Santos, J.C., Nunes, G.F.M., Moreira, A.B.R., Perez, V.H., de Castro, H.F.: Characterization of Candida rugosa lipase immobilized on poly(N-methyloacrylamide) and its application in butyl butyrate synthesis. Chem. Eng. Technol. 30, 1255–1261 (2007)Google Scholar
  107. Sheldon, R.A., van Pelt, S.: Enzyme immobilisation in biocatalysis: why, what and how? Chem. Soc. Rev. 42, 6223–6225 (2013)Google Scholar
  108. Sheldon, R.A.: Enzyme immobilization: the quest for optimum performance. Adv. Synth. Catal. 49, 1289–1307 (2007)Google Scholar
  109. Spinelli, D., Fatarella, E., Di Michele, A., Pogni, R.: Immobilization of fungal (Trametes versicolor) laccase onto Amberlite IR-120 H beads: optimization and characterization. Proc. Biochem. 48, 218–223 (2012)Google Scholar
  110. Tavares, A.P.M., Rodriguea, O., Fernandez-Fernandez, M., Dominguez, A., Moldes, D., Sanroman, M.A., Macado, E.A.: Immobilization of laccase on modified silica: stabilization, thermal inactivation and kinetic bahaviour in 1-ethyl-3-methylimidazolium ethylsulfate inonic liquid. Bioresour. Technol. 131, 405–412 (2013)Google Scholar
  111. Thorn, C., Gustafsson, H., Olsson, L.: Immobilization of feruloyl esterases in mesoporous materials leads to improved transestrification yield. J. Mol. Catal. B 72, 57–64 (2011)Google Scholar
  112. Thudi, L., Jasti, L.S., Swarnalahta, Y., Fadnavis, N.W., Mulani, K., Deokar, S., Ponratham, S.: Enzyme immobilization on epoxy supports in reverse micellar media: prevention of enzyme denaturation. J. Mol. Catal. B 74, 54–62 (2012)Google Scholar
  113. Torres, P., Reyes-Duarte, D., Lopez-Cortes, N., Ferrer, M., Ballesteros, A., Plou, F.J.: Acetylation of vitamin E by Candida antarctica lipase B immobilized on diffrent carriers. Proc. Biochem. 43, 145–153 (2008)Google Scholar
  114. Tran, D.T., Chen, C.L., Chang, J.S.: Immobilization of Brukholderia sp. lipase on a ferric nanocomposite for biodiesel production. J. Biotech. 158, 112–119 (2012)Google Scholar
  115. Tuzmen, N., Kalburcu, T., Denizli, A.: Immobilization of catalase via adsorption onto metal-chelated affinity cryogels. Proc. Biochem. 47, 26–33 (2012)Google Scholar
  116. Vinoba, M., Bhagiyalakshmi, M., Jeong, S.K., Yoon, Y.I., Nam, S.C.: Immobilization of carbonic anhydrase on spherical SBA-15 for hydration and sequestration of CO2. Colloid Surf. B 90, 91–96 (2012)Google Scholar
  117. Wang, S., Su, P., Ding, F., Yang, Y.: Immobilization of cellulase on polyamidoamine dendrimer-grafted silica. J. Mol. Catal. B 89, 35–40 (2013)Google Scholar
  118. Wanjari, S., Pabhu, C., Satyanarayana, T., Vinu, A., Rayalu, S.: Immobilization of carbonic anhydrase on mesoporous aluminosilicate for carbonation reaction. Micropor. Mesopor. Mater. 160, 151–158 (2012)Google Scholar
  119. Weber, E., Sirim, D., Schreiber, T., Thomas, B., Pleiss, J., Hunger, M., Glaser, R., Urlacher, V.B.: Immobilization of P450 BM-3 monooxygenase on mesoporous molecular sives with different pore diameters. J. Mol. Catal. B 64, 29–37 (2010)Google Scholar
  120. Wu, C., Zhou, G., Jiang, X., Ma, J., Zhang, H., Song, H.: Active biocatalysts based on Candida rugosa lipase immobilized in versicular silica. Proc. Biochem. 47, 953–959 (2012)Google Scholar
  121. Wu, H., Wang, J., Kang, X., Wang, C., Wang, D., Liu, J., Aksay, I.A., Lin, Y.: Glucose biosensor based on immobilization of glucose oxidase in pltinum nanoparticles/graphene/chitosan nanocomposite film. Talanta 80, 403–406 (2009)Google Scholar
  122. Xiao, P., Lv, X., Deng, Y.: Immobilization of chymotrypsin on silica beads based on high affinity and specificity aptamer and its applications. Anal. Lett. 45, 1264–1273 (2012)Google Scholar
