Ghasemi Amir, Ghafourian Sobhan, Vafaei Sedighe, Mohebi Reza, Farzi Maryam, Taherikalani Morovat, Sadeghifard Nourkhoda
Department of Microbiology and Immunology, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran.
Clinical Microbiology Research Center, Ilam University of Medical Sciences, Ilam, Iran.
Osong Public Health Res Perspect. 2015 Dec;6(6):336-40. doi: 10.1016/j.phrp.2015.10.003. Epub 2015 Oct 20.
In an attempt α-amylase gene from Pyrococcus woesei was amplified and cloned into a pTYB2 vector to generate the recombinant plasmid pTY- α-amylase.
Escherichia coli BL21 used as a host and protein expression was applied using IPTG. SDS-PAGE assay demonstrated the 100 kDa protein. Amylolytic activity of proteins produced by transformed E. coli cells was detected by zymography, and the rate of active α-amylase with and without the intein tag in both soluble conditions and as inclusion bodies solubilized by 4M urea were measured.
Amylolytic activity of ∼185,000 U/L of bacterial culture was observed from the soluble form of the protein using this system.
These results indicate that this expression system was appropriate for the production of thermostable α-amylase.
尝试扩增来自沃氏嗜热栖热菌的α-淀粉酶基因，并将其克隆到pTYB2载体中以构建重组质粒pTY-α-淀粉酶。
以大肠杆菌BL21作为宿主，使用异丙基-β-D-硫代半乳糖苷（IPTG）进行蛋白质表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析显示出100 kDa的蛋白质。通过酶谱法检测转化后的大肠杆菌细胞产生的蛋白质的淀粉分解活性，并测定在可溶性条件下以及经4M尿素溶解的包涵体形式中带有和不带有内含肽标签的活性α-淀粉酶的比率。
使用该系统从蛋白质的可溶性形式中观察到细菌培养物的淀粉分解活性约为185,000 U/L。
这些结果表明该表达系统适用于生产耐热性α-淀粉酶。
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