Modification of two amino groups with polyethylene glycol causes a unique activity change of an alkaline proteinase from alkalophilicBacillus sp.

Abstract

An alkaline proteinase fromBacillus sp. NKS-21 (ALPase I) was modified by activated polyethylene glycol (PEG2) with an Mr of 10,000. The modified preparation was purified with the isoelectric focusing method. Two amino groups out of the total five amino groups of ALPase I were modified. This modification gave rise to an 85% loss of the activity towards milk casein as a substrate, while the modification caused a 20% loss of binding ability towards anti-ALPase I antiserum. The kinetic parameters of PEG2-ALPase I towards peptidyl-p-nitroanilides (pNA-substrates) and peptidyl-7-methyl-coumaryl-4-amides (MCA-substrates) suggested that at least one of the two amino groups modified with PEG2 was near the active site of ALPase I. The stability of the modified enzyme against heat treatment was increased. The PEG2-ALPase I could express enzymatic activity in the organic solvent benzene.