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Review
Application of Immobilized Enzymes in Food Industry
Ekaterina D. Yushkova, Elena A. Nazarova, Anna V. Matyuhina, Alina O. Noskova, Darya
O. Shavronskaya, Vladimir V. Vinogradov, Natalia N. Skvortsova, and Elena Krivoshapkina
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04385 • Publication Date (Web): 25 Sep 2019
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Page 1 of 65 Journal of Agricultural and Food Chemistry
1 Application of Immobilized Enzymes in Food Industry
3 Ekaterina D. Yushkova, Elena A. Nazarova, Anna V. Matyuhina, Alina O. Noskova,
4 Darya O. Shavronskaya, Vladimir V. Vinogradov, Natalia N. Skvortsova*,
5 Elena F. Krivoshapkina*
6
7 ITMO University, Lomonosova Street 9, 191002, St. Petersburg, Russian Federation
8
9 *Corresponding authors:
10 Skvortsova N.N.
11 ITMO University,
12 Lomonosova Street 9, 191002,
13 St. Petersburg, Russian Federation
14 E-mail address: [email protected]
15 Tel: +79119413267
16
17 Krivoshapkina E.F.
18 ITMO University,
19 Lomonosova Street 9, 191002,
20 St. Petersburg, Russian Federation
21 E-mail address: [email protected]
22 Tel: +79819511892
23
24
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ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry Page 2 of 65
25 Abstract: Enzymes are macromolecular biocatalysts, widely used in food industry. In
26 applications, enzymes are often immobilized on inert and insoluble carriers, which increase their
27 efficiency due to multiple reusability. The properties of immobilized enzymes depend on the
28 immobilization method and the carrier type. The choice of the carrier usually concerns the
29 biocompatibility, chemical and thermal stability, insolubility under reaction conditions,
30 capability of easy regeneration and reusability, as well as cost efficiency. In this review, we
31 provide an overview of various carriers for enzyme immobilization, with the primary focus on
32 food industry.
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34 Keywords: Enzyme immobilization, Carriers, Food industry, Enzyme activity.
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51 Introduction
52 Immobilized enzymes are heterogeneous catalysts simply separable from the reaction medium.
53 They allow impeding the reaction at any time, obtaining the pure product without contamination.
54 Furthermore, they can be applied in technological process more than once. Enzymes catalytic
55 activity, thermo- and pH-stability depend on the choice of the support, appropriate
56 immobilization method and technological process conditions.
57 All carriers for enzyme immobilization need to be non-toxic, biocompatible, and insoluble under
58 reaction conditions, have high affinity for protein, required functional groups, and possess
59 thermal stability. Regeneration simplicity, high adsorption capacity, and support availability for
60 the technological production are especially important. Meanwhile, the choice of the carriers is
61 limited considering the safety of their components, the chemical stability, resistance to
62 microorganisms, and other factors essential for food industry and biotechnology.
63 In this review we overview and analyze various carrier materials, which are used for enzyme
64 immobilization, and their influence on biocatalytic properties of the enzymes, applied in food
65 technology and in technology of biological active substances production.
66 Enzyme immobilization methods
67 Many techniques of enzyme immobilization have been developed during few past decades. The
68 most common methods are presented below.
69 Adsorption
70 Adsorption is probably the simplest immobilization method. It involves various non-covalent
71 interactions, including Van der Waals forces, hydrophobic interactions, hydrogen bonds, and salt
72 linkages1. The carrier may be immersed in enzyme solution for physical adsorption, which is
73 based on enzyme-ligand principles of interaction, either the protein may be dried on an electrode
74 surface2,3. This method is cheap, does not require an activation reagent, and also protects
75 enzymes against proteolysis, aggregation, and interaction with hydrophobic interfaces4. The key
76 advantages are easy contact between a support and enzyme, resulting with immobilization in a
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77 few minutes and therefore minimal enzyme conformational changes that leads to the high
78 immobilized molecule activity retention3,5. Among disadvantages of adsorption, there is enzyme
79 leaching from the support because of weak links between the carrier and the enzyme that leads to
80 the substrate contamination6,7. Physical adsorption may also cause the enzyme denaturation
81 depending on the support material surface chemistry.
82 Entrapment and encapsulation
83 These methods involve caging or covering the enzyme in fiber or gel of synthetic polymeric or
84 natural support8–10. Such methods are effective and low cost, provide easy contact between
85 enzymes and substrate along with increase mechanical stability11. On the other hand, entrapment
86 and encapsulation reduce mass transfer of substrate to enzyme because of small matrix pores
87 size12, but big pores allow enzyme leaking from the carriers13. Another disadvantage is enzyme
88 deactivation during the procedure of immobilization, support material abrasion during use, and
89 low loading capacity3. Thus, this problem may be solved by cross-linking agent addition14.
90 Covalent binding/Cross-linking
91 Covalent binding is one of the most investigated and often used methods of enzyme
92 immobilization, which results from covalently bonding of proteins to an insoluble support
93 material (matrix or carrier)4,15. Covalent binds are formed between functional groups of carrier
94 surface and enzymes amino acid residues16. The amino acid functional groups have interactions
95 with amino (NH2), carboxyl (COOH), hydroxyl (OH), and sulphydryl (SH) groups. The reactive
96 functional groups may be added to the carrier directly; either its surface may be modified in
97 order to provide active groups by using functionalizing and activating agents. The first one
98 includes organosilanes, gelatin, and polyethyleneimine (PEI). Among activating agents the most
99 popular are glutaraldehyde (GA), carbonyldiimidazole (CDI), and N-hydroxysuccinimide (NHS)
100 and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)17–19. Without a
101 doubt, glutaraldehyde is the most popular cross-linking agent. It dissolves in aqueous solvents,
102 therefore, it may form stable intra- and inter-subunit covalent bonds17,20–22. This method allows
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103 to reuse the enzymes in more cycles than other immobilization methods13,23, prevent enzymes
104 leakage into reaction environment providing pure products, and increase their thermal
105 stability24,25. However, the covalent binding method decreases the enzyme movement degree and
106 contributes their conformational changes that may lead to the loss of enzyme activity26.
107 Enzymes applied in food industry
108 Immobilized enzymes have wide using in food industry in order to fabricate various products
109 and goods, including the production of flavors, syrups, confectionaries, milk foods, alcoholic and
110 fruit beverage, yeasts for baked goods, whey lactose hydrolysates2,9. Immobilized enzymes
111 application in dairy industry is very important. A lot of people have lactose intolerance and they
112 cannot consume the milk. This problem can be solved by using immobilized lactase, which
113 catalyzes the lactose hydrolysis, for lactose free milk production. Immobilized enzymes are also
114 used for fruit juice clarification and removing the bitterness from citrus fruit juice27. Other main
115 application of immobilized enzymes is using them as biosensors in order to determine the
116 various components content and control the quality of products4,28,29. Immobilized enzymes are
117 also used in food packaging, which is the perspective application, for prolonging the shelf life
118 and improving the packed food quality19,30. Their application also includes production of
119 biodiesel, biofuel cells, polyester, micro-pollutant mitigators, and amino acids27,31. The last one
