PURIFICATION, PROPERTIES AND STRUCTURAL ASPECTS OF A THERMOACIDOPHILIC ALPHA-AMYLASE FROM ALICYCLOBACILLUS-ACIDOCALDARIUS ATCC-27009 - INSIGHT INTO ACIDOSTABILITY OF PROTEINS

The alpha-amylase from the thermoacidophilic eubacterium Alicyclobacil lus (Bacillus) acidocaldarius ius strain ATCC 27009 was studied as an example of an acidophilic protein. The enzyme was purified from the cu lture fluid. On an SDS/polyacrylamide gel, the protein exhibited an ap parent molecular mass of 160 kDa, which is approximately 15% higher th an that predicted from the nucleotide sequence. The difference is due to the enzyme being a glycoprotein. Deglycosylation or synthesis of th e enzyme in Escherichia coli gave a product with the mass expected for the mature protein. The amylase hydrolyzed starch at random and from the inside, and its main hydrolysis products were maltotriose and malt ose. It also formed glucose from starch (by hydrolysing the intermedia te product maltotetraose to glucose and maltotriose) and exhibited som e pullulanase activity. The pH and temperature optima were pH 3 and 75 degrees C, respectively, characterizing the enzyme as being thermoaci dophilic. Alignment of the sequence of the enzyme with that of its clo sests neutrophilic relatives and with that of alpha-1,4 or alpha-1,6 g lycosidic-bond hydrolyzing enzymes of known three-dimensional structur e showed that the acidophilic alpha-amylase contains approximately 30% less charged residues than do its closests relatives, that these resi dues are replaced by neutral polar residues, and that hot spots for th ese exchanges are likely to be located at the surface of the protein. Literature data show that similar effects are observed in three other acidophilic proteins. It is proposed that these proteins have adapted to the acidic environment by reducing the density of both positive and negative charges at their surface, that this effect circumvents elect rostatic repulsion of charged groups at low pH, and thereby contribute s to the acidostability of these proteins.