Mechano-chemical and biological energetics of immobilized enzymes onto functionalized polymers and their applications

Figures & data

Figure 1. Schematic illustration of various types of immobilizations method. Reproduced with permission from rogriguez-abetxuko et al. 2020., frontier bioengineering biotechnology, 8, 830 [17].

Figure 2. Immobilization of enzymes onto functionalized polymeric matrices and the advantages in terms of improving the physicochemical properties of immobilized enzymes.

Table 1. Advantages/disadvantages of polymeric matrices

S.No	Polymeric matrices	Advantages	Disadvantages
1.	Synthetic polymer	Presence of several functional groups Strong binding of an enzyme Immobilization of greater amount of enzyme	Time-consuming Nonrenewable High production cost
2.	Biopolymer	Nontoxic Cheap High affinity for enzymes Biodegradable Presence of several functional groups Reuseable	The necessity of clearing and proper preparation
3.	Inorganic polymers	High stability Mechanical resistance Good sorption capacity Easy surface functionalization Relatively cheap Inert	Possibility of unspecific interaction Limited biocompatibility Weak enzyme-matrices interaction without crosslinking agents
4.	Composites	Strong affinity for the enzyme Highly stable Limited enzyme leakage	Expensive Limited reusability

Figure 3. The overall scheme of A) immobilization of acrylamidase on chitosan. Reproduced with permission from Bedade et al. 2019., food chemistry, 275, 95–104 .[58]. B) immobilization of lignin peroxidase on sodium alginate beads. Reproduced with permission from Bilal et al. 2019, biocatalysis and agricultural biotechnology, 20,101,205 [59].

Figure 4. Schematic illustration for the immobilization of pectinase on alginate-montmorillonite (MMT) beads b) lineweaver burk plot of free and immobilized pectinase. Reproduced with permission from Mohammadi et al. 2019, international journal of biological macromolecules, 137, 253–260 [71].

Table 2. Kinetic parameters for the enzymatic reactions

S.No	Enzyme	Polymeric matrices	Km (mM)	Kcat (s^−1)	Kcat/Km (mM^−1s^−[1])	Vmax	Reference
1	Laccase	Chitosan	0.33	55.8	169.2	0.14^a	[75]
2	Acid phosphatase	Gelatin	0.3	-	-	2.1 ^b	[76]
3	β-glucosidase	Chitin funcmethanoltionalized nanoparticles	3.31	25.8	7.8	-	[68]
4	Formate dehyrogenase	Polyethylenimine grafted graphene oxide	6.07	3.2	0.5	0.0123^a	[77]
5.	Urease	Carboxymethyl cellulose	14.0	-	-	2.0^b	[60]
6	Laccase	Chitosan	0.12	-	-	597.0^c	[70]
7	Acid phosphatase	Agarose	0.23	-	-	2.3 ^b	[78]
8	Carbonic anhydrase	Polyurethane	12.2	2.0	166.4	-	[79]
9	Lipase	Polymer-grafted silica nanoparticles	2.04	296.0	145.0	15.1^d	[80]
10	Lipase	Magnetic cellulose nanocrystal	12.4	-	-	0.12 ^d	[81]
11	Carbonic anhydrase	Mesoporous aluminosilicates	0.15	1.9	-	2.3 ^e	[82]
12	Invertase	Chitosan	61.3	-	-	177.7 ^b	[83]
13	β-galactosidases	Collagen	6.9	8456.0	1224.0	8670.0^d	[76]
14	Maltase	Agarose	1.91	-	-	6214.0^f	[84]
15	Lactase	Agarose-carboxymethyl cellulose	107.24	36.6	-	-	[74]
16	Laccase	Chitosan-clay composite	0.5	-	-	96.0 ^f	[85]
17	Tyrosinase	Polyamide	1.56	-	–	-	[86]
18	Lipase	Agarose	261.7	1,806,241.8	6900.0	46.3^g	[87]

^aµMmin⁻¹ ml⁻¹

^bµmol⁻¹min⁻¹mg

^cUmL⁻[1]

^dmMs⁻[1]

^emolmin⁻¹ml⁻¹

^fU ml⁻¹ min⁻¹

^gIUml⁻[1]

Figure 5. A) Schematic representation for the synthesis of enzyme inorganic nanoflower/alginate beads. reproduced with permission from Zhao et al. 2017., process biochemistry, 57, 87–94 [114]. B) immobilization of manganese peroxidase on glutaraldehyde-activated gelatin for fruit juice clarification. Reproduced with permission from Bilal et al. 2016., Catalysis letter, 146, 2221–2228 [123].

