Enzyme and Microbial Technology 35 (2004) 126–139 Application of chitin- and chitosan-based materials for enzyme immobilizations: a review Barbara Krajewska∗ Jagiellonian University, Faculty of Chemistry, 30-060 Kraków, Ingardena 3, Poland Received 11 September 2003; received in revised form 24 December 2003; accepted 24 December 2003 Abstract As functional materials, chitin and chitosan offer a unique set of characteristics: biocompatibility, biodegradability to harmless products, nontoxicity, physiological inertness, antibacterial properties, heavy metal ions chelation, gel forming properties and hydrophilicity, and remarkable affinity to proteins Owing to these characteristics, chitin- and chitosan-based materials, as yet underutilized, are predicted to be widely exploited in the near future especially in environmentally benign applications in systems working in biological environments, among others as enzyme immobilization supports This paper is a review of the literature on enzymes immobilized on chitin- and chitosan-based materials, covering the last decade One hundred fifty-eight papers on 63 immobilized enzymes for multiplicity of applications ranging from wine, sugar and fish industry, through organic compounds removal from wastewaters to sophisticated biosensors for both in situ measurements of environmental pollutants and metabolite control in artificial organs, are reviewed © 2004 Elsevier Inc All rights reserved Keywords: Chitin; Chitosan; Enzyme immobilization; Applications; Review Why enzymes? While conventional methodologies of chemical processes have been developed in the past decades to a level allowing production, separation and analytical determination of an enormous range of sophisticated products, alternative methodologies that are not only efficient and safe but also environmentally benign and resource- and energy-saving, are being increasingly sought One of the most promising strategies to achieve these goals is the utilization of enzymes [1–5] Enzymes exhibit a number of features that make their use advantageous as compared to conventional chemical catalysts Foremost among them are a high level of catalytic efficiency, often far superior to chemical catalysts, and a high degree of specificity that allows them to discriminate not only between reactions but also between substrates (substrate specificity), similar parts of molecules (regiospecificity) and between optical isomers (stereospecificity) These specificities warrant that the catalyzed reaction is not perturbated by side-reactions, resulting in the production of one wanted end-product, whereas production of undesirable by-products is eliminated This provides substantially higher reaction yields reducing material costs In addition, ∗ Tel.: +48 12 6336377; fax: +48 12 6340515 E-mail address: krajewsk@chemia.uj.edu.pl (B Krajewska) 0141-0229/$ – see front matter © 2004 Elsevier Inc All rights reserved doi:10.1016/j.enzmictec.2003.12.013 enzymes generally operate at mild conditions of temperature, pressure and pH with reaction rates of the order of those achieved by chemical catalysts at more extreme conditions This makes for substantial process energy savings and reduced manufacturing costs Also, enzymes practically not present disposal problems since, being mostly proteins and peptides, they are biodegradable and easily removed from contaminated streams This unique set of advantageous features of enzymes as catalysts has been exploited since the 1960s and several enzyme-catalyzed processes have been successfully introduced to industry, e.g in the production of certain foodstuffs, pharmaceuticals and agrochemicals, but now also increasingly to organic chemical synthesis Why immobilize enzymes? In addition to the unquestionable advantages, there exists a number of practical problems in the use of enzymes To these belong: the high cost of isolation and purification of enzymes, the instability of their structures once they are isolated from their natural environments, and their sensitivity both to process conditions other than the optimal ones, normally narrow-ranged, and to trace levels of substances that can act as inhibitors The latter two result in enzymes’ short operational lifetimes Also, unlike conventional heteroge- B Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139 neous chemical catalysts, most enzymes operate dissolved in water in homogeneous catalysis systems, which is why they contaminate the product and as a rule cannot be recovered in the active form from reaction mixtures for reuse Several methods have been proposed to overcome these limitations, one of the most successful being enzyme immobilization [1–6] Immobilization is achieved by fixing enzymes to or within solid supports, as a result of which heterogeneous immobilized enzyme systems are obtained By mimicking the natural mode of occurence in living cells, where enzymes for the most cases are attached to cellular membranes, the systems stabilize the