120 is the main part of food supplements. In food industry, immobilized enzymes can be applied in
121 both continuous and batch processes that is one of their advantages over soluble enzymes2,27.
122 Some examples of immobilized enzymes, which are reported in this review, their food
123 substrates/food products are presented in Table 1.
124 Carrier materials
125 Various materials are used as matrix, carriers, or supports for enzyme immobilization. The
126 choice of them depends on immobilization method and enzyme application. All carrier materials
127 may be classified into organic natural, organic synthetic, inorganic, and hybrid supports (Fig.
128 1)18.
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129 Organic carriers
130 Biopolymers
131 Among the supports for enzyme immobilization, biopolymers have advantages due to their “bio”
132 properties, including bio-functionality, biocompatibility, bio-stability, and biodegradability44.
133 Besides this, biopolymers have high structural and chemical variety, mild synthesis conditions,
134 and they are also available45.
135 Gel biopolymers are the most interesting for enzyme immobilization. Gels are dispersed systems
136 with liquid dispersion medium (water, lower mono- and oligo-alcohol, hydrocarbons), where
137 particles of the dispersion phase form a spatial structural grid. Three-dimensional polymer frame
138 imparts mechanical properties of solids to the gels such as lack of fluidity, ability to keep shape,
139 strength, plasticity, and elasticity.
140 Some early studies were dedicated to the inulinase immobilization, which catalyzes the reaction
141 of inulin hydrolysis into fructose and fructooligosaccharides, on polysaccharides gelling agents
142 such as alginate and carrageenan and by inclusion in polyacrylamide gel (Fig. 2a). Inulinase
143 immobilization allows obtaining of fructose syrup, widely used in confectionery industry and as
144 a component of functional nutrition, reducing the risk of diabetes, caries and obesity. Inulinase
145 may be also applied in the production of butanol, acetone, and ethanol in industries close to food
146 production.
147 In 1977, the experiments of inulinase immobilization, which was selected from mold fungi
148 Penicillium sp., into alginate and carrageenan were carried out. Heterogeneous biocatalysts
149 based on carageenan saved 11.7% of free enzyme activity, while a preservation of inulinase
150 catalytic activity, included into alginate gel, was 35%. A temperature optimum of enzyme
151 activity increased by 5°С46.
152 There are known immobilization methods of β-galactosidase47, α-amylase48,49, laccase50,
153 pectinase51 and glucose oxidase52 into calcium alginate beads. In all cases, enzymes showed 70-
154 90% of initial free enzyme activity and higher thermal stability compared with free enzymes.
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155 Immobilized a-amylase lost only 25% of activity after 10 cycles, glucose oxidase kept 37% after
156 7 cycles, laccase and pectinase saved 70% of initial activity after 3 cycles and 40% after 6 using
157 cycles, respectively.
158 On the other hand, an addition of pullulan to glucoamylase and a-amylase before immobilization
159 on alginate beads leads to a higher immobilization yield (85%) than the free enzymes (25%).
160 Such system also shows better thermal and pH stability, and enhances the reusability of enzymes
161 entrapped in alginate beads53.
162 Gelatin may be also applied as a carrier for enzyme immobilization because of its cheapness,
163 availability, and abundance. It also can stabilize the immobilized enzyme by being a protein
164 itself 54. In 1992, Bayramoglu et al. studied α-amylase immobilization on photographic gelatin
165 films. They observed that immobilized enzymes kept 46.8 and 38.8% of activity after 14 cycles
166 for film strips with chromium sulphate and chromium acetate, which were used as cross-linkers,
167 respectively55,56. Using glutaraldehyde as a cross-linking agent leads to 82.5% of enzyme
168 immobilization. This optimum may be achieved with 10% glutaraldehyde, 20% gelatin, and 1 h
169 hardening time. Higher concentrations of gelatin and glutaraldehyde decrease the percent of
170 immobilization due to steric hindrance57.
171 Higher encapsulation efficiency of β-galactosidase (near 63%) may be received with
172 immobilization in carrageenan beads, which were prepared by an injection-gelation method with
173 polysaccharide (k-carrageenan), used as the gelling agent, and a potassium, which is applied as a
174 cross-linker (Fig. 2b). In addition, enzyme, encapsulated in potassium-carrageenan beads,
175 showed a higher activity than the free enzyme because of the K+ ions stabilization effects on
176 enzyme structure58.
177 Hybrid organic carrier of alginate-gelatin-calcium phosphate was used for β-galactosidase
178 immobilization. Studies have shown that the gelatinous film and calcined shell significantly
179 increase the immobilization efficiency and mechanical stability, reduce the capsule swelling
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180 degree, and prevent the β-galactosidase loss. Preparation, immobilized in capsules of alginate,
181 gelatin and calcium phosphate, showed higher stability to pH and temperature changes11.
182 In order to stabilize fungal α-amylase, enzyme covalent immobilization on chitosan-containing
183 cellulose is used. Thermal stability of immobilized α-amylase compared to native form has
184 increased 3.5 times; resistance to inactivation pH has also increased. The decrease in the
185 inactivation rate constant of the immobilized enzyme indicates a stability increase, comparing
186 with the native enzyme, due to steric factors caused by the formation of azomethine bonds with
187 chitosan and cellulose. It is observed, immobilized enzyme use instead of free α-amylase leads to
188 the barley malt hydrolyzates yield increase in 1.5 times59, which indicates the prospects for its
189 practical application in food industry.
190 Biocompatibility, nontoxicity, and biodegradability allow using chitosan (2-amino-2-deoxy-β-D-
191 glucan) as an immobilization carrier60. However, chitosan fibers and beads are relatively faint
192 and suffer from weak mechanical stability61. In order to improve mechanical properties, chitosan
193 beads were obtained by ionotropic gelation with sodium tripolyphosphate (TPP) that was similar
194 to physical cross-linking. After this step, Na2CO3, a porogen, and glutaraldehyde, chemical
195 crosslinker, were added. It leads not only to increasing mechanical stability, but to a higher
196 activity of β-galactosidase immobilized in this support. Reusability study showed that enzyme
197 kept 59.1% of its initial activity after the 13 work cycles62.
198 An adsorption study of lipase from the fungus Rhizopus niveus on chitosan was carried out.
199 Chitosan is formed by deacetylation of the linear polysaccharide chitin. It is shown that low
200 molecular weight chitosan binds better to lipase and allows saving 41% of the soluble enzyme
201 activity. Adsorption on a high molecular weight carrier (10 kDa) leads to the preservation only
202 24% of the activity. When the biocatalyst is immobilized on a high molecular weight chitosan,
203 the temperature optimum shifts by 8°C to the region of high values and becomes 45°С.
204 However, this effect is not observed with using low molecular weight chitosan. In this way, the
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205 optimum temperature retains the value equaled to 37°С, which is similar to the native enzyme.
206 Both free and immobilized enzymes exhibit the greatest catalytic activity at pH 7.063.
207 Alginate beads, coated with chitosan, were used as carriers for acrylamidase immobilization in
208 order to remove acrylamide from roasted instant coffee. The immobilized enzymes performed
209 higher pH and temperature optimums and provided the preservation 80% of activity after four
210 cycles64.
211 Synthetic polymers
212 Synthetic polymers are ion-exchange resins that are insoluble in nature and have a porous
213 surface. These polymers may attach the enzyme very strong due to their porous structure. Such
214 materials are characterized by the availability, regeneration ease, and matrix resistance to
215 microorganisms. Ion-exchange materials derived from polysaccharides, especially cellulose,
