Bioconjugation as a smart immobilization approach for α-amylase enzyme using stimuli-responsive Eudragit-L100 polymer: a robust biocatalyst for applications in pharmaceutical industry

Abstract

Enzymes are powerful versatile biocatalysts, however, industrial application of enzymes is usually hampered by their susceptibility. Bio-inspired Eudragit-a-amylase conjugate (E-AC) was proposed as a biocatalyst for various pharmaceutical and industrial applications. In this study, a-Amylase (E.C. 3.2.1.1) was immobilized by covalent conjugation to Eudragit L-100 under mild conditions. The effect of polymer, carbodiimide and enzyme concentrations on optimization of (E-AC) was investigated. In addition, characterization of the free a-Amylase and E-AC with regard to pH, temperature, kinetic parameters, reusability and operational and storage conditions was carried out. Results showed a shift of the optimum pH of E-AC towards the alkaline side whereas, E-AC exhibited higher thermal stability at all tested temperatures. The kinetic parameters, K m values were 2.87 mg/ml and 3.15 mg/ml and V max values were 8.35 mg/ml/min and 8.98 mg/ml/min for free and E-AC, respectively. E-AC retained 85% of the initial activity after five consecutive amylolytic cycles, thus emphasizing its powerful potentials. Operational storage and thermal stability were highly improved as well for E-AC conjugate with an 11.6 stabilization factor in comparison to the free a-amylase. In this study, Eudragit L-100 polymer was successfully used as smart immobilization support to create a reversibly soluble-insoluble enzyme biocatalyst to enforce and extend biotechnological applications of a-amylase in the pharmaceutical industry.

Bio-inspired trypsin-chitosan cross-linked enzyme aggregates: a versatile approach for stabilization through carrier-free immobilization

“…Also, the catalytic efficiency (V max /K m ) of immobilized trypsin was higher than that of the free enzyme (Table 1). Comparable results have been reported by other authors (Yu et al, 2013;Abdel-Mageed et al, 2019).…”

“…The reason for observed loss in enzymatic activity could be due to subjecting the trypsin enzyme to mechanical stress during consecutive centrifugation cycles which might have caused conformational and morphological changes (Dalal et al, 2007;Mahmod et al, 2016). An observed enhancement of enzyme re-usability was reported earlier for different enzymes using various immobilization techniques such as catalase (Abdel-Mageed et al, 2018), α-amylase (Talekar et al, 2012;Abdel-Mageed et al, 2019), and trypsin (Sun et al, 2013;Mahmod et al, 2016).…”

“…As the K m values were of the same magnitude, we concluded that the immobilization process employed did not impair the catalytic function of the enzyme. The results obtained for the kinetic study indicated lower affinity of the enzyme towards the substrate (Mahmod et al, 2016;Abdel-Mageed et al, 2019).…”

Metal Affinity Immobilization of the Processive Endoglucanase EG5C-1 from Bacillus subtilis on a Recyclable pH-Responsive Polymer

“…A major advantage of such a smart bioconjugate is that it can not only achieve hydrolysis of insoluble cellulose and separation with a residual insoluble substrate in a soluble state but also achieve recovery from a resultant reaction medium in an insoluble state by simple pH adjustments. Previously, Eudragit polymers have been used to immobilize various enzymes including cellulase, amylase, lipase, and xylanase via covalent and noncovalent methods. − However, Eudragit polymers do not bind cellulase strongly, resulting in low loading capacity and excessive leakage . In contrast, the covalent immobilization of cellulase on Eudragit S100 by carbodiimide coupling improves enzyme loading and reusability significantly but leads to uncontrolled conformational changes and a concomitant major loss of catalytic activity …”

Antioxidant-biocompatible and stable catalase-based gelatin–alginate hydrogel scaffold with thermal wound healing capability: immobilization and delivery approach

“…The t 1/2 values at 50 °C demonstrate the preserving effect of immobilization on CAT activity with t 1/2 values of 46 and 100 min for free CAT and CAT–Gel–Alg, respectively. Thermal inactivation and degradation of enzymes regularly take place at higher temperatures thus exhibiting lower t 1/2 values (Abdel-Mageed et al 2019b ). Extensive application opportunities are expected for immobilized enzymes with improved thermal stability.…”