structure of enzymes, hence their activities Thus, as compared to free enzymes in solution immobilized enzymes are more robust and more resistant to environmental changes More importantly, the heterogeneity of the immobilized enzyme systems allows easy recovery of both enzyme and product, multiple reuse of enzymes, continuous operation of enzymatic processes, rapid termination of reactions and greater variety of bioreactor designs Enzymes may be immobilized by a variety of methods, which may be broadly classified as physical, where weak interactions between support and enzyme exist, and chemical, where covalent bonds are formed with the enzyme [1–4,6,7] To the physical methods belong: (i) containment of an enzyme within a membrane reactor, (ii) adsorption (physical, ionic) on a water-insoluble matrix, (iii) inclusion (or gel entrapment), (iv) microencapsulation with a solid membrane, (v) microencapsulation with a liquid membrane, and (vi) formation of enzymatic Langmuir-Blodgett films The chemical immobilization methods include: (i) covalent attachment to a water-insoluble matrix, (ii) crosslinking with use of a multifunctional, low molecular weight reagent, and (iii) co-crosslinking with other neutral substances, e.g proteins Numerous other methods which are combinations of the ones listed or original and specific of a given support or enzyme have been devised However, no single method and support is best for all enzymes and their applications This is because of the widely different chemical characteristics and composition of enzymes, the different properties of substrates and products, and the different uses to which the product can be applied Besides, all of the methods present advantages and drawbacks Adsorption is simple, cheap and effective but frequently reversible, covalent attachment and crosslinking are effective and durable, but expensive and easily worsening the enzyme performance, and in membrane reactor-confinment, entrapment and microencapsulations diffusional problems are inherent Consequently, as a rule the optimal immobilization conditions for a chosen enzyme and its application are found empirically by a process of trial and error in a way to ensure the highest possible retention of activity of the enzyme, its operational stability and durability Advantageous though it is, the immobilization involves a number of effects worsening the performance of enzymes [1–4,6,7] Compared with the free enzyme, most commonly 127 the immobilized enzyme has its activity lowered and the Michaelis constant increased These alterations result from structural changes introduced to the enzyme by the applied immobilization procedure and from the creation of a microenvironment in which the enzyme works, different from the bulk solution The latter is strongly dependent on the reaction taking place, the nature of the support and on the design of the reactor Furthermore, being two phase systems, the immobilized enzyme systems suffer from inevitable mass transfer limitations, producing unfavourable effects on their overall catalytic performances These, however, may be reduced by applying appropriate reactor designs For the implementation in a commercial process all beneficial and detrimental effects of whether a chemical catalyst or an enzyme is chosen, and whether a free or immobilized enzyme is used, have to be weighed taking into account all relevant aspects, health and environmental included, in addition to obvious economical viability To date, several immobilized enzyme-based processes have proved economic and have been implemented on a larger scale, mainly in the food industry, where they replace free enzyme-catalyzed processes, and in the manufacture of fine speciality chemicals and pharmaceuticals, particularly where asymmetric synthesis or resolution of enantiomers to produce optically pure products are involved [1–5,8] A selection of currently used immobilized-enzyme processes, in the approximate order of the decreasing scale of manufacture, is given in Table The scale of the processes ranges from about 106 t per year for high-fructose corn syrup, arguably one of the most commercially important immobilized enzyme-based process, to about 102 t per year for enantiopure l-DOPA [5] Areas of present and potential future applications of immobilized enzyme systems other than industrial (Table 1) include: laboratory scale organic synthesis, and analytical and medical applications [1–5,7] Having been shown to be able to catalyze reactions not only in aqueous solutions but also in organic media, enzymes offer great potential for assisting organic synthesis [9] They can simplify the chemical procedures by reducing the number of synthetic steps, they can enhance the purity of the products, and most importantly, they can catalyze regio- and stereoselective synthesis giving, otherwise unobtainable compounds with the desired properties In analytical applications immobilized enzymes are used chiefly in biosensors [3,10–12] and