Boosting the stability of β-galactosidase immobilized onto soy-protein isolate-glutaraldehyde-functionalized carrageenan beads

Abstract

Uncontrolled enzyme-immobilizer interactions were evident after immobilizing β-galactosidase onto soy-protein isolate-glutaraldehyde-functionalized carrageenan beads. Such interactions triggered shortcomings in the immobilized β-galactosidase (iβGL) thermal and storage stabilities. The thermal stability of the iβGL was somewhat lesser than that of the free βGL. Moreover, the iβGL suffered an initial sharp fall-off in its activity after storing it. Thus, approaches were adopted to prevent the occurrence of such uncontrolled enzyme-immobilizer interactions, and accordingly, boost the stability of the iβGL. These approaches involved neutralizing the covalently reactive GA entities via glycine and also altering the functionalizing GA concentrations. Nonetheless, no improvement was recorded in the iβGL thermal stability and this indicated that the uncontrolled enzyme-immobilizer interactions were not mediated via GA. Another approach was then attempted which involved treating the iβGL with lactose. The lactose-treated iβGL (LT-iβGL) presented superior thermal stability as was verified from its smaller kd and bigger t1/2 and D-values. The LT-iβGL t1/2 values were 5.60 and 3.53 fold higher than those presented by the free βGL at 62 and 65 °C, respectively. Moreover, the LT- iβGL presented loftier ΔG than did the free βGL. The storage stability of the LT- iβGL was also superior as it offered 100.41% of its commencing activity on its 43rd storage day. Thus, it could be concluded that lactose prevented the uncontrolled enzyme-immobilizer interactions. Finally, advantageous galacto-oligosaccharides (GOS) were prepared via the iβGL. The GOS were then analyzed with mass spectrometry, and it was shown that their degree of polymerization reached up to 7.

Propelling of Enzyme Activity by Using Different Triggering Strategies: Applications and Perspectives

“…8,9 Also, linking all multimeric enzyme subunits to an immobilizer (easily obtained in the case of dimeric enzymes) will prevent their dissociation and prevent their inactivation. 8,9 The immobilization strategy facilitates the separation and reuse of the enzyme, thereby minimizing or eliminating protein contamination in the reaction medium, which significantly reduces the cost, thus ensuring its continued industrial applicability. 3 This article is licensed under CC-BY 4 Another benefit of utilizing an immobilized enzyme is that it avoids product contamination by the enzyme, which can be very undesirable especially in food technology.…”

“…Disruptions caused by immobilization can increase K i (inhibitory constant) to a greater extent than K m (Michaelis−Menten constant), reducing the effect of inhibition on the enzymatic reaction. 9 According to Garcia-Galan et al, 11 immobilization on a solid immobilizer may also lead to blockage of the enzyme inhibition zone and completely prevent this inhibition. Although immobilization has in many cases been associated with a decrease in enzyme activity or worsening of other catalytic properties, some reports in the literature have reported that immobilization of an enzyme also increases its activity (reaction rate per milligram of enzyme).…”

“…The structure of such enzymes may be disrupted secondary to immobilization, and their mobility may be diminished. In addition, immobilization can change the microenvironment of an enzyme. ,− On the other hand, the immobilization technique can be combined with enzyme purification. Immobilization, for instance, can be used to purify large proteins (>200 kDa) and separate them from smaller proteins (<100 kDa).…”

“…However, to enable protein purification, covalent binding should only be carried out following preferred adsorption. , In addition, immobilization can inhibit enzyme inhibition. Disruptions caused by immobilization can increase K i (inhibitory constant) to a greater extent than K m (Michaelis–Menten constant), reducing the effect of inhibition on the enzymatic reaction . According to Garcia-Galan et al, immobilization on a solid immobilizer may also lead to blockage of the enzyme inhibition zone and completely prevent this inhibition.…”

“…Formation of multipoint covalent bonds between the enzyme and a solid immobilizer will serve to solidify the enzyme structure. Such structural solidification will increase the enzyme stability against disturbing factors such as solvents and increased temperatures. , Also, linking all multimeric enzyme subunits to an immobilizer (easily obtained in the case of dimeric enzymes) will prevent their dissociation and prevent their inactivation. , The immobilization strategy facilitates the separation and reuse of the enzyme, thereby minimizing or eliminating protein contamination in the reaction medium, which significantly reduces the cost, thus ensuring its continued industrial applicability …”