Acidostable and acidophilic proteins: The example of the alpha-amylase from Alicyclobacillus acidocaldarius

Abstract

Acidophilic microorganisms grow optimally at pH values between 1-4. They have adapted to the acid condition by maintaining their cytoplasmic pH at a value close to neutrality. Hence, only those (macro)molecules, which face the acid medium, have had to adapt to this extreme condition. Literature data show that several exoproteins from thermoacidophilic prokaryotes are characterized by a low charge density. It is proposed that this property contributes to the stability of these proteins both below and above the pK(a)-values of their glutamate and aspartate residues. As an example of an acidophilic protein, the alpha-amylase from the Gram-positive Alicyclobacillus acidocaldarius ATCC27009 was studied. The enzyme is thermoacidophilic, with optima of temperature and pH of 75 degrees C and pH 3, respectively. The nucleotide sequence of the cloned gene (8) indicates that the alpha-amylase belongs to a large family of starch-degrading enzymes with a characteristic catalytic (beta alpha)(8)-domain. Three essential and probably catalytic acidic residues have been conserved, suggesting that the acidophilic alpha-amylase degrades starch with essentially the same mechanism as do its neutrophilic relatives. Still, the acidophilic protein contains three exchanges in residues uniformally or almost uniformally conserved among all members of the enzyme family. In order to test whether these exchanges contribute to the acidic pH optimum, the alpha-amylase gene was expressed in Escherichia coli. Sonication of the enzyme-producing cells released alpha-amylase activity associated with a 140 kDa protein. The optima of temperature and pH for the protein produced in E. coli were similar to those of the native enzyme. Experiments are underway in which it is tested which residues contribute to the acid pH optimum of the alpha-amylase. (C) 1997 Elsevier Science Inc.