to a lesser extent, in diagnostic test strips Biosensors are constructed by integrating biological sensing systems, e.g enzyme(s), with transducers These obtain a chemical signal produced by the interaction of the biological system with an analyte and transduce it into a measurable response Different kinds of transducers have been employed in biosensors, viz potentiometric, amperometric, conductometric, thermometric, optical and piezo-electric, most of the current research being placed on the first two Enzymes for the most cases are immobilized either directly on a transducer’s working tip or in/on a polymer 128 B Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139 Table Some of the more important industrial applications of immobilized enzyme systems [1–3,5] Enzyme (EC number) Substrate Product Glucose isomerase (5.3.1.5) ␤-Galactosidase (3.2.1.23) Lipase (3.1.1.3) Nitrile hydratase (4.2.1.84) Aminoacylase (3.5.1.14) Raffinase (3.2.1.22) Invertase (3.2.1.26) Aspartate ammonia-lyase (4.3.1.1) Thermolysin (3.4.24.27) Glucoamylase (3.2.1.3) Papain (3.4.22.2) Hydantoinase (3.5.2.2) Penicillin amidase (3.5.1.11) Glucose Lactose Triglycerides Acrylonitrile 3-Cyanopyridine Adiponitrile d,l-Aminoacids Raffinose Sucrose Ammonia + fumaric acid Peptides Starch Proteins d,l-Amino acid hydantoins Penicillins G and V ␤-Tyrosinase (4.1.99.2) Pyrocatechol Fructose (high-fructose corn syrup) Glucose and galactose (lactose-free milk and whey) Cocoa butter substitutes Acrylamide Nicotinamide 5-Cyanovaleramide l-Amino acids (methionine, alanine, phenylalanine, tryptophan, valine) Galactose and sucrose (raffinose-free solutions) Glucose/fructose mixture (invert sugar) l-Aspartic acid (used for production of synthetic sweetener aspartame) Aspartame d-Glucose Removal of “chill haze” in beers d,l-Amino acids 6-Aminopenicillanic acid (precursor of semi-synthetic penicillins, e.g ampicillin) l-DOPA membrane tightly wrapping it up In principle, due to enzyme specificity and sensitivity biosensors can be tailored for nearly any target analyte, and these can be both enzyme substrates and enzyme inhibitors Advantageously, their determination is performed without special preparation of the sample Meeting the demand for practical, cost-effective and portable analytical devices, enzyme-based biosensors have enormous potential as useful tools in medicine, environmental in situ and real time monitoring, bioprocess and food control, and in biomedical and pharmaceutical analysis Their use, impaired as yet by not quite satisfactory reliability, is predicted to become widely accepted once their storage and operational stabilities have been improved The most extensively studied enzymes for the application in enzyme-based biosensors are presented in Table Of these, glucose sensors are the most widely studied constituting ca 1/3 of the enzyme-biosensors literature, the subsequent ten sensors occupy another 1/3 of the literature and the other sensors the remaining 1/3 [11] From a practical and commercial point of view, four of the sensors listed, namely glucose, lactate, urea and glutamate have been widely used [12] Medical applications of immobilized enzymes include [1,4,13] diagnosis and treatment of diseases, among those enzyme replacement therapies, as well as artificial cells and organs, and coating of artificial materials for better biocompatibility Offering a great potential in this area, real application of immobilized enzymes has as yet suffered from serious problems from their toxicity to the human organism, allergenic and immunological reactions as well as from their limited stability in vivo Examples of potential medical uses of immobilized enzyme systems are listed in Table Table Some of the most frequently studied enzymes for enzyme-based biosensors [3,10–12] Enzyme (EC number) Substrate Application Glucose oxidase (1.1.3.4) Horseradish peroxidase (1.11.1.7) Glucose H O2 Lactate oxidase (1.13.12.4) Tyrosinase (1.14.18.1) Lactate Phenols, polyphenols Glutamate oxidase (1.4.3.11) Urease (3.5.1.5) Alcohol dehydrogenase (1.1.1.1) Acetylcholinesterase (3.1.1.7) Glutamate Urea Ethanol Acetylcholine, acetylthiocholine Choline oxidase (1.1.3.17) Lactate dehydrogenase (1.1.1.27) Cholesterol oxidase (1.1.3.6) Penicillinase (3.5.2.6) Alliinase (4.4.1.4) Choline lactate Cholesterol Penicillins Cysteine sulfoxides Diagnosis and treatment of diabetes, food science, biotechnology Biological and industrial applications, inhibition-based determination of heavy metal ions and pesticides Sports medicine, critical care, food science, biotechnology Determination of phenolic compounds in foods, inhibition-based determination of carbamate pesticides Food science, biotechnology Medical diagnosis, artificial kidney, environmental monitoring Food science, biotechnology Inhibition-based determination of organophosphorus and carbamate pesticides Enzyme used in conjunction with acetycholinesterase Sports medicine, critical care, food science, biotechnology