Influence of different silica derivatives in the immobilization and stabilization of a Bacillus licheniformis protease (Subtilisin Carlsberg)

Abstract

Alcalase 2T, a commercial preparation of Subtilisin Carlsberg, was covalent immobilized onto physiochemically characterized silica supports. The effect of mean pore diameter and surface chemistry on enzyme activity in the hydrolysis of casein has been examined. Two sets of chemically distinct silica supports were used presenting terminal amino (S APTES) or hydroxyl groups (S TESPM-pHEMA). The percentage of immobilized protein was smaller in S APTES (31-39%) than in S TESPM-pHEMA (62-71%), but presented higher total and specific activity. Silicas with large pores (S 1000 , 130/1200 Å) presented higher specific activities relative to those with smaller pore sizes (S 300 , 130/550 Å). The influence of glutaraldehyde concentration and the time of enzyme coupling to the S 1000 S APTES supports was examined. The apparent K m value for the S 1000 S APTES immobilized enzyme is lower than the soluble one which may be explained by the partitioning effects of the substrate. No intraparticle diffusion limitations were observed for the immobilized enzyme and therefore the substrate diffusion does not influence the observable kinetics. Finally, the optimum pH, optimum temperature, thermal stability, operational stability, and storage stability of the immobilized and freely soluble enzymes were compared.

Covalent attachment of cholesterol oxidase and horseradish peroxidase on perlite through silanization: Activity, stability and co-immobilization

“…Different procedures have been used for support preparation and enzyme immobilization on silica supports by using 3-APTS and glutaraldehyde (Blasi et al, 2005;Ettalibi and Baratti, 2001;Ferreira et al, 2003;Kim et al, 2004;Singh et al, 1999). In this study our method is slightly different with previous studies.…”

“…In the first one glutaraldehyde acts as a simple linker and one molecule link amine groups of protein and 3-APTS as shown in Fig. 6 (left) (Blasi et al, 2005;Facci et al, 2002;Ferreira et al, 2003;Paolo Facci et al, 2002;Zhao, 2004). In the second mechanism glutaraldehyde polymerized on the surface and attached to the 3-APTS (Fig.…”

Water‐soluble carbon nanotube‐enzyme conjugates as functional biocatalytic formulations

“…conjugation onto the MWNTs is not surprising given the well-known stabilization of enzymes on solid supports. The increased stabilization can be due to multi-point attachment of the enzyme to the support (Blanch and Clark, 1997;Ferreira et al, 2003) and/or because of decreased proteinprotein interactions on the highly curved surface of carbon nanotubes (Asuri et al, 2006). What is particularly interesting is that the enzyme retains high intrinsic activity along with its increased thermostability while in a soluble form.…”

Purification, immobilization and characterization of linoleic acid isomerase on modified palygorskite

“…However, the activity of the immobilized enzyme decreased at higher glutaraldehyde concentrations. This may be due to the reseau among enzyme favored by the use of glutaraldehyde, which is a bifunctional cross-linking agent and consequently affecting the enzyme activity [30]. a Total enzyme activity and total protein are an average of three parallel replicates and is repented as mean standard deviation b Purification fold is the ratio of specific activity measured at each successive step divided by specific activity of the crude extract…”