  123. Xie, W., Wang, J.: Immobilized lipase on magnetic chitosan microspheres for transestrification of soybean oil. Biomass Bioenergy 36, 373–380 (2012)Google Scholar
  124. Xu, F., Wang, W.H., Tan, Y.J., Bruening, M.L.: Facile trypsin immobilization in polymeric membranes for rapid, efficient, protein digestion. Anal. Chem. 82, 10045–10051 (2010)Google Scholar
  125. Yang, J., Hu, Y., Jiang, L., Zou, B., Jia, R., Huang, H.: Enhancing the catalytic properties of porcine pancreatic lipase by immobilization on SBA-15 modified by functionalized ionic liquid. Biochem. Eng. J. 70, 46–54 (2013)Google Scholar
  126. Yu, J., Ju, H.: Preparation of porous titania sol–gel matrix for immobilization of horseradish peroxidase by a vapor deposition method. Anal. Chem. 74, 3579–3583 (2002)Google Scholar
  127. Yu, W., Fang, M., Tong, D.S., Shao, P., Xu, T., Zhou, C.: Immobilization of Candida rugosa lipase on hexagonal mesoporous silca and selective estrification in nonaqueous medium. Biochem. Eng. J. 70, 97–105 (2013)Google Scholar
  128. Yucel, Y.: Optimization of immobilization conditions of Thermomyces lanuginosus lipase on olive pomace powder using response methodology. Biocatal. Agric. Biotechnol. 1, 39–44 (2012)Google Scholar
  129. Zaidan, U.H., Rahman, M.B.A., Othman, S.S., Basr, M., Abdulmalek, E., Rahman, R.N.Z.R.A., Salleh, A.B.: Biocatalytic production of lactose ester catalysed by mica-based immobilised lipase. Food Chem. 131, 199–205 (2012)Google Scholar
  130. Zhai, R., Zhang, B., Liu, L., Xie, Y., Zhang, H., Liu, J.: Immobilization of enzyme biocatalyst on natural halloysite nanotubes. Catal. Commun. 12, 259–263 (2010)Google Scholar
  131. Zhang, B., Xing, J.M., Liu, H.Z.: Synthesis and characterization of superparamagnetic poly(urea-formaldehyde) adsorbents and their use for adsorption of flavonoids from Glycyrrhiza uralensis Fisch. Adsorption 14, 65–72 (2008)Google Scholar
  132. Zhang, Y.T., Zhi, T.T., Zhang, L., Huang, H., Chen, H.L.: Immobiliation of carbonic anhydrase by embedding and covalent coupling into nanocomposite hydrogel containig hydrotalcite. Polymer 50, 5693–5700 (2009)Google Scholar
  133. Zhao, H.: Methods for stabilizing and activating enzymes in ionic liquids: a review. J. Chem. Technol. Biotechnol. 85, 891–907 (2010)Google Scholar
  134. Zheng, M.M., Lu, Y., Dong, L., Guo, P.M., Deng, Q.C., Li, W.L., Feng, Y.Q., Huang, F.H.: Immobilization of Candida rugosa lipase on hydrophobic/strong cation-exchange functional silica particles for biocatalytic synthesis of phytosterol esters. Bioresour. Technol. 115, 141–146 (2012)Google Scholar
  135. Zhou, G., Fung, K.K., Wong, L.W., Chen, Y., Renneberg, R., Yang, S.: Immobilization of glucose oxidase on rod-like and vesicle-like mesoporous silica for enhancing current responses of glucose biosensors. Talanta 84, 659–665 (2011)Google Scholar
  136. Zhu, H., Pan, J., Hu, B., Yu, H.L., Xu, J.H.: Immobilization of glycolate oxidase from Medicago falcata on magnetic nanoparticles for applications in biosynthesis of glyoxylic acid. J. Mol. Catal. B 61, 174–179 (2009)Google Scholar
  137. Zhu, Y., Kaskel, S., Shi, J., Wage, T., Van Pee, K.H.: Immobilization of Tramtes versicolor laccase on magnetically separable mesoporous silica spheres. Chem. Mater. 19, 6408–6413 (2007)Google Scholar

Copyright information

© The Author(s) 2014

Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

Authors and Affiliations

  • Teofil Jesionowski
    • 1
  • Jakub Zdarta
    • 1
  • Barbara Krajewska
    • 2
  1. 1.Faculty of Chemical Technology, Institute of Chemical Technology and EngineeringPoznan University of TechnologyPoznańPoland
  2. 2.Faculty of ChemistryJagiellonian UniversityKrakówPoland

Personalised recommendations