Medical applications Pharmaceutical applications Food industry (garlic-, onions- and leek-derived products) B Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139 Table Selected potential medical uses of immobilized enzymes [1,4,13] Enzyme (EC number) Condition Asparaginase (3.5.1.1) Arginase (3.5.3.1) Urease (3.5.1.5) Glucose oxidase (1.1.3.4) Carbonate dehydratase (4.2.1.1) + catalase (1.11.1.6) Catalase (1.11.1.6) Glucoamylase (3.2.1.3) Glucose-6-phosphate dehydrogenase (1.1.1.49) Xanthine oxidase (1.1.3.22) Phenylalanine ammonia lyase (4.3.1.5) Urate oxidase (1.7.3.3) Heparinase (4.2.2.7) Leukemia Cancer Artificial kidney, uraemic disorders Artificial pancreas Artificial lungs Acatalasemia Glycogen storage disease Glucose-6-phosphate dehydrogenase deficiency Lesch–Nyhan disease Phenylketonuria Hyperuricemia Extracorporeal therapy procedures OH HO O NH C=O CH3 OH HO NH2 OH HO O O OH 129 OH O HO Chitin O NH C=O CH3 OH O HO O NH2 OH O HO OH O OH O HO OH O O NH2 OH O HO O NH C=O CH3 Chitosan O HO O O O OH Cellulose Why immobilize enzymes on chitin- and chitosan-based materials? The properties of immobilized enzymes are governed by the properties of both the enzyme and the support material [4,6] The interaction between the two lends an immobilized enzyme specific physico-chemical and kinetic properties that may be decisive for its practical application, and thus, a support judiciously chosen can significantly enhance the operational performance of the immobilized system Although it is recognized that there is no universal support for all enzymes and their applications, a number of desirable characteristics should be common to any material considered for immobilizing enzymes These include: high affinity to proteins, availability of reactive functional groups for direct reactions with enzymes and for chemical modifications, hydrophilicity, mechanical stability and rigidity, regenerability, and ease of preparation in different geometrical configurations that provide the system with permeability and surface area suitable for a chosen biotransformation Understandably, for food, pharmaceutical, medical and agricultural applications, nontoxicity and biocompatibility of the materials are also required Furthermore, to respond to the growing public health and environmental awareness, the materials should be biodegradable, and to prove economical, inexpensive Of the many carriers that have been considered and studied for immobilizing enzymes, organic or inorganic, natural or synthetic, chitin and chitosan are of interest in that they offer most of the above characteristics Chitin and chitosan are natural polyaminosaccharides [14–28], chitin being one of the world’s most plentiful, renewable organic resources A major constituent of the shells of crustaceans, the exoskeletons of insects and the cell walls of fungi where it provides strength and stability, chitin is estimated to be synthesized and degraded in the biosphere in the vast amount of at least 10 Gt each year Chemically, chitin is composed of ␤(1 → 4) linked 2-acetamido-2-deoxy-␤-d-glucose units Fig Structure of chitin, chitosan and cellulose (or N-acetyl-d-glucosamine) [14], forming a long chain linear polymer (Fig 1) It is insoluble in most solvents Chitosan, the principal derivative of chitin, is obtained by N-deacetylation to a varying extent that is characterized by the degree of deacetylation, and is consequently a copolymer of N-acetyl-d-glucosamine and d-glucosamine Chitin and chitosan can be chemically considered as analogues of cellulose, in which the hydroxyl at carbon-2 has been replaced by acetamido and amino groups, respectively Chitosan is insoluble in water, but the presence of amino groups renders it soluble in acidic solutions below pH about 6.5 It is important to note that chitin and chitosan are not single chemical entities, but vary in composition depending on the origin and manufacture process Chitosan can be defined as chitin sufficiently deacetylated to form soluble amine salts, the degree of deacetylation necessary to obtain a soluble product being 80–85% or higher Commercially, chitin and chitosan are obtained at a relatively low cost from shells of shellfish (mainly crabs, shrimps, lobsters and krills), wastes of the seafood processing industry [15,18,20,22–24] Basically, the process consits of deproteinization of the raw shell material with a dilute NaOH solution and decalcification with a dilute HCl solution To result in chitosan, the obtained chitin is subjected to N-deacetylation by treatment with a 40–45% NaOH solution, followed by purification procedures Thus, production and utilization of chitosan constitutes an economically attractive means of crustacean shell wastes disposal sought worldwide Chitosan possesses distinct chemical and biological properties [14–28a] In its linear polyglucosamine chains of high molecular weight, chitosan has reactive amino and hydroxyl groups, amenable to chemical modifications [14,18,19,23] Additionally, amino groups make chitosan a cationic polyelectrolyte (pKa ≈ 6.5), one of the few found 130 B Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139 in nature This basicity gives chitosan singular properties: chitosan is soluble in aqueous acidic media at pH < 6.5 and when dissolved possesses high positive charge on –NH3 + groups, it adheres to negatively charged surfaces, it aggregates with polyanionic compounds, and chelates heavy metal ions Both the solubility in acidic solutions and aggregation with polyanions impart chitosan with excellent gel-forming properties Along with unique biological properties that include biocompatibility, biodegradability to harmless products, nontoxicity, physiological inertness, remarkable affinity to proteins, hemostatic, fungistatic, antitumoral and anticholesteremic properties, chitin and chitosan, as yet underutilized, offer an extraordinary potential in a broad spectrum of applications which are predicted to grow rapidly once the standardized chitinous materials become available Crucially, as bio- and biodegradable polymers chitin/chitosan materials are eco-friendly, safe for humans and the natural environment Increasingly over the last decade chitin- and chitosanbased materials have been examined and a number of potential products have been developed for areas such as [14,17,19,23,24,27,28b] wastewater treatment (removal of heavy metal ions, flocculation/coagulation of dyes and proteins, membrane purification processes), the food industry (anticholesterol and fat binding, preservative, packaging material, animal feed additive), agriculture (seed and fertilizer coating, controlled agrochemical release), pulp and paper industry (surface treatment, photographic paper), cosmetics and toiletries (moisturizer, body creams, bath lotion) But owing to the unparalleled biological properties, the most exciting uses of chitin/chitosan-based materials are those in the area of medicine and biotechnology [16,20–22,28a] In medicine they may be employed as bacteriostatic and fungistatic agents, drug delivery vehicles, drug controlled release systems, artificial cells, wound healing ointments/dressings, haemodialysis membranes, contact lenses, artificial skin, surgical sutures and for tissue engineering In biotechnology on the other hand, they may find application as chromatographic matrices, membranes for membrane separations, and notably as enzyme/cell immobilization supports As enzyme immobilization supports chitin- and chitosanbased materials are used in the form of powders, flakes and gels of different geometrical configurations Chitin/chitosan powders and flakes are available as commercial products among others from Sigma-Aldrich and chitosan gel beads (Chitopearl) from Fuji Spinning Co Ltd (Tokyo, Japan) Otherwise the chitinous supports are laboratorymanufactured Preparation of chitosan gels is promoted by the fact that chitosan dissolves readily in dilute solutions of most organic acids, including formic, acetic, tartaric and citric acids, to form viscous solutions that precipitate upon an increase in pH and by formation of water-insoluble ionotropic complexes with anionic polyelectrolytes In this way chitosan gels in the form of beads, membranes, coatings, capsules, fibres, hollow fibers and sponges can be manufac- tured Commonly, different follow-up treatments and modifications are applied to improve gel stability and durability The methods of chitosan gel preparation described in the literature can be broadly divided into four groups: solvent evaporation method, neutralization method, crosslinking method and ionotropic gelation method [15,20,21,23–27] 3.1 Solvent evaporation method The method is mainly used for the preparation of membranes and films, the latter being especially useful in preparing minute enzymatically active surfaces deposited on tips of electrodes A solution of chitosan in organic acid is cast onto a plate or an electrode tip and allowed to dry, if possible at elevated temperature (ca 65 ◦ C) Upon drying the membrane/film is normally neutralized with a dilute NaOH solution and crosslinked to avoid disintegration in solutions of pH < 6.5 A crosslinking agent may also be mixed with the initial chitosan solution before drying Enzymes may be immobilized on such prepared membranes either on their surfaces by adsorption, frequently followed by crosslinking (reticulation), or covalent binding, commonly preceded by chemical activation of the surface, or included into chitosan solution to achieve inclusion Spray drying is a variant of the solvent evaporation method allowing the preparation of beads smaller in size than those prepared with the other methods [44] 3.2 Neutralization method If an acidic chitosan solution is mixed with alkali, an increase in pH results in precipitation of solid chitosan This method is exploited to produce chitosan precipitates, membranes, fibers, but foremost spherical beads of different sizes and porosities These