216 have become high popular. For example, cation exchanger carboxymethylcellulose (KM-
217 cellulose) and anion exchanger diethylaminoethylcellulose (DEAE-cellulose) were obtained by
218 treating cellulose with epichlorohydrin and triethylamine65.
219 Covalent immobilization on DEAE A-500 cellulose was performed for the inulinase enzyme.
220 Free inulinase use allows obtaining of 90-95% fructose syrup by the hydrolysis of inulin-
221 containing fructans raw materials. The hydrolysis product of a 5% solution of dahlia inulin with
222 Aspergillus niger mutant 817 inulase covalently immobilized on A-500 DEAE-cellulose is a
223 mixture of 97% D-fructose and 3% D-glucose. Immobilized enzyme exhibits stability at pH 4.5-
224 6.5 and at 30°C, pH 5.0-6.0 at 50°C, and keeps its activity until 60°C, when the optimum
225 conditions of the native enzyme are determined by pH 5.2 and 50°C. Complete inactivation of
226 both immobilized and free inulinase was observed at 75°C46. Covalent immobilization of
227 inulinase from Kluyveromyces sp. Y-85 on macroporous polystyrene beads provides a possibility
228 to hydrolytically decompose a 4.5% solution of Jerusalem artichoke fructans to form a mixture,
229 which has 85% of D-fructose and 15% of D-glucose, with 32 days of enzyme half-life66.
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230 Use of the DEAE cellulose with glutraldehyde as a crosslinking agent for α-amylase
231 immobilization provides maximum (68%) of enzyme efficiency and reusability during 6 cycles
232 keeping 49% of the initial activity. It also leads to increase the temperature optimum and thermal
233 stability of the enzymes from 60° to 70°C67. Similar results with 84% efficiency of α-amylase
234 immobilized on DEAE cellulose were reported by Kikani et.al68.
235 Synthetic polymers are often applied for lipase immobilization. There are studies of support
236 influence on enzyme properties at the same physical adsorption mechanism. As a result, higher
237 reaction conversion was obtained when lipase was adsorbed on the octadecyl methacylate (OM)
238 carrier than on the octadecyl methacrylate (OMC) support. It was explained by the more
239 considerable enzymes amount adsorbed on the OM support (92%) than on the OMC support
240 (56%)69. On the other hand, styrene methacrylate gave the highest specific activity hydrolysis of
241 methyl phenylacetate and triacetin for the most of lipases (from Rhizomucor miehie (RML), from
242 Thermomyces lanuginosus (TLL), the forms A and B from Candida antarctica (CALA and
243 CALB), and of the phospholipase Lecitase Ultra™ (LU))70. Candida antarctica (CALB) lipase
244 was also immobilized on divinyl benzene/acrylate polymer, which possesses high mechanical
245 stability and may be used for multiple times in reactors. This system performed high enzyme
246 activity and provided immobilizations yields more than 90% of the biocatalyst71.
247 Ionites have a framework with an excess charge and a layer of mobile counter-ions in their base.
248 Ion-exchange resins matrix consists of a high-molecular hydrophobic spatial grid where
249 functionally active hydrophilic groups are fixed. The chemisorption cation exchange weak-acid
250 fiber VION KN-1 based on polyacrylonitrile fiber is used for inulase immobilization from
251 Kluyveromyces marxianus Y-303 (Fig. 2c). VION is characterized by an increased rate of
252 sorption and regeneration in comparison with granular materials, high capacity, selectivity, and
253 hydrolytic resistance to the action of acids, alkalis, and other agents. As a result, inulase
254 immobilized on VION KN-1 fibrous ion exchanger retained 27.5% of the free enzyme activity.
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255 During adsorption immobilization on the AM-21-A anion exchanger, this system performed
256 14.5% of activity; and the activity was 17.8% with using strongly basic resin AV-16 GS72.
257 Strong acid sulfonic cation exchanger KU-2, based on polystyrene crosslinked with
258 divinylbenzene, has high chemical and thermal stability, and it does not dissolve in the most
259 organic solvents. Inulinase immobilized on the cation resin KU-2 retained 61.7% of the native
260 enzyme activity73.
261 A strongly basic macroporous anion exchanger AV-17-2P, obtained by reacting styrene and
262 divinylbenzene chloromethyl copolymer with trimethylamine, is characterized by mechanical
263 strength, chemical and biological stability, environmental safety, low prime cost, and sufficient
264 permeability for enzyme and substrate molecules. Inulinase bound to the anion exchanger AV-
265 17-2P possessed 75.5% of the initial native enzyme activity73.
266 When inulase was immobilized on the above carriers, the temperature optimum range shifted by
267 20°C towards higher temperatures and became 70°C. On the other hand, 80% of the enzyme
268 maximum catalytic activity was preserved at a temperature of free enzyme complete inactivation,
269 which is 80°C. However, the immobilized inulase pH optimum preserved 4.5-4.774.
270 Afterwards, the experiment was carried out on the immobilization of inulinase selected from
271 Helianthus tuberosus on KU-2, KU-2-8h and AV-17-2P. It was shown that heterogeneous
272 biocatalyst retained its activity in 10 cycles with using these carriers. Specific catalytic activity
273 of the immobilized enzymes insignificantly changed during the year75.
274 The Duolite A568 anion exchange resin, produced by The Dow Chemical Company, with
275 immobilized Kluyveromyces marxianus YS-1 inulase after partial purification with ethanol and
276 gel chromatography on Sephadex G-100 made it possible to save 90% of the initial enzyme
277 activity at 60°C for 3 hours. The native enzyme activity decreased to 10% in the same
278 conditions. The temperature optimum of the immobilized enzyme was 55°C, which is 5°C higher
279 than soluble enzyme had, and the pH optimum was 5.576.
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280 Synthetic polymer gels are also applied for enzyme immobilization. Immobilization of inulinase
281 from fungi Fusarium oxysporum (optimal temperature is 37°C and pH is 6.0) by inclusion into
282 polyacrylamide gel provided the preservation more than 45% of free enzyme activity at
283 temperature of 45°C and pH 6.2. Immobilized enzyme retained 58% of the activity after storage
284 at 25°C during 96 h. Thermal stability of insoluble inulinase enhanced in the presence of inulin77.
285 In order to optimize immobilization method, inulinase was immobilized in polyvinyl-alcohol
286 hydrogel (capsules) with using glutaraldehyde (GA). It was shown that the presence of GA
287 enhances the biocatalyst thermal stability. The obtained immobilized enzyme showed 80% of the
288 initial activity after 3 months keeping at 4°C78.
289 One of the important directions of β-galactosidase application is a synthesis of galacto-
290 oligosaccharides (GOS), which are related to a group of prebiotics. Prebiotics are indigestible
291 food components stimulated the growth and livelihoods of the symbiotic microflora of the
292 human large intestine. Thus, they have a beneficial effect on the body. PVA-gel is inexpensive,
293 shows excellent physical and chemical properties, and possesses high stability, so it may be used
294 for these purposes. In 2008, the research of β-galactosidase from Aspergillus oryzae inclusion in
295 polyvinyl alcohol capsules of a lenticular form, which allowed keeping 32% of the soluble
296 enzyme catalytic activity, was conducted. Immobilization did not lead to expansion of enzyme
297 pH range, but allowed keeping β-galactosidase stability during 530 h of lactose hydrolysis at
298 45°C. Immobilized enzyme kept the original activity during 14 months at 4°C and pH 4.579.
299 In 2012, the activity of β-galactosidase, enclosed in hydrogel polyvinyl alcohol capsules of a
300 lenticular form and sol-gel carriers obtained from tetramethyl orthosilicate (TMOS), was
301 compared with the free enzyme activity. Encapsulation in PVA alcohol matrix allowed keeping
302 95% of free enzyme activity after 7 using cycles and 51% after 3 months of storage. On the other
303 hand, sol-gel method provided only 39% of catalytic activity and led to a lower lactose
304 conversion rate80,81.
