Steyn A J, Marmur J, Pretorius I S
Department of Microbiology, University of Stellenbosch, South Africa.
Gene. 1995 Dec 1;166(1):65-71. doi: 10.1016/0378-1119(95)00633-0.
The yeast Lipomyces kononenkoae (Lk) secretes a highly active raw starch-degrading alpha-amylase (alpha Amy) that liberates reducing groups from glucose polymers containing both alpha-1,4 and alpha-1,6 bonds. The LKA1 gene encoding this industrially important alpha Amy was cloned as a 2261-bp cDNA fragment from a glucose-derepressed mutant (IGC4052B) of Lk and characterized. The nucleotide (nt) sequence of the cDNA fragment was determined, revealing an open reading frame of 1872 bp, encoding a 596 amino-acid (aa) mature protein (LKA1) with a calculated M(r) of 65,706. The similarity between the aa sequence of LKA1 and those of other alpha Amy showed four common conserved regions characteristic of the alpha Amy protein family: (A) 264DIVVNH269, (B) 349GLRIDTVKH357, (B') 376GEVFD380 and (C) 439FLENQD444. The deduced aa sequence revealed significant homology to the aa sequences of the Aspergillus oryzae, Schwanniomyces occidentalis and Saccharomycopsis fibuligera alpha Amy, various bacterial cyclomaltodextrin glucanotransferases, a beta-amylase and the 5'-region of a glucoamylase. LKA1 was expressed in Saccharomyces cerevisiae (Sc) under the control of the phosphoglycerate kinase (PGK1) promoter and Northern blot analysis showed the presence of a single 2.3-kb transcript. The 28-aa signal peptide of the LKA1 protein efficiently directed its secretion into the medium when expressed in Sc.
酵母科诺宁科酵母（Lipomyces kononenkoae，Lk）分泌一种高活性的生淀粉降解α-淀粉酶（α Amy），该酶能从含有α-1,4和α-1,6键的葡萄糖聚合物中释放出还原基团。编码这种具有重要工业价值的α Amy的LKA1基因，是从Lk的葡萄糖去阻遏突变体（IGC4052B）中克隆得到的一个2261 bp的cDNA片段，并对其进行了表征。测定了该cDNA片段的核苷酸（nt）序列，发现一个1872 bp的开放阅读框，编码一个596个氨基酸（aa）的成熟蛋白（LKA1），计算所得的分子量（M(r)）为65706。LKA1的氨基酸序列与其他α Amy的氨基酸序列之间的相似性显示出α Amy蛋白家族的四个常见保守区域：（A）264DIVVNH269，（B）349GLRIDTVKH357，（B'）376GEVFD380和（C）439FLENQD444。推导的氨基酸序列与米曲霉、西方施万酵母和扣囊复膜孢酵母的α Amy、各种细菌环糊精葡聚糖转移酶、一种β-淀粉酶以及一种糖化酶的5'区域的氨基酸序列具有显著同源性。LKA1在磷酸甘油酸激酶（PGK1）启动子的控制下在酿酒酵母（Sc）中表达，Northern印迹分析显示存在一个单一的2.3 kb转录本。当在Sc中表达时，LKA1蛋白的28个氨基酸的信号肽有效地将其分泌到培养基中。
