Covalent Immobilization of Invertase on Chemically Activated Poly (Styrene-2-Hydroxyethyl Methacrylate) Microbeads

Abstract

A carrier for invertase enzyme was synthesized from styrene (S) and 2‐ hydroxyethyl methacrylate (HEMA) in the form of microbeads. These poly (styrene‐2‐hydroxyethyl methacrylate), P(S‐HEMA) microbeads were activated by epichlorohydrin (ECH) treatment for covalent immobilization. The free and immobilized invertase were assayed in the hydrolysis of sucrose to glucose, and the obtained results were compared. The optimum pH was 4.5 for free and 5.5 for immobilized invertase. The optimum temperature of invertase shifted from 45C to 55C upon immobilization. For free and immobilized enzymes, kinetic parameters were calculated as 4.1 × 10−3 mol L−1and 9.2 × 10−3 mol L−1for Km, and 6.6 × 10−2 mol L−1 min−1and 4.1 × 10−1 mol L−1 min−1for Vmax, respectively. After 1 month of storage at 4C, free enzyme retained 36% of its initial activity, while for the ECH‐activated P(S‐HEMA) immobilized enzyme, P(S‐HEMA)‐E, this value was observed as 67%. In repeated batch use, i.e., 20 times in 3 days, 78% retention of the initial activity was observed for P(S‐HEMA)‐E system. PRACTICAL APPLICATIONS Immobilization of enzymes are very important for many industrial applications, e.g., food, medicine, pharmacology, etc. Invertase converts sucrose to glucose and fructose, which have wide applications in food industry especially as sweeteners. Glucose–fructose mixture has much lower crystallinity compared to sucrose and therefore used in the production of noncrystallizing jams and creams. They are also used as liquid sweeteners. Immobilization enables repeated use, provides significant reduction in the operation costs, facilitates easy separation and speeds up recovery of enzyme and extends the stability of enzyme by protecting the active material from deactivation. Industrial application of immobilized invertase may decrease the production cost of glucose–fructose mixture because it could be used repeatedly for long periods. Although invertase is not a very expensive enzyme, the technique can also be applied to expensive ones for biotechnological productions.

High sucrolytic activity by invertase immobilized onto magnetic diatomaceous earth nanoparticles

“…At 45 °C the free form was inactivated at a much faster rate than the immobilized form but both enzymatic preparations at 55 °C lost their initial activity after 120 min treatments. We conclude that thermal stability of the mDE-APTES-invertase was better than the free invertase probably due to the covalent bond formation that might reduce the conformational flexibility of the enzyme and make it more stable against temperature changes [28] . Other authors also observed that after immobilization there was an improvement in thermal stability of enzyme [29] , [30] , [31] .…”

“…The mDE-APTES-invertase presented a retained activity around 83% for 120 days storage period whereas the free enzyme lost all of its activity in the same period. The results showed a good storage stability of the immobilized derivative, and this can be attributed to the stability of the enzyme being enhanced by reducing its denaturation rate after immobilization [28] . Akgöl et al [32] also observed an increase of storage stability of the immobilized invertase onto magnetic polyvinylalcohol microspheres.…”

Enzymes in Food Processing: A Condensed Overview on Strategies for Better Biocatalysts

“…However, this often brings along loss of activity during the process of immobilization, due to support binding to critical residues for enzyme activity, and steric hindrance, among others. Examples include the immobilization of α -amylase [ 177 ] and of levansucrase [ 178 ] on glutaraldehyde-treated chitosan beads, through the glutaraldehyde reaction between the free amino groups of chitosan and the enzyme molecule; the immobilization of pectinase onto Amberlite IRA900 Cl through glutaraldehyde cross-linking [ 179 ]; glucoamylase onto dried oxidized bagasse [ 180 ], onto polyglutaraldehyde-activated gelatin [ 181 ], or onto macroporous copolymer of ethylene glycol dimethacrylate and glycidyl methacrylate through the carbohydrate moiety of the enzyme [ 182 ]; glucoamylase or invertase immobilized onto montmorillonite K-10 activated with aminopropyltriethoxysilane and glutaraldehyde [ 183 , 184 ]; and invertase immobilized on nylon-6 microbeads, previously activated with glutaraldehyde and using PEI as spacer [ 185 , 186 ]; on polyurethane treated with hydrochloric acid, polyethylenimine and glutaraldehyde [ 187 ]; on poly(styrene-2-hydroxyethyl methacrylate) microbeads activated with epichlorohydrin [ 188 ]; or on poly(hydroxyethyl methacrylate)/glycidyl methacrylate films [ 189 ]. Within this methodology for immobilization, highlight should be given to the introduction of commercial supports (namely, Eupergit, Sepabeads) with a high density of epoxide functional groups aimed at multipoint attachment, typically with the ε -amino group of lysine, to confer high rigidity to the enzyme molecule, hence enhancing stabilization [ 190 , 191 ].…”

Synthesis and characterization of stimuli-responsive hydrogels: evaluation of external stimuli influence on L929 fibroblast viability

“…One of the most widely studied hydrogel material employed in biomaterial engineering is polymers of 2-hydroxyethyl methacrylate (HEMA) monomer, a kind of thermosetting polymers, that has proven its value with having resistance to degradation and hydrolysis in acidic or basic media [4,5]. Poly(2-hydroxyethyl methacrylate) (p(HEMA))is an intriguing biomaterial displaying significant properties, such as tissue-like elasticity, high diffusion capacity and high water content [6,7] as well as low toxicity [8] and polar properties of HEMA monomer [9]. In this context, p(HEMA) surface modifications have been used to enhance surface biocompatibility by conjugating biorecognition elements.…”