Table 15.

Supports for carrier‐bound attachment of plant proteases.[ ²⁸⁰ , ²⁸¹ ]

Support	Features	Examples
Agarose beads	• Commercial availability (well‐defined pore size: 45 to 620 nm), inert support. • Compatible with mechanical stirring. • Transparent support (enzyme fluorescence). • Possibility of rigorous control during immobilization process.	• Papain [282] • Ficin [283] • Cardosin A [284] • S. origanifolia protease [285]
Cellulose beads	• Biological polymer (non‐porous nanoparticles or macroporous particles). • Support activation by direct oxidation (sodium periodate) forming di‐aldehyde that reacts with primary amino groups of proteins. • Immobilization process at 45 °C and pH 7. • High proteolytic stability. • Glutaraldehyde or succinic anhydride are used to covalently immobilize the enzyme to the support.	• Papain [286] • N. benthamiana protease [287]
Cotton fabric	• Previous oxidation with sodium periodate is required. • Low loss of enzyme activity. • Low stability through consecutive uses, specially at alkaline conditions or in the presence of detergents. • Optimal pH shifted compared to native enzyme (commonly optimal pH is increased during immobilization).	• Papain [288]
Chitosan	• Polysaccharide derived from chitin rich is hydroxyl groups and glucosamine (weak anion exchanger). • Activating agents are required to obtain a covalent enzyme immobilization (e. g., glutaraldehyde, epichlorohydrin, divinylsulfone, genipin). • High enzyme stability. • High microbial resistance. • Chitosan matrix acts as enzyme photoprotector.	• Mungbean protease [289] • Procerain B [290] • Papain [291] • Bromelain [292] • Ficin [293]
Alginate	• Small pore size of alginate beads of enzyme immobilization is required (low commercial availability). • Alginate can adsorb metal ions from the reaction medium, protecting the enzyme properties. • Low loss of catalytic activity.	• Papain [294] • Araujiain [295] • N. tabacum protease [296] • M. oleifera protease [296] • M. koenigii protease [296] • C. sativum protease [296]
Synthetic organic supports	• The chemical structure of the matrix could be designed (e. g., nylon grafted with polyacrylamide, monofunctional acrylate, polyoxyethylene dimethacrylate, ionic resin exchange, poly‐L‐lactic acid polymeric beads, polyacrylamide) • Activating agents are required (glutaraldehyde, polyethylene glycol, succinic anhydride) • High proteolytic activity. • High thermal stability of immobilized enzyme with remarkable microbial resistance.	• Papain (p(HEMA‐EGDMA)) [297] • Ficin (PVA) [298] • Ficin (Poly(α‐hydroxyacids)) [299]
Polymeric membranes	• Commonly used for industrial applications: pharmaceutics (drug preparation), wastewater treatment (toxic compounds adsorption), biorefinery (hydrolysis reaction), biomedicine (drug delivery) and food processing (food preservation). • Great design reactor flexibility. • Materials support: vinyl alcohol/vinyl butyral copolymer (PVAB), hydroxyethyl cellulose coated with polyethersulfone (PES) hollow fibers • Higher specificity towards substrate than free enzyme. • High thermal stability over a wide range of pH. • Remarkable storage properties and stability. • High‐cost immobilization.	• Papain (PVAB) [300] • Papain (PES) [301]
Smart polymers	• These materials change the solubility/insolubility status depending on the reaction conditions (pH and temperature). • Easy enzyme recovery and reuse. • Support materials: polymethyl methacrylate/N‐isopropylacrylamide/methacrylic acid, poly(styrene/N‐isopropylacrylamide/methacrylic acid. • High bioactivity and affinity towards substrate. • Remarkable enzyme stability after consecutive uses.	• Papain (polymethyl metha crylate/N‐isopropylacrylamide/ methacrylic acid) [302]
Mesoporous silicates	• Well‐defined pore geometry, narrow pore size distribution and large surface area (spherosil, aminoorganosilica activated and porous silica). • High thermal and mechanical stability. • Remarkable dispersion in water and storage stability. • Abundant amount of hydroxyl groups on the surface, thus facilitating the binding of enzymes. • Surface modifying agents are required (e. g., trimethoxy‐derivatives, glutaraldehyde). • Better pH and thermal stability of immobilized protein than the source enzyme. • Low costs associated to materials purchase.	• S. melongena (ZSM‐5 zeolite) [303] • A. curassavica protease (octyl‐glyoxyl‐silica) [304] • Antiacanthain (glyoxyl‐silica) [305] • Granulosain f (glyoxyl‐silica) [305] • Papain (mesoporous silica) [306]
Inorganic oxide supports	• Oxide‐based materials: titanium, aluminum, and zirconium oxides are commonly used for enzyme immobilization. • Attractive material properties, such as: high stability, resistant mechanical strength, good adsorption capacity and high hydrophilicity. • Low‐cost processing.	• Araujiain (TiO2) [307] • C. Linamarase (clay of kaolin) [308] • Papain (Al2O3) [309]
Magnetic particles	• Easy recovery of the biocatalyst after the enzymatic tests using a magnetic field. • Large particles could lead to considerable diffusional limitations. • The surface of ferrite particles (Fe3O4) is commonly activated using thionylchloride to generate reactive chloride groups that interact with the free amino groups of enzymes to form amide bonds. • The immobilized biocatalysts exhibit higher pH, thermal and storage stabilities as well as environmental adaptability and reusability compared to the free enzyme. • Low immobilization costs. • Slight activity loss of biocatalyst during consecutive uses. • Valuable alternative for substrate hydrolysis in suspension.	• Bromelain [310] • C. cardunculus extract (CoFe2O4) [311] • Papain (Ag/CuFe2O4) [312]