Screening of Supports for the Immobilization of β-Glucosidase

Abstract

A set of supports were screened for the immobilization of a partially purified extract of β-glucosidase from Aspergillus sp. These supports, namely, Eupergit, Amberlite, alginate, gelatin, polyvinyl alcohol- (PVA-) based matrices (Lentikats), and sol-gel, have proved effective for the implementation of some other enzyme-based processes. The initial criterion for selection of promising supports prior to further characterization relied on the retention of the catalytic activity following immobilization. Based on such criterion, where immobilization in sol-gel and in Lentikats outmatched the remaining approaches, those two systems were further characterized. Immobilization did not alter the pH/activity profile, whereas the temperature/activity profile was improved when sol-gel support was assayed. Both thermal and pH stability were improved as a result of immobilization. An increase in the apparent K M (Michaelis constant) was observed following immobilization, suggesting diffusion limitations.

Biochemical studies on immobilized fungal β-glucosidase

“…The optimal pH for both immobilized and free forms of the enzyme was 4.0 (data not shown). Roughly similar patterns, where optimum pH is not changed with immobilization, were reported previously (D'Souza and Kubal, 2002;Tu et al, 2006;Figueira et al, 2011). AbdElGhaffar and Hashem, (2010) found that the maximum activity was at pH 7.0 for free cellulase and cellulase immobilized on chitosan-L-glutamic acid-GDA (1%), pH 8.0 for cellulase immobilized on chitosan-4-aminobutyric acid-GDA (1%).…”

“…Calsavara et al (2001) reported that t ½ of free and immobilized cellobiase at 65 ºC were 2.1 and 21.3 h, respectively. This is probably due to the formation of multiple covalent bonds between β-glucosidase and the support that reduce conformational flexibility and thermal vibration, thus preventing the immobilized protein from unfolding and denaturing (Mateo et al, 2000;Wang et al, 2009;Figueira et al, 2011). The deactivation rate constant at 65 ºC for the immobilized β-glucosidase is 9.5x10 -3 / min, which is lower than that of the free form (12.53x10 -3 / min).…”

“…At 65 ºC, free enzyme retained 20% of its initial activity after 1h, whereas the immobilized enzyme conserved 42% of its activity in the same conditions. The immobilization support can have a protecting effect from heat when enzyme inactivation occurs (Chang et al, 2008;Figueira et al, 2011). Figueira et al (2011) reported that immobilization causes an increase in enzyme rigidity, which is commonly reflected by an increase in stability towards denaturation upon raising the temperature ( Table 2).…”

“…The immobilization support can have a protecting effect from heat when enzyme inactivation occurs (Chang et al, 2008;Figueira et al, 2011). Figueira et al (2011) reported that immobilization causes an increase in enzyme rigidity, which is commonly reflected by an increase in stability towards denaturation upon raising the temperature ( Table 2). The time required for the enzyme to come to 50% of its initial activity is indicated by t ½ , which can be calculated from the slope (K) of a semi-log plot as t ½ = 0.693/K.…”

Hybrid Nanomaterials of Magnetic Iron Nanoparticles and Graphene Oxide as Matrices for the Immobilization of β-Glucosidase: Synthesis, Characterization, and Biocatalytic Properties

“…A similar improvement of the stability of bgl has been reported previously, where different materials were used for the covalent immobilization of the enzyme (Ahmed et al, 2013). The observed improvement in the thermal stability of immobilized bgl comparing to the free enzyme may result from the combined action of a reduced molecular mobility and an improved conformational stabilization of the enzyme induced by the covalent bonding of bgl to the nanomaterials (Figueira et al, 2011;Patila et al, 2016a). It is interesting to note that bgl/GO-OA-γFe exhibits significant higher halflife constant than bgl/GO-γFe.…”