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305 Immobilization of glucoamylase from the mold fungus Aspergillus niger into PVA capsules led
306 to an increment in the enzyme pH optimum and allowed keeping the enzymatic activity for 63
307 days at a temperature of 45°C. Immobilized enzyme kept long-term stability in continuous and
308 batch hydrolysis modes82.
309 Synthetic chromatographic carrier is another example of support for enzyme immobilization.
310 Non-ionogenic sorbents with macroporous structure based on super-stitched polystyrene of the
311 “Syrosorb” series were created by V.А. Davankov and M.P. Zyurupa in the early 70s of the 20th
312 century. They were initially used as materials for chromatography. Styrosorbs possess high
313 sorption capacity compared to other types of organic sorbents, high kinetic characteristics and
314 easy regeneration.
315 The activity of Aspergillus awamori inulases immobilized on Styrosorb was 90% of the free
316 enzyme activity. On the other hand, using styrosorb allowed increasing the optimum range of pH
317 from 4.7-5.0 for free glucoamylase to 4.7-6.2 for immobilized enzyme. Sorption on Styrosorb
318 led to a higher thermal stability, which is reflected with higher deactivation temperature83–85.
319 Simultaneously, the most successful result of Aspergillus awamori glucoamylase
320 (“Glucoavamorin” preparation) was achieved by using Sibunit brand mesoporous carbon carrier,
321 where activity was 94% at temperature of 65-75°С and constant pH 4.5-5.0. The enzyme half-
322 life was 250 hours (30 working days) at 60°C, which indicates the possibility of using this
323 biocatalyst in industrial technologies86.
324 Inorganic carriers
325 Inorganic sol-gel materials
326 The enzyme entrapment in the process of inorganic materials sol-gel condensation can be
327 considered as a special class of biomolecules immobilization in an inorganic matrix. Proteins
328 retain the native structure in the biocomposite, exhibiting enzymatic activity, and the range of
329 conditions for catalytic action are also expanded87.
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330 These matrices are characterized by preparation simplicity, optical transparency, mechanical,
331 chemical, and thermal resistance. The sol-gel technology is a drainless method of nanocomposite
332 materials obtaining, thus, it is also environmentally safe. This method allows excluding
333 numerous washing steps, because compounds used as a raw materials do not pollute the final
334 product with impurities88,89.
335 The important advantages of sol-gel matrices are:
336 • Strong enzymes fixation in the gel structure;
337 • Almost full preservation of the enzymes conformational mobility and their catalytic properties;
338 • The polymer network protective action (e.g. from biodegradation) provided a longer
339 biocatalysts half-life;
340 • Pores size controlled by changing the components ratio and conditions of the reaction;
341 • Permeability, which allows low molecular weight compounds to diffuse through the material
342 (without enzyme loss);
343 • The possibility of modifying the matrix by introducing various compounds.
344 Sol–gel systems based on silicon-containing compounds are the best studied and the most widely
345 used. They were the historical beginning of the sol-gel chemistry processes90. In 1845, Ebelmen
346 obtained a transparent material by slow hydrolysis of silicic ester for the first time. Scientists
347 knew about gel formation during the alkali metal silicates acidification before, but this process
348 did not find the practical use91. Alumina and other compounds are also basically applied for sol-
349 gel carrier creation87.
350 The formation principle of a silicon sol-gel matrix for enzyme immobilization consists in the
351 transition of a silicon alkoxide liquid solution (sol) into a gel in consequence of hydrolysis and
352 polycondensation reactions, with the subsequent transformation into a monolithic composite,
353 powder, or thin film coating. Sol is the colloidal dispersion of solid particles in a liquid. Particles
354 of the dispersed phase are aggregates, consisted of many molecules, which interact with each
355 other due to the dispersion forces of attraction, forming the inorganic polymer structure.
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356 Gradually, a cluster is formed from the polymerizing branched oligomers, and when it reaches of
357 macroscopic sizes, a sol passes into the gel. As a result, the gel consists of a continuous
358 structural polymer network (a solid skeleton), and a continuous liquid phase, which has colloidal
359 particles size (1-1000 nm). For instance, the process of obtaining a sol-gel matrix based on
360 tetraalkoxysilane is the sequential formation of the hydroxyalkoxy derivatives hydrolysis
361 products at the initial stage, and then the products of oxoalkoxy derivatives during
362 polycondensation92.
363 In order to create a bimodal sol-gel network with micro- and nanopores, it is necessary to add
364 organic polymers, which are pore-forming agents (polyethylene glycol (PEG)), to avoid
365 excessive pores reduction during gel aging. Organic functional groups in a silica grid provide
366 structure flexibility by decreasing the degree of cross-linking. There are two reasons for it.
367 Firstly, it occurs due to an increase in the rate of silicon alkoxide precursors hydrolysis with
368 increase of the methyl groups number attached to it. Secondly, the amount of reactive Si-OR
369 bonds in alkylalkoxysilane decreases, which also leads to the cross-linking degree reduction93.
370 The polycondensation processes of silane precursors TEOS (tetraethoxysilane) and MTES
371 (methyltriethoxysilane) are characterized by high speed under conditions of basic catalysis with
372 sodium fluoride in the presence of polyethylene glycol (PEG) in the system. The maximum
373 amount of formed Si–O–Si bonds is observed in the structure obtained with using 50 vol. %
374 MTES, because in this case the quantities of the most active nucleophiles (Si(OR)3CH3) and the
375 most active substrates (Si(OR)4) are equal. At 60°C, it is possible to synthesize ordered silica
376 using MTES, TEOS, and NaOH as a catalyst without adding alcohol to the reaction mixture. On
377 the other hand, with the MTES amount increase amorphous materials are formed. Hybrid silicon
378 dioxide spherical particles can be obtained as a co-solvent from MTES and TEOS in the
379 presence of ethanol under alkaline conditions at room temperature94.
380 Influence of sol-gel carriers entrapment on enzymes stability increase
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381 Enzymes inclusion in sol-gel structures allows increasing their resistance for different physical
382 and chemical factors such as temperature, pH, radiation, aggressive compounds action.
383 For instance, most enzymes in the native form are very sensitive to UV radiation even with a
384 short exposure time. Photodegradation is associated with the effect on enzymes chromophores of
385 phenylalanine, histidine, tyrosine, tryptophan, and cystine in the range of 200-400 nm. Herewith,
386 the radical formation, photoionization, and disulfide bonds rupture occur. The primary,
387 secondary and tertiary protein structures change, fragmentation and aggregation of enzymes
388 occur and, as a result, enzymatic activity decreases. Sol-gel carriers can be effectively used as
389 materials, which prevent UV inactivation and denaturation of biocatalysts95.
390 Photoprotection is based on the action of several factors:
391 • the rigid polymer structure prevents the denaturation development;
392 • the hydration shell, surrounding the enzymes, is an additional protective layer;
393 • the inorganic matrix is impenetrable for short-wave and medium-wave UV radiation;
394 • photoprotective substances (2-hydroxybenzophenone, 2,2'-dihydroxybenzophenone and 2,2'-
395 dihydroxy-4-methoxybenzophenone (2.2'-DH-4-MBP)) can be included in the carrier.
396 Photoprotective molecules can absorb the energy of UV radiation and transform it into thermal
397 energy, which is less destructive for the enzymes, through the photophysical process. The
398 experiment with the three different enzymes (coal anhydrase, acid phosphatase and horseradish
399 peroxidase) confirmed a significant enzymes stability increase in regards to UV radiation (UV-A
