Characteristics of Two Forms of α-Amylases and Structural Implication

Complete (Ba-L) and truncated (Ba-S) forms of α-amylases from Bacillus subtilis X-23 were purified, and the amino- and carboxyl-terminal amino acid sequences of Ba-L and Ba-S were determined. The amino acid sequence deduced from the nucleotide sequence of the α-amylase gene indicated that Ba-S was produced from Ba-L by truncation of the 186 amino acid residues at the carboxyl-terminal region. The results of genomic Southern analysis and Western analysis suggested that the two enzymes originated from the same α-amylase gene and that truncation of Ba-L to Ba-S occurred during the cultivation of B. subtilis X-23 cells. Although the primary structure of Ba-S was approximately 28% shorter than that of Ba-L, the two enzyme forms had the same enzymatic characteristics (molar catalytic activity, amylolytic pattern, transglycosylation ability, effect of pH on stability and activity, optimum temperature, and raw starch-binding ability), except that the thermal stability of Ba-S was higher than that of Ba-L. An analysis of the secondary structure as well as the predicted three-dimensional structure of Ba-S showed that Ba-S retained all of the necessary domains (domains A, B, and C) which were most likely to be required for functionality as α-amylase.

本研究从枯草芽孢杆菌X-23（Bacillus subtilis X-23）中纯化得到α-淀粉酶（α-amylase）的完整形式（Ba-L）与截短形式（Ba-S），并测定了二者的氨基端与羧基端氨基酸序列。通过该α-淀粉酶基因的核苷酸序列推导得到的氨基酸序列显示，Ba-S是由Ba-L在羧基端区域截除186个氨基酸残基后产生的。基因组Southern印迹分析（genomic Southern analysis）与蛋白质免疫印迹分析（Western analysis）的结果表明，两种酶均源自同一α-淀粉酶基因，且Ba-L向Ba-S的截短过程发生在枯草芽孢杆菌X-23的培养过程中。尽管Ba-S的一级结构比Ba-L短约28%，但二者具备完全一致的酶学特性，包括摩尔催化活性、淀粉水解模式、转糖基化能力、pH对稳定性与活性的影响、最适温度以及生淀粉结合能力，仅Ba-S的热稳定性高于Ba-L。对Ba-S的二级结构以及预测的三维结构分析显示，Ba-S保留了α-淀粉酶发挥功能所必需的全部结构域（结构域A、B与C）。