Effect of Different Additives on the Structure and Activity of β-Galactosidase Immobilized on a Concanavalin A–Modified Silica-Coated Titanium Dioxide Nanocomposite

Abstract

Interpreting the relationship between the activity and structure of β-galactosidase is necessary to perceive the impact of the enzyme’s conformation on its catalysis. The current study thoroughly explains the effects of additives such as ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), dithiothreitol (DTT), and urea on β-galactosidase activity and structure. β-Galactosidase activity was determined at various ionic strengths and temperatures as a function of time. Structural studies evaluating changes in the secondary and tertiary structures of the enzyme in the presence of the additives were conducted using ultraviolet (UV)-visible and intrinsic fluorescence spectroscopy. The immobilized enzyme showed enhanced stability under different environmental conditions. Activity assays demonstrated concentration-dependent inactivation of β-galactosidase in the presence of SDS and urea, which suggests that hydrophobic and charged residues are present near the active site. In the presence of EDTA, loss in activity was noted, which confirms that β-galactosidase is a metalloenzyme. Enhancement in enzyme activity in the presence of DTT suggests the presence of a cysteine residue near the catalytic center. In UV-visible and intrinsic fluorescence spectroscopy studies, the native enzyme showed significant conformational transitions in the presence of DTT, SDS, and urea and very few changes in the presence of EDTA. However, the immobilized enzyme could resist significant structural changes. In conclusion, this study provides a detailed description of the association between the activity and conformational stability of β-galactosidase.

Glutaraldehyde-pea protein grafted polysaccharide matrices for functioning as covalent immobilizers

“…The K m and V max of A. oryzae β-GLs were also reduced after the covalent linking to GA-copper gelled 28 . Moreover, reduction in K m was also observed after the adsorption of A. oryzae β-GL onto graphene-iron-oxide nano-composites 43 and onto concanavalin A-layered-silica-coated titanium-dioxide 35 . The reduction in K m implied that the β-GL affinity for its substrate increased.…”

“…The β-GL temperature optimum was not affected by its immobilization via the GA-PP-Carr beads. Analogously, the A. oryzae β-GLs temperature optima were unaltered following the covalent binding to GA-whey protein isolate grafted Carr beads 22 and the bio-affinity adsorption onto concanavalin A-layered-silica-coated titanium-dioxide 35 . Moreover, the β-glucosidase-Zn 3 (PO 4 ) 2 hybrid nanoflower and its free compeer manifested similar temperature optima 36 .…”

“…The β-GLs stabilities were also explored in presence of EDTA, a known metal ion chelator 35 . The free β-GL was significantly inhibited via EDTA (P-value < 0.05).…”

Assessing the production of galactooligosaccharides in batch and continuous mode by using β‐galactosidase immobilised on mesoporous silicon dioxide nanoparticles

“…Similarly, the β‐galactosidase immobilised on glutaraldehyde activated SiO 2 NPs retained 87% of original enzyme activity after exposure to 35 °C for 12 h. However, the free enzyme lost its activity completely within 10 h incubation time (Verma et al ., 2012). Shafi & Husain (2022) reported that β‐galactosidase immobilised on concanavalin‐A modified silica‐coated titanium dioxide nanoparticles showed better thermal stability than free enzyme. Contradictorily, Beniwal et al .…”

“…The study also reported a shift in the optimum pH from 6.5 to 7. Verma et al (2012) and Shafi & Husain (2022) also reported a shift in the optima pH from 6.0 to 7.0 and 4.5 to 5.0 after nano-immobilisation. The nanoimmobilised enzyme retained higher activity (82.87% and 39.5%) than its free counterpart (50.26% and 8.7%) at acidic (4.5) and alkaline (8.5) pH, respectively (Selvarajan et al, 2015).…”