Natalia Dessy, Vidilaseris Keni, Ismaya Wangsa T, Puspasari Fernita, Prawira Iman, Hasan Khomaini, Fibriansah Guntur, Permentier Hjalmar P, Nurachman Zeily, Subroto Toto, Dijkstra Bauke W, Soemitro Soetijoso
Biochemistry Research Division, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology, Jalan Ganesa No 10, Bandung 40132, Indonesia; Center for Life Sciences, Bandung Institute of Technology, Jalan Ganesa No. 10, Bandung 40132, Indonesia.
Biochemistry Research Division, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology, Jalan Ganesa No 10, Bandung 40132, Indonesia.
J Biotechnol. 2015 Feb 10;195:8-14. doi: 10.1016/j.jbiotec.2014.12.002. Epub 2014 Dec 20.
Native enzyme and a mutant containing an extra disulphide bridge of recombinant Saccharomycopsis fibuligera R64 α-amylase, designated as Sfamy01 and Sfamy02, respectively, have successfully been overexpressed in the yeast Pichia pastoris KM71H. The purified α-amylase variants demonstrated starch hydrolysis resulting in a mixture of maltose, maltotriose, and glucose, similar to the wild type enzyme. Introduction of the disulphide bridge shifted the melting temperature (TM) from 54.5 to 56 °C and nearly tripled the enzyme half-life time at 65 °C. The two variants have similar kcat/KM values. Similarly, inhibition by acarbose was only slightly affected, with the IC50 of Sfamy02 for acarbose being 40 ± 3.4 μM, while that of Sfamy01 was 31 ± 3.9 μM. On the other hand, the IC50 of Sfamy02 for EDTA was 0.45 mM, nearly two times lower than that of Sfamy01 at 0.77 mM. These results show that the introduction of a disulphide bridge had little effect on the enzyme activity, but made the enzyme more susceptible to calcium ion extraction. Altogether, the new disulphide bridge improved the enzyme stability without affecting its activity, although minor changes in the active site environment cannot be excluded.
粟酒裂殖酵母R64 α-淀粉酶的天然酶和含有额外二硫键的突变体（分别命名为Sfamy01和Sfamy02）已成功在毕赤酵母KM71H中过表达。纯化后的α-淀粉酶变体表现出淀粉水解，产生麦芽糖、麦芽三糖和葡萄糖的混合物，这与野生型酶相似。二硫键的引入使解链温度（TM）从54.5℃变为56℃，并使该酶在65℃下的半衰期增加了近两倍。这两个变体具有相似的kcat/KM值。同样，阿卡波糖对其抑制作用仅受到轻微影响，Sfamy02对阿卡波糖的IC50为40±3.4μM，而Sfamy01的IC50为31±3.9μM。另一方面，Sfamy02对EDTA的IC50为0.45 mM，比Sfamy01的0.77 mM低近两倍。这些结果表明，二硫键的引入对酶活性影响不大，但使该酶更容易被钙离子提取。总之，新的二硫键提高了酶的稳定性而不影响其活性，尽管不能排除活性位点环境有微小变化。
