Candussio A, Schmid G, Böck A
Lehrstuhl für Mikrobiologie der Universität München, Federal Republic of Germany.
Eur J Biochem. 1990 Jul 20;191(1):177-85. doi: 10.1111/j.1432-1033.1990.tb19108.x.
Two amylases have been purified from the culture fluid of an alkaliphilic bacterium. Amylase A-60 consists of a single type of polypeptide chain of 60 kDa and exhibits an alpha-amylase-type of starch cleavage. Amylase A-180 is approximately 180 kDa in size, represents the largest exoenzyme so far identified in prokaryotes and in the initial enzyme reaction cleaves starch exclusively to maltopentaose. A-60 and A-180 are immunologically unrelated enzymes. The structural gene for amylase A-180 has been cloned and its nucleotide sequence was determined. An open reading frame was identified for a putative protein of 182 kDa whose amino-terminal sequence, deduced from the nucleotide sequence, was identical in 23 out of 25 positions to that determined for the protein. The amino-terminus of the mature protein, at the gene level, is preceded by a sequence segment showing all the characteristics of a signal peptide from Gram-positive bacteria. Analysis of the deduced amino acid sequence revealed that the 70-kDa N-terminal part is similar to classical alpha-amylases. The C-terminal part contains three repeated sequence blocks of 99 amino acid residues each which are also present in two bacterial beta-amylases. It appears, therefore, that A-180 has arisen by gene fusion events.
已从一种嗜碱细菌的培养液中纯化出两种淀粉酶。淀粉酶A - 60由一条60 kDa的单一类型多肽链组成，具有α - 淀粉酶类型的淀粉裂解活性。淀粉酶A - 180大小约为180 kDa，是迄今为止在原核生物中鉴定出的最大的胞外酶，在初始酶促反应中仅将淀粉裂解为麦芽五糖。A - 60和A - 180是免疫不相关的酶。已克隆出淀粉酶A - 180的结构基因并确定了其核苷酸序列。鉴定出一个编码推定的182 kDa蛋白质的开放阅读框，从核苷酸序列推导的其氨基末端序列，在25个位置中的23个与该蛋白质测定的序列相同。在基因水平上，成熟蛋白质的氨基末端之前有一个序列片段，显示出革兰氏阳性菌信号肽的所有特征。对推导的氨基酸序列分析表明，70 kDa的N末端部分与经典的α - 淀粉酶相似。C末端部分包含三个每个99个氨基酸残基的重复序列块，这在两种细菌β - 淀粉酶中也存在。因此，似乎A - 180是由基因融合事件产生的。
