Optimal Immobilization ofβ-Galactosidase ontoκ-Carrageenan Gel Beads Using Response Surface Methodology and Its Applications

Abstract

β-Galactosidase (β-gal) was immobilized by covalent binding on novel κ-carrageenan gel beads activated by two-step method; the gel beads were soaked in polyethyleneimine followed by glutaraldehyde. 22 full-factorial central composite experiment designs were employed to optimize the conditions for the maximum enzyme loading efficiency. 11.443 U of enzyme/g gel beads was achieved by soaking 40 units of enzyme with the gel beads for eight hours. Immobilization process increased the pH from 4.5 to 5.5 and operational temperature from 50 to 55°C compared to the free enzyme. The apparent K m after immobilization was 61.6 mM compared to 22.9 mM for free enzyme. Maximum velocity V max was 131.2 μmol·min−1 while it was 177.1 μmol·min−1 for free enzyme. The full conversion experiment showed that the immobilized enzyme form is active as that of the free enzyme as both of them reached their maximum 100% relative hydrolysis at 4 h. The reusability test proved the durability of the κ-carrageenan beads loaded with β-galactosidase for 20 cycles with retention of 60% of the immobilized enzyme activity to be more convenient for industrial uses.

Chemical characterization of Levan and optimization of immobilized Bacillus tequilensis levansucrase onto 𝜅-Carrageenan –CMC Gel Beads.

“…The observed R 2 was in reasonable agreement with the adjusted R 2 of 0.887. This result confirmed a satisfactory adjustment of the quadratic model of the experimental data [19]. All the previous results reflected the applicability and accuracy of the central composite design for optimization of levansucrase immobilization process.…”

“…The K m value for free and immobilized levansucrase was 2.5 and 2.85 mg/ ml, respectively. An increase in K m after enzyme immobilization could be back to the low substrate diffusion rate to the enzyme active site [19]. The increase in K m value after immobilization was mentioned by many other investigators [30].…”

“…Optimization of loading capacity and loading time of partially purified levansucrase on the Carr. CMC gel beads were carried out by using 2 2 fullfactorial central composite design [19,25] with four-star points (±∞) and three replicates at the center point. Design matrix of 11 trials experiment (Table 2) shows the coded and actual values.…”

“…The RSM was identified as a proper method detecting the optimal conditions or the region that fits the operation specification. The optimum conditions for enzyme immobilization have been reported previously [19].…”

Role of Glutaraldehyde in Imparting Stability to Immobilized β-Galactosidase Systems

“…Km value for immobilized enzyme increased from 22.9 mM (for free enzyme) to 61.6mM while _max value was decreased from 177.1 _mol/min (for free enzyme) to 131.2mol/min. The full conversion experiment showed that the developed IβGS was active as compared to the free enzyme, and achieved 100% lactose hydrolysis after 4h of cycle operation 29 .…”