400 (320–400 nm), UV-B (280–320 nm), and UV-C (200–280 nm)) after entrapment into a doped
401 sol-gel aluminum oxide matrix. Thus, the photostability of carbonic anhydrase in the boehmite
402 matrix with 2.2'-DH-4-MBP increased 5 times compared with the boehmite matrix without
403 additives and 32 times compared with the native enzyme solution95.
404 Thereby, it becomes possible to apply UV sterilization of enzyme systems used in microbiology,
405 biotechnology and medicine that allows avoiding microbial contamination. Also, protection from
16
406 the destructive action of sunlight may increase the shelf life and biomolecules effectiveness in
407 the composition of biopharmaceuticals and catalysts in food technology.
408 Thermal stability studies of proteins entrapped in an aluminum oxide matrix were carried out
409 with the method of differential scanning calorimetry (DSC). The temperature of ovalbumin
410 denaturation changes by 30°C due to entrapment after three days of aging and reaches 100°C.
411 Similar stabilization is observed for bovine serum albumin. High stabilization is obtained for
412 human serum albumin since the temperature of 54°C to a denaturation temperature of 120°C.
413 The thermal stability study of the therapeutically important (acid phosphatase, asparaginase) and
414 industrial enzymes (proteinase and xylanase) confirmed the high thermostability of the enzyme-
415 alumina system. Thus, when asparaginase is heated in a solution to 75°C, the activity decreases
416 by 72%. On the other hand, it decreases only by 1.9% after entrapment procedure. The activity
417 of xylanase, entrapped in aluminum oxide, enhances with a temperature increase to 80°C, while
418 soluble and entrapped in silica xylanase almost completely loses their activity at this
419 temperature. Denaturation of acid phosphatase in solution completely occurs at temperatures
420 over 70°C. Acid phosphatase, entrapped into aluminum oxide, exhibits an activity increase to a
421 temperature of 60°C, after that activity begins to decrease. However, even at 75°C the activity is
422 still higher than at room temperature87.
423 The source of observed thermal stabilization of enzymes entrapped into sol-gel matrix is the
424 ceramic frame rigidity, which is an obstacle to the polypeptide chains denaturing movement, and
425 density of water molecules retention by walls nanopores, protected the protein from destructive
426 dehydration96.
427 Sol−gel magnetite matrix is a perspective stabilizing magnetic biocarrier for enzyme
428 immobilization. Different enzymes, such as carbonic anhydrase, xylanase, proteinase, were
429 entrapped in iron oxide structure (Fig. 3a). As a result, denaturation temperature of all
430 immobilized enzymes enhanced by 20-27°C compared with free enzymes. One of the main
431 properties of enzyme-ferria matrices is their magnetic responsiveness, due to this they may be
17
432 easily removed from the reaction mixture95. Entrapped amylase showed high thermal and storage
433 stability with losing only 9% of initial enzyme activity after 21 days. It was also observed that
434 immobilized α-amylase activity decreases by only 4% after 10 uses, reflecting a high composite
435 material stability and a prolonged profile of action97.
436 Silochromes and other silicate materials may be applied as supports for enzyme immobilization.
437 The first research of silochrome use for this purpose was reported in 1976, and their application
438 had been developing during two decades after that98,99.
439 Silochromes (aerosilogels, aerosil) are a pure and geometrically homogeneous form of porous
440 amorphous silica with a specific surface area of 70-150 m2/g, which are obtained by suspending
441 pyrogenic silica (aerosil) in water. The particles strength is small, but due to its purity, they are
442 convenient for the development of various modifying methods. The detailed knowledge of these
443 materials, their low cost, and availability provide prerequisites for their use in large-scale
444 industrial processes, including their application as carriers for enzyme immobilization.
445 The lactase, selected from Aspergillus oryzae fungi, on the silochrome surface retained 13.7% of
446 free enzyme activity and 20-22% of activity after immobilization on silica with a bimodal pore
447 distribution at pH 4.5 and 10°C with an equally high adsorption degree. Biporous adsorbent
448 synthesis was carried out in the presence of cationic compounds, which were
449 cetyltrimethylammonium bromide (CTAB) and trimethylbenzene, sodium silicate was used as a
450 source of silicon100.
451 β-galactosidase of the bacterium Bacillus circulan, encapsulated in the matrix of colloidal silicon
452 dioxide during sol-gel process in the presence of nonionic surface-active substance (SAS) Triton
453 X-100 and fructose, was characterized by a stability increase by 28.4 times in comparison with
454 the free form. Thereby, it was concluded that it is rational to use this method in the synthesis of
455 galacto-oligosaccharides101. It was also observed that the stability of enzyme/silica system in the
456 operative conditions of 37 °C and pH 7.4 is clearly increased in comparison with the native
457 enzyme102.
18
458 Flow-through catalytic bioreactors, which were based on mesoporous monoliths, obtained by
459 sol-gel method, were created. Unlike with packed columns, the stationary phase based on
460 monolithic type sorbents is a single homogeneous material with a system of interconnected flow
461 pores. The hydrolytic sucrose decomposition by an inulase in a monolithic bioreactor based on a
462 silica sorbent was characterized by an increase in speed of 1000 times compared with a
463 suspension system, based on a mesoporous silica foam with cellular pore morphology (MCF),
464 and a high affinity for the substrate relative to the native enzyme. This method allowed achieving
465 continuous operation constancy of the microreactor with the initial activity for 2 weeks, and
466 made it also possible to return to the same catalytic activity after 6 weeks at a storage
467 temperature of 4°C103,104.
468 Nanoparticles
469 In 1973, Robinson et al. were the first who applied a magnetic material as a support for enzyme
470 immobilization105. Magnetic nanomaterials allow easy and efficiency removing of immobilized
471 enzymes from the reaction medium with using magnet106,107. Furthermore, modification of
472 magnetic nanoparticles surface by functional groups provides better enzyme attachment to the
473 support, increases the nanoparticles stability, and decreases the accumulation of nanoparticles108.
474 Since then, magnetic nanoparticles have been increasingly using for enzyme immobilization109.
475 Covalent immobilization of Bacillus subtilis lipase A with modified aldehyde tags on Fe3O4
476 monodispersed microspheres leads to higher thermal and operational stability and reusability in
477 comparison with the native enzymes110. The hydrolyzing temperature optimum of immobilized
478 enzymes was 70°C, whereas for free lipase it was 50°C. The recycling of immobilized lipase was
479 also investigated. It was shown that enzyme kept 90% of activity after 10 working cycles. Using
480 of magnetic carriers allows conveniently collecting them in order to simple reuse111. In similar
481 research, Wang et al. achieved 65% of immobilized lipase activity after 7 cycles112. Using starch
482 dialdehyde instead of glutaraldehyde as a cross-linker led to higher storage stability and
19
483 recycling rate compared with glutaraldehyde. Lipase immobilized on such support also showed
484 higher acid-base tolerance and thermal stability113.
485 In other research, inulinase was immobilized on magnetic nanoparticles, prepared by coating
486 Fe3O4 nanoparticles with gluten hydrolysates, which were used as functionalized agents.
487 Immobilized and free enzyme activity was measured by photocolorimetric method with using
488 dinitrosalicylic acid (DNS) and inulin as a substrate. The decrease in the flexibility of the
489 immobilized inulinase due to covalent binding114,115 leads to a higher temperature optimum
490 (45°C) versus 40°C of the free enzyme. Immobilized enzymes also showed great reusability,
491 retaining 70% of initial activity after 12 hydrolysis cycles21.
