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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 altered by substituting amino acids that modify the surface charge or hydrophobicity of the native enzyme. This effect is most dramatic when the changes were made at surface-exposed sites around the binding pocket/active site of the enzyme. One mechanism that is consistent with the data is based on the relationship between the level of adsorption and the enzyme's affinity for the surface. In this mechanism weakly adsorbed enzymes are postulated to move more rapidly from site to site on the surface, thereby increasing substrate hydrolysis.

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We describe the adsorption and catalytic behavior of the serine protease subtilisin BPN′ on controlled pore glass (CPG) beads with a short (aminopropyl) or a long (aminoalkyl CH2 > 12) chain covalent link separating the reporter peptide succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (sAAPFpNA) from the surface. The propyl-linked sAAPFpNA modified glass surface (aminopropyl CPG:sAAPFpNA) showed a 2-fold increase in protease adsorption over an aminopropyl-glass surface. In contrast, the sAAPFpNA surface with the long chain connector showed a 2-fold drop in adsorption relative to an aminoalkyl surface. BPN′-catalyzed hydrolysis rates showed an inverse relationship to adsorption. Water-soluble polymers [poly(vinylpyrrolidone) (PVP), poly(ethylene oxide) (PEO), poly(4-vinylpyridine-N-oxide) (PVPO) and a copolymer of 1-vinyl-2-pyrrolidone and 1-vinylimidazole (PVPVI)] neutralize the 2-fold increase in BPN′ adsorption and provide more than a 3-fold increase in the initial rate of hydrolysis for BPN′-catalyzed cleavage of pNA. Another water-soluble polymer, poly(vinyl alcohol) (PVA), causes only a slight adsorption decrease and hydrolysis increase for the BPN′, aminopropyl CPG:sAAPFpNA system. None of the polymers causes a significant change in BPN′-catalyzed hydrolysis of, or adsorption on, aminoalkyl (CH2 > 12) CPG:sAAPFpNA. The apparent mechanism behind these effects is one in which the long alkyl chains and adsorbed polymers decrease the amount of adsorbed enzyme and increase the amount available for reaction in solution. A model is presented which describes the relationship between adsorption and surface hydrolysis. Materials and MethodsEnzyme Solution. The steps used to isolate, purify, and prepare a stock solution of the serine protease used here, subtilisin BPN′ (MW ) 27 534 g/mol), from Bacillus amyloliquefaciens are outlined elsewhere. 14 Prior to use, the enzyme stock solution is