Subtilisin BPN' variants: increased hydrolytic activity on surface-bound substrates via decreased surface activity

Biochemistry
P F BrodeD N Rubingh

Abstract

Site-directed mutagenesis and random mutagenesis were used to produce variants of subtilisin BPN' (Bacillus amyloliquefaciens) protease with variable surface adsorption properties. Protease adsorption and peptide hydrolysis rate were measured for these variants using a model substrate consisting of a peptide covalently bound to a surface. While most variants adsorb at a level very similar to that of native BPN', several variants were identified which adsorb either more or less. For surface-bound substrates we report a linear dependence between the concentration of adsorbed protease enzyme and substrate hydrolysis, similar to the linear dependence between enzyme solution concentration and hydrolysis of soluble substrates. On the basis of this knowledge we hypothesized that variants designed to adsorb at a higher level on a surface-bound peptide substrate would hydrolyze that surface-bound substrate faster. Contrary to our original expectations, the variants that adsorb more on the covalently bound peptide surface hydrolyze this substrate slower. In addition, variants of BPN' which adsorb at a lower level than native BPN' hydrolyze the surface-bound substrate faster. Enzyme adsorption and the subsequent peptide hydrolysis are alt...Continue Reading

Citations

Nov 28, 2012·Applied Microbiology and Biotechnology·Felix JakobUlrich Schwaneberg
Jan 11, 2001·Biochimica Et Biophysica Acta·P N Bryan
Jan 1, 2014·MicrobiologyOpen·Alexander PelzerKarl-Erich Jaeger
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