Immobilization and Activity of Pepsin in Silicone Elastomers

Abstract

Pepsin [EC, 3.4.23.1] from Porcine stomach mucosa was immobilized in silicone elastomers utilizing condensation-cure room temperature vulcanization (RTV) of silanol-terminated poly(dimethylsiloxane) (PDMS). Two network precursor chain molar masses were used in this investigation: in pepsin-silicone (A), M n ∼26,000 g mol −1 and in pepsin-silicone (B) M n ∼750 g mol −1 . Tetraethyl orthosilicate (TEOS) was used as the cross-linking agent and dibutyltin dilaurate was used as the catalyst. The activity and stability of free pepsin and pepsin immobilized in PDMS were studied with respect to pH, temperature, cross-link density, solvents and storage time using a hemoglobin assay. A notable finding is that free pepsin has zero activity in neutral buffer solution (pH 7) after incubation for 5 h, while pepsin immobilized in the silicone elastomers was found to retain more than 70% of its maximum normalized activity. There was no marked improvement in the thermal stability of the PDMS immobilized pepsin when compared to free pepsin and all the three systems showed no activity at and above 70°C. From the Lineweaver-Burk kinetic analyses, the apparent K m (g L −1 hemoglobin) for free pepsin was 4.5, for pepsinsilicone (A) was 5.1, and for pepsin-silicone (B) was 3.9, the V max (U/mg of pepsin) for free pepsin was 14,000, for pepsin-silicone (A) was 11,710, and for pepsin-silicone (B) was 8,510, respectively after incubation in buffer solution at pH 2 and 37°C. The activity of the free and the PDMS immobilized pepsin in six different organic solvents was also studied. The pepsin retained high activity in non-polar solvents such as n-hexane, isooctane and toluene, but the enzyme performed poorly in methanol, ethanol and tetrahydrofuran. The degree of swelling of the pepsin immobilized silicone elastomers in these solvents had no impact on the activity of the pepsin. When stored at room temperature for time periods up to 6 months, pepsin immobilized in silicone elastomers was observed to retain its full activity. The results reported herein demonstrate that cross-linked PDMS is a promising support material for the immobilization of hydrolytic enzymes such as pepsin.

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