Table 3. Polymeric matrices immobilized enzyme and biotechnological applications

S No	Enzyme	Source	Renewable matrix	Mode of immobili- -zation	Kcat Free enzyme	Kcat Immobilized enzyme	Enzymatic performance	Application	References
1.	β-galactosidase	Cicer arietinum	Agarose	Adsorption	3.13 × 10^−[^4]	4.15 ×10^−[^4]	High tolerance in acidic and alkaline pH and improved reusability	Lactose hydrolysis	[126]
2.	Pectinase	Bacillus licheniformis	Agar-agar	Entrapment	NA	NA	Retained 80% activity at 40°C and exhibited high reusability for pectin hydrolysis	Fruit juice industry	[127]
3.	Alcohol dehydrogenase	Saccharomyces cerevisiae	Gelatin	Encapsulation	NA	NA	Improved pH tolerance, recycling, and storage stability	Brewing industries	[128]
4.	Phytase	Lactarius volumes	Modified chitosan	Covalent binding	NA	NA	Enhanced catalytic activity and resistance to metal ions	Hydrolysis of phytic acid in cereals	[129]
5.	Lipase	Candida rugosa	Agarose	Covalent binding	NA	NA	Improved stability and retained recycling activity up to four cycles of the reuse	Hydrolysis of fat	[130]
6.	Pectinase	Acinetobacter calcoaceticus	Cellulose	Covalent binding	NA	NA	Enhanced efficiency and reusability	Fruit juice clarification	[111]
7.	β-glucosidase	Bacillus subtilis	Alginate	Entrapment			Improved reusability and storage stability	Sugarcane juice	[88]
8.	Inulinase	Aspergillus tubingensis	Chitosan	Covalent binding	NA	NA	Enhanced stability and more than 70% chicory inulin were hydrolyzed to fructose at 60°C	Production of fructose syrup	[112]
9.	Acrylamidase	Cupriavidusoxalaticus	Chitosan coated calcium alginate	Covalent binding	NA	NA	Better pH and thermal stability	Removal of acrylamide from roasted coffee	[58]
10.	Maltase	B. licheniformis	Agar-agar	Entrapment	NA	NA	Retained 100% of activity after 2 hr at 50°C	Maltose hydrolysis	[124]
11.	Amylase	Bacillus amyloliquefaciens	Alginate	Entrapment	NA	NA	Retained 60% of initial activity after 5 cycles of reuse	Starch hydrolysis	[122]
12.	Pectinase	Bacillus licheniformis	Chitosan	Covalent binding	NA	NA	Enhanced pH and thermal stability	Pectin polymer degradation	[131]
13.	Inulinase	Aspergillus Niger	Chitosan	Covalent binding	NA	NA	Improved pH and temperature tolerance	Inulin hydrolysis	[132]
14.	Glucose oxidase	Aspergillus Niger	Alginate	Encapsulation	NA	NA	Retained 92% of activity at pH 4.0 and reused up to 7 cycles	Reduction of fermentable sugar in the must	[133]
15.	Transglutaminase	Serratia plymuthica	Alginate	Cross-linking	NA	NA	Enhanced stability and isomaltulose yield	Sucrose conversion into isomaltulose	[134]
16.	Pectinase	Aspergillus aculeatus	Calcium alginate beads	Entrapment	16.45	6.07	Excellent operational stability retained 80% of activity up to three cycles of reuse	Fruit juice clarifiation	[135]
18.	Manganese peroxidase	Ganoderma lucidum	Gelatin	Encapsulation	NA	NA	Improved thermal stability and reusability	Fruit Juice Clarification	[123]

Figure 6. Illustration of N-succinyl chitosan preparation and its application strawberry preservation. Reproduced with permission from Niu et al. 2020., Food Control, 106, 829. [125].

Figure 7. a) The scheme of ginger peroxidase (GP) immobilization in ANGG/AGG b) Effluent decolorization in stirred batch reactor and reusability of immobilized GP in ANGG/AGG c) Schematic representation of continuous reactor. Reproduced with permission from Ali et al. 2018., International Journal of Biological Macromolecules, 116, 463–471 [145].

Figure 8. Schematic illustration of hydrogel preparation containing itaconic acid immobilized laccase. Reproduced with permission from Horn et al. 2021., applied polymer materials, 3, 2823–2834 [151].

Figure 9. A) Conversion of CO₂ to methanol using co-immobilization and sequential immobilization methods. B) The schematic representation of the sequential immobilization system. C) production of methanol using a free and immobilized enzyme. Reproduced with permission from Luo et al. 2015., New Biotechnology, 32, 319–327; .[163] and Cen et al. 2019., Advance synthesis and Catalysis, 361, 5500–5515 [164].

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