Dec 1, 1975

Some properties of a protease (subtilisin BPN') immobilized to porous glass

Biotechnology and Bioengineering
J D Chapman, H O Hultin

Abstract

Subtilisin BPN' was immobilized to porous glass via isothiocyanate coupling. The pH optimum of the enzyme was shifted to the alkaline side on binding. This effect was more pronounced with ethyl lactate than with N-tosyl arginine methyl ester (TAME). Presumably, the shift is a reflection of the negative charge on the surface of the glass. The Michaelis constant and Vmax of soluble subtilisin BPN' with TAME were two and one orders of magnitude, respectively, lower than with ethyl lactate. Vmax, calculated per g of active enzyme, with TAME as the substrate was not affected by immobilization, while Vmax with ethyl lactate decreased greater than tenfold. The apparent KM decreased on immobilization with ethyl lactate as substrate and increased with TAME. Results are explained in terms of diffusional resistance and a possible attraction of ethyl lactate to the glass surface. Active site titration indicated that about 25% of the immobilized enzyme was active.

  • References10
  • Citations4

Mentioned in this Paper

Surface Properties
Glass (Substance)
Lactates
Subtilisin 72
Hydrogen-Ion Concentration
Tosylarginine Methyl Ester

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