are obtained by adding a chitosan solution dropwise to a solution of NaOH, most frequently prepared in water-ethanol mixtures, where ethanol, being a non-solvent for chitosan, facilitates the solidification of chitosan beads Following the preparation, the beads are commonly subjected to crosslinking Enzyme immobilization, similar to the solvent evaporation method, is achieved by binding onto the gel surface by adsorption, reticulation or covalent binding, or by inclusion if the enzyme is dissolved in the initial chitosan solution 3.3 Crosslinking method In this method an acidic chitosan solution is subjected to straightforward crosslinking by mixing with a crosslinking agent, which results in gelling Gels obtained in bulk solution are later crushed into particles To obtain gel membranes, the chitosan solution cast on a plate is immersed in a crosslinking bath, and to obtain beads the solution is added dropwise therein In the case of electrodes, crosslinking treatment is frequently done upon covering the tip of the electrode with chitosan solution Clearly, immobilization of Table Enzymes immobilized on chitin- and chitosan-based materials Application Support (preparation method) Immobilization Reference Acid phosphatase (3.1.3.2) F Hydrophobic interaction chromatography; I Mercapto-chitin powder Chitosan beads (b) Chitosan precipitate (b) I III, IV III [29] [30,31] [32] Alanine dehydrogenase (1.4.1.1) E Determination of l-alanine (medicine) Chitosan beads III [33] Alkaline phosphatase (3.1.3.1) F Hydrophobic interaction chromatography C Molecular cloning Chitosan precipitate (b) Chitosan beads (c) III III [32] [34] Alkaline protease (3.4.21.62) B Production of laundry detergents Chitin powder Chitosan powder III (78%)a I (15%), III [35] [35] H Ester and peptide synthesis; transesterification Chitosan beads I [36] Alcohol dehydrogenase (1.1.1.1) I Chitosan beads Chitosan membrane (a) III (25%) III, IV [37] [38] Alcohol oxidase (1.1.3.13) Aminoacylase (3.5.1.14) E Determination of ethanol B Production of l-phenylalanine Chitosan beads Chitosan-coated alginate beads (d) III V (>100%) [39] [40] ␣-Amylase (3.2.1.1) A Hydrolysis of starch for glucose syrup and E for BOD analysis in waters F Hydrophobic interaction chromatography Chitin powder Chitosan beads Chitosan precipitate (b) Chitosan microbeads (a) III (38%) [41] I III V [41,42] [43] [32] [44] ␤-Amylase (3.2.1.2) A Production of high maltose syrup from starch Chitosan beads I [45] ␣-l-Arabinofuranosidase (3.2.1.55) A Aromatization of musts, alcoholic beverages and fruit juices Bromelain (3.4.22.32) Carbonic anhydrase (4.2.1.1) I I Chitosan powder Glyceryl-chitosan powder Chitosan particles (c) Chitosan beads Chitosan-coated alginate beads (d) I, II (3.2%) [47], III II V IV V [46–48] [46] [49] [50,51] [52] Catalase (1.11.1.6) A Removal of H2 O2 from food C Treatment of hyperoxaluria I Chitosan powder Chitosan film (a) Chitosan membrane (a) Chitosan-organosilane particles (c) Chitosan beads (c) I, IV, II V III (4%) I V [53a,b] [54] [55] [56] [57] Cellulase (3.2.1.4) A Decrease in viscosity of fruit/vegetable juices F Affinity chromatography Chitin powder Chitosan beads (b) Chitosan solution IV (15%) I protective additive [58] [59] [60] Chitosanase (3.2.1.132) I Chitin powder III [61] ␣-Chymotrypsin (3.4.21.1) H Ester and peptide synthesis F Preparation of trypsin-free chymotrypsin Chitin film Chitosan beads Chitosan-magnetite beads II I I [62] [36] [63] Creatinine deaminase (3.5.4.21) D Creatinine biosensor (medical diagnosis) Chitosan membrane (a) I, III [64] B Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139 Enzyme (EC number) 131 132 I Chitosan powder I (3.5%), IV(5.2%) [65] Dextranase (3.2.1.11) C Partial hydrolysis of dextran for preparation of blood substitutes and B of dentifrices Chitin powder and colloidal chitin Chitosan powder I, III I, III (63%) [66] [66] endo-1,4-␤-Xylanase (3.2.1.8) C Conversion of hemicelluloses (pulp industry) Chitosan powder Chitosan beads Chitosan-xanthan beads (d) I (24%) III (20%) V (180%) [70] [67] [67] [68–70] Ficin (3.4.22.3) Galactose oxidase (1.1.3.9) I D Galactose biosensor Chitosan beads Chitosan membrane (a) IV III [50] [71a] ␣-Galactosidase (3.2.1.22) A Raffinose hydrolysis in beet molasses C Blood group specificity; Fabry disease Chitin powder IV (67%) [71b,c] ␤-Galactosidase (3.2.1.23) A Hydrolysis of lactose (lactose-free dairy products) Chitin powder Chitosan powder Chitosan beads (b) Chitosan beads Chitosan-polyphosphate beads (d) Chitosan precipitate (b) III III III (100%) [75] I, III V II [72,73] [74] [75,76] [77–79] [80] [81] Glucoamylase (3.2.1.3) A Hydrolysis of starch (ethanol production) Chitin powder Chitosan magnetite beads (c) Chitosan powder Chitosan beads III I I I, III [42] [63] [82] [83] Glucose oxidase (1.1.3.4) D Glucose biosensors E Determination of glucose Chitin powder ␤-Chitin membrane (coagulation) Chitin film (coagulation) Chitosan beads Chitosan membrane (a) Chitosan membrane (a, c, d) Sol–gel/chitosan membrane (c) Chitosan-organosilane particles (c) Chitosan beads-liposomes I V I III III V V I III [84] [85,86] [87] [88] [71a,89a,89b] [90–93a] [93b] [56,93c] [94] ␣-Glucosidase (3.2.1.20) A Hydrolysis of maltose (food/feed additives) Chitosan beads III [95] ␤-Glucosidase (3.2.1.21) A Wine making and juice processing F Hydrophobic interaction chromatography Chitosan Chitosan Chitosan Chitosan Chitosan Chitosan Chitosan III, II (29%) [98] V III (60%), IV I (90%) III, IV III I [48,96–98] [49,99] [100] [101] [31] [32] [63] Glutamate dehydrogenase (1.4.1.2) E Glutamate determination (food industry and medicine) Chitosan membrane (a) Succinyl-, glutaryl-, phtalyl-chitosan membranes (a) III IV [102] [102] Glutamate oxidase (1.4.3.11) D Glutamate biosensor Chitosan membrane (a) III [71a] powder particles (c) flakes solution beads (b) precipitate (b) magnetite beads (c) B Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139 Cyclodextrin glycosyltransferase (2.4.1.19) Table (Continued ) Enzyme (EC number) Application Support (preparation method) Immobilization Reference ␤-Glycosidase (3.2.1.group) A Cellobiose hydrolysis for glucose production Chitosan powder Chitosan precipitate (b) II II [103] [104,105] Horseradish peroxidase (1.11.1.7) D H2 O2 biosensor; E determination of H2 O2 B Oxidative polymerization of aniline G Removal of phenols from petroleum refinery wastewaters E Inhibition-based determination of Hg(II) Chitosan powder Chitosan beads Chitosan membrane (c) III, IV (62%) [106b] III III [106a,b] [39,88,107] [108] Chitosan film (a) Chitosan solution Silica sol–gel chitosan film (c) Chitosan-carbon film (a) V Protective additive I I [54,109,110] [111] [112–114] [115] Chitosan powder Chitosan solution Chitosan microbeads (a) Chitosan-organosilane particles (c) Chitosan-magnetite beads (c) I (91%), III (44%), IV (70%) Protective additive V I I [116] [117] [44] [56] [63] Isoamylase (3.2.1.68) A Hydrolysis of starch (glucose and maltose) Chitin powder III (46%) [118,119] Laccase (1.10.3.2) B Pulp and paper industry G Removal of phenols from effluents Chitosan precipitate (b) V, II (45%) [121] [120,121] Lactate oxidase (1.13.12.4) Leucine dehydrogenase (1.4.1.9) D Lactate biosensor E Determination of l-leucine (medicine) Chitosan-enzyme beads (d) Chitosan beads V III [122] [33] Limonoid glucosyltransferase (2.4.1.210) A Debittering of citrus juice Chitosan powder Chitosan precipitate (b) III III [123] [123] Lipase (3.1.1.3) H Esterifications and transesterifications B Hydrolysis of olive oil Chitosan flakes Chitosan beads Chitosan beads Chitosan-polyphosphate beads (d) Chitosan membrane (a) Chitosan-PVA membrane (a) Chitosan-xanthan beads (d) I (7.1%) I (14.7%) [124], IV IV + II (91.5%) V (42–50%) V, III (47%) [130] V V (90–99%) [124] [124–126a,c] [126b] [127,128] [129,130] [129] [131–133] Lysozyme (3.2.1.17) F Affinity membrane chromatography A Cheesemaking Microporous chitin membrane (a) Chitosan powder PHEMA-chitosan membranes microporous chitin membrane (a) I I (10%) I [134] [135] [136–138] Neutral proteinase (3.4.24.28) Nucleoside phosphorylase (2.4.2.1) -Nucleotidase (3.1.3.5) Octopine dehydrogenase (1.5.1.11) Oxalate oxidase (1.2.3.4) A Hydrolysis of soybean protein E Determination of fish and shellfish freshness E Determination of fish and shellfish freshness E Determination of shellfish freshness C Treatment of hyperoxaluria Chitosan Chitosan Chitosan Chitosan Chitosan II III III III II [139] [140–142] [140,142] [143] [53b] Papain (3.4.22.2) A Removal of “chill haze” in beers; I B Hydrolysis of collagen/keratin (cosmetics) Chitin powder Chitosan beads Chitosan precipitate (b) II IV II (82%) [144] [50,145,146] [147] Pectin lyase (4.2.2.10) A Reduction of fruit/vegetable juices’ viscosity Chitin powder III (26%) [58] precipitate (b) beads beads beads powder 133 A Hydrolysis of sucrose (production of invert sugar) B Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139 Invertase (3.2.1.26) Chitosan beads I (15%) [148] Chitosan precipitate (b) III [32] Pepsin (3.4.23.1) Phospholipase A2 (3.1.1.4) I C Lowering plasma cholesterol level Succinylated chitosan powder Chitosan beads IV (80%) IV (50%) [149] [150] Proteases (3.4.groups) A Casein hydrolysate debittering; I Chitin powder Chitin film Chitosan-xanthan beads (d) III II V [151] [62] [68,133] Pullulanase (3.2.1.41) A Hydrolysis of starch (glucose/maltose syrup) Chitin powder Chitosan-magnetite particles (c) Chitosan powder Chitosan beads III IV I, III I [152] [153] [152] [45] Putrescine oxidase (1.4.3.10) E Determination of meat freshness Chitosan beads III [154] ␣-l-Rhamnopyranosidase (3.2.1.40) A Aromatization of musts, alcoholic beverages and fruit juices Chitin powder Chitosan powder Chitosan particles (c) II III, II V [155] [48,155] [49] Sulfite oxidase (1.8.3.1) D Sulfite biosensor Chitosan-PHEMA membrane (b) I [156,157] Tannase (3.1.1.20) A Hydrolysis of tea tannins Chitin powder and colloidal chitin Chitosan