492 Silica nanoparticles are often used for enzyme immobilization116–118. Goel et al. presented the
493 report of β-galactosidase immobilization on such carriers using glutaraldehyde for support
494 activation. Uniform spherical particles of 15 nm size had increased to 35 nm after silica NPs-β-
495 gal conjugates formation. However, the silica nanoparticles shape did not change upon
496 immobilization process. In order to determine β-galactosidase activity, 2-nitrophenyl-β-D-
497 galactopyranoside was used as a substrate. Immobilized enzyme showed higher stability over
498 native enzyme, retaining 50% of initial activity after 6 h. The silica NPs-β-gal conjugates were
499 also reused 14 cycles with preservation 50% of activity after 9 cycles119. Benival et al. obtained
500 71% of immobilized β-galactosidase activity after 13 cycles, and it slightly decreased after 14
501 and 15 cycles. They also observed the temperature optimum range increase from 30-37°C to 35-
502 42°C for free and immobilized enzyme, respectively117,120.
503 Silica shell, which coats magnetic nanoparticles, does not only protect them from degradation,
504 but may also be applied for following functionalization by different functional groups (Fig. 3d).
505 Fe3O4–SiO2 nanoparticles were amino-functionalized with aminopropyletriethoxysilane
506 (APTES) and then activated by glutaraldehyde. At last, α-amylase was attached to the
507 AFSMNPs by covalent bonds. Immobilized enzymes showed better stability at high temperature,
508 because the structure of such enzyme was largely preserved by temperature changes121. The pH
20
509 optimum of immobilized α-amylase increased comparing with the free enzymes that may be
510 caused by the basic nature of the non-modified free amino groups presented on the silica
511 nanoparticles surface. Immobilized enzymes on such carriers provided great reusability retaining
512 82% and 74% of initial activity after 10 and 20 using cycles, respectively122.
513 Silver nanoparticles have a big potential for biotechnological application. Using glutaraldehyde
514 for support activation leads to retaining 93% of free β-galactosidase activity. This system also
515 shows great storage stability (93% after 2 months), and reusability, keeping 88% of original
516 activity after 6 repeated uses123.
517 Porous ceramic
518 Porous materials (membranes) including Al2O3, ZrO2, SiO2 and TiO2 possess high mechanical
519 and chemical stability124–126. Their production is easy and inexpensive because low cost mineral
520 materials are used as raw ones. Such materials provide a large surface area per volume unit
521 because of the porous structure and presence of microchannels127,128. They are applied in many
522 industries, including biomedicine, pharmaceuticals, and others129,130. Due to the special
523 properties of porous materials such as high pore density, uniform pore size, low cost, and
524 resistance to organic solvents, they are used as supports for enzyme immobilization131. These
525 carriers may be applied in membrane reactors in food industry as a good alternative to batch
526 reactors132–136.
527 Zirconia possesses high thermal, chemical, and pH stability, it is also resistant to mechanical
528 degradation. On the other hand, zirconia contains many hydroxyl groups137, hence it is attractive
529 for fabrication of immobilized enzyme systems138, and biocatalytic reactors139,140. Adsorption
530 method was used for α-amylase immobilization on zirconia (Fig. 3c). Free and immobilized
531 enzymes activity in starch hydrolysis reaction were tested in a batch reactor. Immobilized α-
532 amylase showed the highest stability at pH 7 in comparison with pH 6 for free enzyme. It may be
533 explained by pH optimum increase, reflecting the amphoteric properties of zirconia139. The
534 decrease of sensitivity to pH changes was also observed as a result of immobilization.
21
535 Porous alumina materials with various length and pore size are easily fabricated by high purity
536 aluminum anodization. They are widely used including their application as carrier for enzyme
537 immobilization124. Porous surface possesses great configuration for enzyme molecules
538 multipoint attachment and increases binding strength that improves enzyme immobilization in
539 comparison with a flat surface141,142.
540 Yang et al. studied immobilization of urease contained in soy43,143 by four methods including
541 electrostatic adsorption, electrostatic adsorption and reticulation by glutaraldehyde, electrostatic
542 adsorption followed by chitosan coating, and electrostatic adsorption with reticulation by
543 glutaraldehyde followed by a chitosan coating on nanoporous alumina membranes. The system,
544 obtained by physical adsorption, showed less stability and enzymatic activity due to leaching of
545 enzymes. While chitosan and glutaraldehyde prevented urease leaching and prohibited the urease
546 inactivation by cross-linking effect, respectively144.
547 Alumina membranes may also be applied for lipase immobilization145. There is a method of
548 lipase from Candida Antarctica immobilization on alumina microporous tubular supports with
549 using gelatin/PEI and glutaraldehyde as fuctionalization and activation agents, respectively
550 (Fig. 3b). It was observed that immobilized enzymes showed loss the activity due to
551 conformational changes in enzymes structure, induced by covalent bonds. However, these
552 supports have not the significant changes after 10 uses, thus, there are no regeneration
553 problems22,146.
554 Mesoporous silica is the most studied mesoporous material for enzyme immobilization
555 application. Silica dissolves in bases solutions like KOH and NaOH, and does not dissolve in all
556 acids excluding hydrogen fluoride. Pore size, particle morphology and structure of porous silica
557 materials can be tailored with a high precision degree. They also have large surface area and
558 pores volume that provide the immobilization of a large enzymes quantity. Furthermore, the
559 silica surface may be easily modified with various functional groups that may optimize the
560 enzyme-support interaction124,127.
22
561 Bacillus circulan β-galactosidase, covalently immobilized on meso- and macroporous silica,
562 showed 40-65% of the native enzyme activity depending on the glyoxyle groups concentration
563 on the support. The effect of the carrier surface functional groups number on the heterogeneous
564 catalyst activity and thermal stability was studied, and it was found that the stability of one of the
565 modified samples exceeded 307 times the soluble enzyme stability at pH 6 and 55°C147.
566 Hybrids
567 Hybrids carriers including organic and inorganic parts are alternative systems for enzyme
568 immobilization. Inorganic materials like silica nanostructures are environmentally more
569 admissible, mechanical and thermal more stable, and provide the resistance to microbial
570 contamination148. However, in order to minimize toxicity and overcome the agglomeration or
571 precipitation of magnetite nanoparticles, their surface is needed to be modificated149. Organic
572 compounds as chitosan are antibacterial, biocompatible, and environmentally friendly
573 polyelectrolytes, which also have many free reactive hydroxyl and amino groups on the support
574 surface that provides an opportunity for enzyme immobilization. It makes them perspective
575 carrier material for application in food industry60,150–152. Application of inorganic-organic
576 composites for enzyme immobilization leads to a higher activity and stability in frequent use of
577 the enzymes122.
578 α-amylase immobilized on chitosan-coated amino-functionalized silica-coated magnetite
579 nanoparticles (AFSMNPs) preserved 84.3% of initial enzyme activity after 40 days against 1.2%
580 of free enzyme activity and 82.9% of AFSMNPs without chitosan layer, and 78.6% after 80 days
581 compared with 65.7% of AFSMNPs. Chitosan-coated AFSMNPs also provide better enzyme
582 reusability, showing 91% of initial activity after 10 uses and 85% after 20 cycles122.
583 Glucoamylase immobilization on chitosan covered magnetic nanoparticles (CSMNPs) improved
584 the enzyme loading capacity, because the size of CSMNPs is smaller (50 nm) than chitosan
585 nanoparticles (CSNPs – 258 nm), and the surface area is greater, so the amount of loaded
