Info

Fig. 5. 12% SDS-PAGE analysis of the P-galactosidase immobilized on boronic-epoxy-Sepabeads. Lane 1: molecular weight marker; lane 2, desorption of the protein subunits not covalently immobilized on Sepabeads-boronic-epoxy support; lane 3, desorption of proteins from Sepabeads-epoxy-boronate-P-galactosidase optimal derivative and cross-linked with polyaldehyde-dextran. Arrows indicate the position of the P-galactosidase. Experiments were carried out in triplicate. All other specifications were described in Subheadings 2. and 3.

However, this treatment resulted in a decrease of almost 80% in the enzyme activity after 1 h of incubation (see Fig. 6). To reduce the activity decrease, the cross-linking process was carried out in the presence of different agents that could protect the active site of the enzyme, such as lactose (substrate) or galactose (competitive inhibitor). Interestingly, both compounds proved to have a protective effect, but the best result was achieved in the presence of 1Mlactose (see Fig. 7). Nevertheless, even in the presence of this protective reagent, the enzyme activity dropped by about 30% after this chemical modification. Therefore, other parameters such as dextran size, temperature, and incubation times were further optimized. Finally, it was observed that, using a dextran of 71,400 Da for 6 h at 4°C, 95% of the activity was recovered after chemical modification of the derivative. Moreover, this treatment succeeded in stabilizing the quaternary structure of all immobilized multimeric enzymes because no protein band, even using unpurified extracts (neither from the target enzyme nor from other multimeric enzymes), was detected when the cross-linked Sepabeads-boronic-epoxy derivative was boiled in SDS-PAGE (see Fig. 5, lane 3). This derivative retained a very high thermostability (see Fig. 8) and did not release any contaminant protein during the reaction, satisfying the requirements for their industrial use in food technology.

This is a problem if the biocatalysts were to be used in food technology. The optimization of the cross-linking with aldehyde-dextran permitted the full stabilization of the quaternary structure of the enzyme.

Enzyme Immobilization

Fig. 6. Effect of cross-linking with dextran-aldehyde on the activity of ß-galactosidase immobilized onto Sepabeads-epoxy-boronate support. Time course of inactivation of ß-galactosidase-Sepabeads-epoxy-boronate derivative by incubation with aldehyde-dextran. (•) Sepabeads-epoxy-boronate-ß-galactosidase derivative. (■) Sepabeads-boronic-epoxy-ß-galactosidase derivative incubated with aldehyde-dextran as described in Subheading 3.3.

Fig. 6. Effect of cross-linking with dextran-aldehyde on the activity of ß-galactosidase immobilized onto Sepabeads-epoxy-boronate support. Time course of inactivation of ß-galactosidase-Sepabeads-epoxy-boronate derivative by incubation with aldehyde-dextran. (•) Sepabeads-epoxy-boronate-ß-galactosidase derivative. (■) Sepabeads-boronic-epoxy-ß-galactosidase derivative incubated with aldehyde-dextran as described in Subheading 3.3.

■35 20

Was this article helpful?

0 0
Detoxify the Body

Detoxify the Body

Need to Detoxify? Discover The Secrets to Detox Your Body The Quick & Easy Way at Home! Too much partying got you feeling bad about yourself? Or perhaps you want to lose weight and have tried everything under the sun?

Get My Free Ebook


Post a comment