precipitate (b) Chitosan-triphosphate beads (d) III, I III V [158] [158] [159] Transglutaminase (2.3.2.13) A Deamidation of food proteins Chitosan beads III [160] Trypsin (3.4.21.4) F Affinity purification A Hydrolysis of proteins Chitin flakes Chitosan-magnetite particles (c) II, IV (67%) I [161] [162] Tyrosinase (1.14.18.1) C Production of l-DOPA G Detection and removal of phenols Chitin flakes Chitin powder Chitosan flakes Chitosan beads (b) Chitosan-organosilane film (c) Chitosan membrane (a, b) III I (95%) III V (15%) [163], III IV I [163] [164] [163,165] [163,165] [166] [167,168] Urease (3.5.1.5) C D G A Chitosan-triphosphate beads (d) Chitosan beads Chitosan membrane (a) Chitosan-PVA capsules (d) Chitosan-PGMA precipitate (d) Chitosan-coated alginate beads (d) Chitosan-organosilane particles (c) III (64%) III (100%) I, II, III (94%) [172] V I (82%) V I [169] [170] [171–173] [174] [175] [176] [56] Uricase (1.7.3.3) Xanthine oxidase (1.1.3.22) E Determination of uric acid (medicine) E Determination of fish freshness Chitosan membrane (a) Chitosan beads IV III [177] [140–142] ␤-Xylolidase (3.2.1.37) B Production of lignocellulosic fibers Chitosan powder Chitosan beads I (25%) III (33%) [67] [67] Artificial kidney Urea biosensor Treatment of fertilizer effluents Removal of urea from beverages and food Applications are presented in nine cathegories: (A) food industry; (B) industries other than food; (C) medicine; (D) biosensors; (E) enzyme reactors for biosensing; (F) separation, purification and recovery of enzymes; (G) environmental; (H) chemical synthesis; (I) immobilization studies Support preparation methods are presented as: (a) solvent evaporation method; (b) neutralization method; (c) crosslinking method; (d) ionotropic gelation method Commercial powders, flakes or gel beads are not marked Immobilizations are presented as five techniques: (I) adsorption of enzyme on support; (II) adsorption of enzyme on support followed by cross-linking with glutaraldehyde (reticulation); (III) covalent binding of enzyme to glutaraldehyde-activated support; (IV) covalent binding of enzyme to support activated with agents other than glutaraldehyde; (V) gel inclusion a In brackets activity retention is given, if reported B Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139 C Production of pectate oligosaccharides (inducers of flowering and antibacterial agents) F Hydrophobic interaction chromatography 134 Pectinase (3.2.1.15) B Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139 enzymes on such prepared gels does not require chemical activation, as the crosslinker, normally a bifunctional agent, fullfils two functions, crosslinking and activation The enzyme may also be entrapped in the gel if mixed with chitosan prior to crosslinking Overwhelmingly, as a crosslinking and surface activating agent glutaraldehyde is used This is due to its reliability and ease of use, but more importantly, due to the availability of amino groups for the reaction with glutaraldehyde not only on enzymes but also on chitosan Other less frequently employed difunctional agents include glyoxal [30,31,57], tris(hydroxymethyl)phosphine P(CH2 OH)3 [38,100], hexamethylenediamine [65,153], ethylenediamine [116], carbodiimides [102,106b,126b,149], epichlorohydrin [129] and N-hydroxysuccinimide [50,51] A comparatively newly developed method of chitosan gelling is by use of sol–gel processes resulting in chitosanorganosilane hybrid gels The method employs silylating agents, such as (CH3 O)3 Si–R–NH2 [56], (CH3 O)2 CH3 Si–R–O–CO–CH=CH2 [113,166], (C2 H5 O)3 Si–O– C2 H5 [114], however, often regarded simply as crosslinkers 3.4 Ionotropic gelation method (or coacervation) By virtue of the attraction of oppositely-charged molecules, chitosan, owing to its cationic polyelectrolyte nature, spontaneously forms water-insoluble complexes with anionic polyelectrolytes [22,27,69] The anionic polyelectrolytes used include alginate, carrageenan, xanthan, various polyphosphates and organic sulfates or enzymes themselves [122] The method is utilized chiefly for the preparation of gel beads, which is achieved by adding an anionic polyelectrolyte solution dropwise into an acidic chitosan solution Enzyme immobilization is achieved here by preparing an enzyme-containing anionic polyelectrolyte solution prior to gelation The enzyme is immobilized by inclusion in the interior of the beads/capsules An overview of enzymes immobilized on chitin- and chitosan-based materials, reported in the literature over the last decade, is presented in Table It implies that there continues to be vivid interest in utilizing chitin-based materials, predominantly chitosan, as a promising enzyme immobilization support for a multiplicity of applications ranging from the wine, sugar and fish industries, through organic contaminants removal from wastewaters to sophisticated biosensors for both in situ measurements of environmental 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