23
586 enzyme is higher. On the other hand, such size is too small in order to adequately separate these
587 particles from the product solution153 (Fig. 4b).
588 Composite films based on chitosan and nanoclays, i.e. montmorillonite, bentonite, and sepiolite
589 are applied for covalent immobilization of proteolytic enzymes because of their high availability,
590 low cost, high surface area, strong adsorption ability, and high cation exchange capacity154–156.
591 The immobilized protease amount was significantly higher for all nanocomposite films in
592 comparison with clay-free support157. Nanoclay inclusion also enhanced mechanical properties.
593 On the other hand, its supplementation negatively influenced on the enzyme catalytic properties,
594 caused by lower protease affinity to the synthetic support than to the clay-free carrier158.
595 There are some studies of using silica/chitosan composites for application in enzymatic
596 catalysis159,160. This system possesses a porous microstructure with high mechanical strength and
597 NH2 groups of chitosan structure that provide an environment compatible with biomolecules and
598 eliminate surface functionalization steps161,162. Ricardi et al. reported β-galactosidase
599 immobilization on a silica/chitosan hybrid carrier. Chitosan was included in the micrometric
600 regions of silica, and the enzymes were attached only to the chitosan part. Exclusively silica part
601 formed the pore structure. It was observed that the efficiency of enzyme, immobilized on this
602 carrier, was higher compared with the immobilized on organofunctionalized silica enzyme
603 efficiency. Operational stability was determined by lactose hydrolysis reaction in fixed bed
604 reactor. Enzyme retained 90% of activity after 200 h of continuous using163.
605 A hybrid silica-organic/polyvinyl alcohol (PVA) composition may be applied for production of
606 biosensors, which are sensitive to alcohols and glucose, in order to analyze wine products.
607 Glucose oxidase immobilized on bioelectrodes, which are modified with such hybrid
608 composition, shows high stability and allows increasing the range of detected glucose
609 concentration (1.0-5.9 µM) compared with agar gel electrodes (3.6-6.3 µM) 164.
610 Optimization of silica-based sol-gel lipase encapsulation is achieved by mediated with
611 polyethyleneimine (PEI). PEI coating prevents the direct interaction between enzyme and
24
612 surface, protecting the native structure. It also improves biocatalytic behavior of immobilized
613 enzymes, increases their thermal stability and recovered activity165.
614 Enzyme immobilization efficiency on various carriers
615 The choice of carrier material can influence on enzyme stability and immobilization efficiency.
616 However, it is difficult to predict which matrix will be the most suitable for a particular enzyme.
617 Therefore, in each case, a detailed study of all parameters is necessary, starting from the creation
618 conditions to the conditions of catalytic system functioning. Fig. 5-7 and Table 2 show
619 comparative characteristics of various carriers types for enzyme immobilization.
620 Overall, this review covers recent studies of enzyme immobilization on various carriers, used in
621 food industry. All carriers were classified into organic, separated on biopolymers and synthetic
622 polymers; inorganic, which includes porous ceramic and nanoparticles; inorganic-organic
623 composites. The comparison between various supports was performed in this study, and it was
624 also shown, how enzyme properties depend on immobilization on specific carrier. The type of
625 support influences on enzyme activity, temperature and pH optimum range. Due to the
626 possibility of immobilized enzymes being removed from the reaction mixture they may be used
627 multiple times, retaining most of their initial activity. The choice of the carrier also affects on
628 storage stability of enzymes. While one carrier allows keeping the enzyme activity during
629 several days, another one provides the preservation of activity only for few hours. In order to
630 optimize the characteristics of immobilized enzymes for industrial applications, it is important to
631 choose correct support material and immobilization method.
632 Acknowledgement
633 This work was supported by Russian Foundation for Basic Research according to the research
634 project № 18-38-00905.
635 Notes
636 The authors declare no conflict of interests.
637 References
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1197
1198
1199
1200
1201
1202
1203
1204
1205
1206
1207
1208
1209
47
1210 Figure 1. Classification of materials used as carriers for enzyme immobilization
1211 Figure 2. Types of immobilization on organic carriers
1212 Figure 3. Types of immobilization on inorganic carriers
1213 Figure 4. Immobilization on inorganic-organic composites
1214 Figure 5. Activity of immobilized inulinase on various carriers
1215 Figure 6. Activity of immobilized β-galactosidase on various carriers
1216 Figure 7. Activity of immobilized α-amylase on various carriers
1217
1218
1219
1220
1221
1222
1223
1224
1225
1226
1227
1228
1229
1230
1231
1232
1233
1234
1235
48
1236
1237 Figure 1. Classification of materials used as carriers for enzyme immobilization
1238
1239
1240
1241
1242
1243
1244
1245
1246
1247
1248
1249
1250
1251
1252
1253
1254
1255
1256
1257
1258
1259
1260
1261
1262
1263
1264
1265
1266
1267
1268
1269
1270
49
1271
1272 Figure 2. Types of immobilization on organic carriers
1273
1274
1275
1276
1277
1278 Figure 3. Types of immobilization on inorganic carriers
1279
1280
1281
1282
1283
1284 Figure 4. Immobilization on inorganic-organic composites
1285
1286
1287
1288
1289
1290
1291
50
1292
1293 Figure 5. Activity of immobilized inulinase on various carriers46,72,73,76
1294
1295
1296 Figure 6. Activity of immobilized β-galactosidase on various carriers79,100,123,163,166,167,
1297
1298
1299 Figure 7. Activity of immobilized α-amylase on various carriers48,49,67,68,168,169
51
1300 Table 1. Immobilized enzymes in food production: enzyme, food substrate/product, application
1301 in food industry, and references
1302 Table 2. Results of enzyme immobilization on organic and inorganic carriers, applied in food
1303 industry
1304
1305
1306
1307
1308
1309
1310
1311
1312
1313
1314
1315
1316
1317
1318
1319
1320
1321
1322
1323
1324
1325
52
1326 Table 1. Immobilized enzymes in food production: enzyme, food substrate/product, application
1327 in food industry, and references
Food substrate/food
Enzyme Application References
products
Inulinase Inulin/ fructose and Production of fructose syrup,
fructooligosaccharides ethanol, acetone and butanol
32,33
reducing the risk of diabetes,
caries and obesity
β-galactosidase Lactose/ glucose, low-lactose and lactose-free
34–36
(lactase) galactose products production
α-amylase Corn and potato The production of glucose
starch/ and fructose solutions, in the 2
oligosaccharides confectionery industry
Glucoamylase Starch/ The production of glucose
oligosaccharides and fructose solutions, in the
confectionery industry, in the 2,9
juices production and in
brewing
Lipase Triglyceride/ glycerol Juices and wines
and fatty acids clarification, the
improvement of their taste,
the vegetable oils conversion
into margarines, in the
9,37
production of cheeses and
cheese-like products, and also
in breadmaking to slow down
the process of bread
hardening
β-fructofuranosidase Sucrose/ glucose and Prevent the saccharification
(invertase) fructose of various products (jam, 38
confectionery) during storage
L-Phenylalanine- Amino acid L- Production of phenylalanine-
ammonia-lyase (PAL) phenylalanine/ free products
27
transcoric acid and
ammonia
Acrylamidase Acrylamid/ acrylic In biotest in order to
acid and ammonia determine acrylamide content 39–42
in products
Urease Urea/ carbon dioxide In biotest in order to control
43
and ammonia the quality of milk
1328
1329
1330
53
Table 2. Results of enzyme immobilization on organic and inorganic carriers, applied in food industry
Results of immobilization
Group of
Carrier Enzyme Temperature Reusability/Storage References
carriers Activity, %
optimum, °C stability
Carrageenan Inulinase 11,7 +5 - 46
Inulinase 35,0 +5 - 46
α-amylase 80,0 - 75% after 10 cycles 48,49
Laccase - +10 70% after 3 cycles 50

