Schwermann B, Pfau K, Liliensiek B, Schleyer M, Fischer T, Bakker E P
Abteilung Mikrobiologie, Universität Osnabrück, Germany.
Eur J Biochem. 1994 Dec 15;226(3):981-91. doi: 10.1111/j.1432-1033.1994.00981.x.
The alpha-amylase from the thermoacidophilic eubacterium Alicyclobacillus (Bacillus) acidocaldarius strain ATCC 27009 was studied as an example of an acidophilic protein. The enzyme was purified from the culture fluid. On an SDS/polyacrylamide gel, the protein an apparent molecular mass of 160 kDa, which is approximately 15% higher than that predicted from the nucleotide sequence. The difference is due to the enzyme being a glycoprotein. Deglycosylation or synthesis of the 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 maltose. It also formed glucose from starch (by hydrolysing the intermediate product maltotetraose to glucose and maltotriose) and exhibited some pullulanase activity. the pH and temperature optima were pH3 and 75 degrees C, respectively, characterizing the enzyme as being thermoacidophilic. Alignment of the sequence of the enzyme with that of its closest neutrophilic relatives and with that of alpha-1,4 or alpha-1,6 glycosidic-bond hydrolyzing enzymes of known three-dimensional structure showed that the acidophilic alpha-amylase contains approximately 30% less charged residues than do its closest relatives, that these residues are replaced by neutral polar residues, and that hot spots for these 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 electrostatic repulsion of charged groups at low pH, and thereby contributes to the acidostability of these proteins.
以嗜热嗜酸真细菌嗜酸栖热菌（芽孢杆菌属）菌株ATCC 27009的α-淀粉酶作为嗜酸蛋白的一个实例进行了研究。该酶从培养液中纯化得到。在SDS/聚丙烯酰胺凝胶上，该蛋白的表观分子量为160 kDa，比根据核苷酸序列预测的分子量约高15%。这种差异是由于该酶是一种糖蛋白。对该酶进行去糖基化处理或在大肠杆菌中合成该酶，得到的产物具有成熟蛋白预期的分子量。该淀粉酶随机地从淀粉内部进行水解，其主要水解产物是麦芽三糖和麦芽糖。它还能将淀粉转化为葡萄糖（通过将中间产物麦芽四糖水解为葡萄糖和麦芽三糖），并表现出一些支链淀粉酶活性。其最适pH和温度分别为pH3和75℃，这表明该酶具有嗜热嗜酸的特性。将该酶的序列与其亲缘关系最近的嗜中性酶以及已知三维结构的α-1,4或α-1,6糖苷键水解酶的序列进行比对，结果表明，嗜酸α-淀粉酶的带电荷残基比其亲缘关系最近的酶少约30%，这些残基被中性极性残基取代，而且这些替换的热点可能位于蛋白质表面。文献数据表明，在其他三种嗜酸蛋白中也观察到了类似的效应。有人提出，这些蛋白通过降低其表面正电荷和负电荷的密度来适应酸性环境，这种效应避免了在低pH值下带电荷基团之间的静电排斥，从而有助于这些蛋白的酸稳定性。
