Steyn A J, Pretorius I S
Department of Microbiology, University of Stellenbosch, South Africa.
Curr Genet. 1995 Nov;28(6):526-33. doi: 10.1007/BF00518165.
A highly active alpha-amylase (76,250 Da) secreted by the raw starch-degrading yeast Lipomyces kononenkoae strain IGC4052B was purified and characterized. Using high performance liquid chromatography (HPLC), end-product analysis indicated that the L. kononenkoae alpha-amylase acted by endo-hydrolysis on glucose polymers containing alpha-1,4 and alpha-1,6 bonds, producing mainly maltose, maltotriose and maltotetraose. The following NH2-terminal amino acids were determined for the purified enzyme: Asp-Cys-Thr-Thr-Val-Thr-Val-Leu-Ser-Ser-Pro- Glu-Ser-Val-Thr-Gly. The L. kononenkoae alpha-amylase-encoding gene (LKA1), previously cloned as a cDNA fragment, was expressed in Saccharomyces cerevisiae under the control of the PGK1 promoter. The native signal sequence efficiently directed the secretion of the glycosylated protein in S. cerevisiae. De-glycosylation of the enzyme indicated that post-translational glycosylation is different in S. cerevisiae from that in L. kononenkoae. Zymogram analysis indicated that glycosylation of the protein in S. cerevisiae had a negative effect on enzyme activity. Southern-blot analysis revealed that there is only a single LKA1 gene present in the genome of L. kononenkoae.
对由降解生淀粉的酵母科诺宁科酵母（Lipomyces kononenkoae）菌株IGC4052B分泌的一种高活性α-淀粉酶（76,250道尔顿）进行了纯化和表征。使用高效液相色谱法（HPLC）进行终产物分析表明，科诺宁科酵母α-淀粉酶通过内切水解作用于含有α-1,4和α-1,6键的葡萄糖聚合物，主要产生麦芽糖、麦芽三糖和麦芽四糖。测定了纯化酶的以下N端氨基酸序列：天冬氨酸-半胱氨酸-苏氨酸-苏氨酸-缬氨酸-苏氨酸-缬氨酸-亮氨酸-丝氨酸-丝氨酸-脯氨酸-谷氨酸-丝氨酸-缬氨酸-苏氨酸-甘氨酸。先前作为cDNA片段克隆的科诺宁科酵母α-淀粉酶编码基因（LKA1），在PGK1启动子的控制下在酿酒酵母（Saccharomyces cerevisiae）中表达。天然信号序列有效地指导了糖基化蛋白在酿酒酵母中的分泌。该酶的去糖基化表明，酿酒酵母中的翻译后糖基化与科诺宁科酵母中的不同。酶谱分析表明，酿酒酵母中蛋白的糖基化对酶活性有负面影响。Southern杂交分析显示，科诺宁科酵母基因组中仅存在一个LKA1基因。
