黑曲霉外源表达系统对Dactylellina cionopaga基因PrD Ⅰ的表达

目的:Dactylellina cionopaga是产生黏性分枝的捕食线虫真菌,进行其基因外源表达是鉴定该菌侵染相关因子的途径之一。方法:该文构建了含有组氨酸标签的黑曲霉表达载体pGT21M-HIS,对D.cionopaga中与侵染机制相关的丝氨酸蛋白酶基因PrD Ⅰ进行了外源表达。结果:RT-PCR鉴定结果显示,PrDⅠ在黑曲霉201中获得了转录。SDS-PAGE和酶活分析结果表明,经亲和层析纯化的重组蛋白分子量约为45kDa,在pH 5.0的条件下具有蛋白水解活性。纯酶液的蛋白浓度为30μg/ml。结论:构建的带有组氨酸标签的表达载体pGT21M-HIS能够用于基因在黑曲霉表达系统中的外源表达,并为目的蛋白的纯化和鉴定提供了极大便利。黑曲霉外源表达系统在表达丝状真菌基因方面相对其他表达系统具有优势。     

Degradation of Dextran by Free Radical Systems

Alcohol dehydrogenase (alcohol: NAD oxidoreductase, E.C. 1.1.1.1) from Thea sinensis seeds (variety: Zairai) was isolated and purified about 1,500-fold using preparative disc electrophoresis. The specific activity was about 3.4 units/mg protein against ethyl alcohol.

Its value was 6.96 S and its molecular weight was approximately 150,000 using gel filtration on Sephadex G–200. The physical, chemical and catalytic properties of the enzyme are described. The oxidoreduction products formed by the enzyme were identified by gas chromatography, and for the unsaturated compounds the conversion of double bond and geometrical isomerization was observed.

The substrate specificity of tea enzyme is discussed in comparison with the enzymes from Leuconostoc mesenteroides, and horse and human livers. Paticularly a tendency for reactivity in the oxidoreduction of unsaturated alcohols and aldehydes were discribed by comparing the effects of geometry, the position of the double bond and the length of chain in substrates.