Alginate gel
75% after 48 h, 167
β-galactosidase 47,0 -
36% after 10 days
Pectinase 75,0 - 40% after 6 cycles 51
Biopolymers
Gelatin α-amylase - +5 46,8% after 14 cycles 55,56
59,1% after 13 cycles/ 62

β-galactosidase - +5
94,7% after 28 days
Chitosan 166
β-galactosidase 85,0 - 70% after 9 cycles
Lipase 41,0 - - 63
80% after 4 cycles, 64

Chitosan coated alginate beads Acrylamidase - +5
40% after 7 cycles
α-amylase 68,0 +10 49% after 6 cycles 67
DEAE cellulose
α-amylase 84,0 - 96% after 20 cycles 68
Synthetic
Cation exchanger VION KN-1 Inulinase 27,5 +20 - 72
polymers
Cation resin KU-2 Inulinase 61,7 - - 73
Anion exchanger AV-17-2P Inulinase 75,5 - - 73
54
Anion exchanger Duolite A568 Inulinase 35,6 +5 90% for 3 h 76
Inulinase 45,0 +8 58% for 96 h 77

Polyacrylamide gel
α-amylase 50,0 +20 - 168
60% after 12 cycles/ 78

Inulinase - -
80% after 3 months
β-galactosidase 32,0 - - 79
PVA hydrogel capsules
β-galactosidase - -
51% after 3 months
Glucoamylase 35,0 -20 80% after 100 uses 82
Acid phosphotase
33% after 1 h (AcP), 95
(AcP), carbonic - +20-22
55% after 1 h (CAB)
Sol−gel magnetite matrix anhydrase (CAB)
Inorganic sol-
96% after 10 cycles/
gel α-amylase - - 97
91% after 21 days
materials
Silochrome β-galactosidase 13,7 - - 100
Sibunit Glucoamylase 94,0 +2 70% after 20-40 h 86
Lipase - +20 90% after 10 cycles 111
Magnetite nanoparticles Lipase - - 65% after 7 cycles 112
Inulinase - +5 70% after 12 cycles 21
Inorganic
50% after 9 cycles/
nanoparticles β-galactosidase - - 119
50% after 6 h
Silica nanoparticles 117
β-galactosidase - +5 50% after 11 cycles
β-galactosidase - +2 71% after 13 cycles 120
55
82% after 10 cycles, 74%

Silica shell magnetic 122
a-amylase - +15 after 20 cycles/
nanoparticles
65,7% after 80 days
Silver nanoparticles β-galactosidase 96,0 -
83% after 2 months
Lipase - - 65% after 4 cycles 22
Alumina membranes
Urease - - 50% after 5 cycles 144
Porous β-galactosidase 40-65 - 50% after 18 h 147

ceramic
Mesoporous silica Inulinase - - 90% after 1 month 103
α-amylase 80,0 -10 - 169
91% after 10 cycles, 85%

after 20 cycles/ 122
Chitosan-coated AFSMNPs α-amylase - +15
84,3% after 40 days,
82,9% after 80 days
Hybrids 168
Aminoalkylsilane-alumina α-amylase 13,0 +15 -
Silica/chitosan composites β-galactosidase 45-60 - 90% after 200 h 163
Silica mediated by PEI Lipase 47,0 - - 165
56
For Table of Contents Only
57
Figure 1. Classification of materials used as carriers for enzyme immobilization
170x61mm (299 x 299 DPI)

Figure 2. Types of immobilization on organic carriers
168x60mm (299 x 299 DPI)

Figure 3. Types of immobilization on inorganic carriers
167x55mm (299 x 299 DPI)

Figure 4. Immobilization on inorganic-organic composites
82x42mm (299 x 299 DPI)

Figure 5. Activity of immobilized inulinase on various carriers
83x56mm (299 x 299 DPI)

Figure 6. Activity of immobilized β-galactosidase on various carriers
83x57mm (299 x 299 DPI)

Figure 7. Activity of immobilized α-amylase on various carriers
83x56mm (299 x 299 DPI)

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Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

is published by the American Chemical Society. 1155 Sixteenth Street N.W.,

1 Application of Immobilized Enzymes in Food Industry

3 Ekaterina D. Yushkova, Elena A. Nazarova, Anna V. Matyuhina, Alina O. Noskova,

4 Darya O. Shavronskaya, Vladimir V. Vinogradov, Natalia N. Skvortsova*,

12 Lomonosova Street 9, 191002,

13 St. Petersburg, Russian Federation

14 E-mail address: [email protected]

19 Lomonosova Street 9, 191002,

20 St. Petersburg, Russian Federation

21 E-mail address: [email protected]

25 Abstract: Enzymes are macromolecular biocatalysts, widely used in food industry. In

29 biocompatibility, chemical and thermal stability, insolubility under reaction conditions,

56 immobilization method and technological process conditions.

65 technology and in technology of biological active substances production.

66 Enzyme immobilization methods

68 most common methods are presented below.

70 Adsorption is probably the simplest immobilization method. It involves various non-covalent

78 immobilized molecule activity retention3,5. Among disadvantages of adsorption, there is enzyme

81 depending on the support material surface chemistry.

82 Entrapment and encapsulation

92 immobilization, which results from covalently bonding of proteins to an insoluble support

99 popular are glutaraldehyde (GA), carbonyldiimidazole (CDI), and N-hydroxysuccinimide (NHS)

100 and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)17–19. Without a

107 Enzymes applied in food industry

123 substrates/food products are presented in Table 1.

124 Carrier materials

129 Organic carriers

132 properties, including bio-functionality, biocompatibility, bio-stability, and biodegradability44.

134 and they are also available45.

139 strength, plasticity, and elasticity.

151 activity increased by 5°С46.

152 There are known immobilization methods of β-galactosidase47, α-amylase48,49, laccase50,

157 cycles, respectively.

161 entrapped in alginate beads53.

167 respectively55,56. Using glutaraldehyde as a cross-linking agent leads to 82.5% of enzyme

170 immobilization due to steric hindrance57.

174 cross-linker (Fig. 2b). In addition, enzyme, encapsulated in potassium-carrageenan beads,

176 enzyme structure58.

182 In order to stabilize fungal α-amylase, enzyme covalent immobilization on chitosan-containing

189 practical application in food industry.

190 Biocompatibility, nontoxicity, and biodegradability allow using chitosan (2-amino-2-deoxy-β-D-

211 Synthetic polymers

215 microorganisms. Ion-exchange materials derived from polysaccharides, especially cellulose,

217 cellulose) and anion exchanger diethylaminoethylcellulose (DEAE-cellulose) were obtained by

218 treating cellulose with epichlorohydrin and triethylamine65.

234 immobilized on DEAE cellulose were reported by Kikani et.al68.

260 enzyme activity73.

262 divinylbenzene chloromethyl copolymer with trimethylamine, is characterized by mechanical

265 17-2P possessed 75.5% of the initial native enzyme activity73.

273 of the immobilized enzymes insignificantly changed during the year75.

285 In order to optimize immobilization method, inulinase was immobilized in polyvinyl-alcohol

288 initial activity after 3 months keeping at 4°C78.

289 One of the important directions of β-galactosidase application is a synthesis of galacto-

304 conversion rate80,81.

308 batch hydrolysis modes82.

314 easy regeneration.

319 Simultaneously, the most successful result of Aspergillus awamori glucoamylase

323 biocatalyst in industrial technologies86.