Pluschkell S, Hellmuth K, Rinas U
GBF National Research Center for Biotechnology, Biochemical Engineering Division, Mascheroder Weg 1, 38124 Braunschweig, Germany.
Biotechnol Bioeng. 1996 Jul 20;51(2):215-20. doi: 10.1002/(SICI)1097-0290(19960720)51:2<215::AID-BIT11>3.0.CO;2-L.
The kinetics of glucose oxidase (GOD) excretion by recombinant Aspergillus niger NRRL-3 (GOD3-18) were investigated using enzymatic activity measurements as well as gel electrophoresis techniques. The majority of GOD was produced during rapid growth in the first phase of the cultivation. The high excretion rate during this phase did not prevent the endocellular accumulation of GOD up to 40% of the total soluble cell protein demonstrating that the production rate exceeded the excretion rate of the enzyme into the culture medium. During the second phase of the cultivation, excretion of GOD occurred at a slower rate, although the majority of GOD produced during the first phase was excreted during the second phase of the cultivation. At the end, about 90% of the total GOD produced was recovered from the culture medium. Two-dimensional gel electrophoresis provided evidence that endo- and exocellular GOD were indistinguishable, revealing identical posttranslational modifications (e.g., signal sequence cleavage, glycosylation pattern). The results demonstrate that the initial steps of the secretory pathway are fast and that the excretion of the enzyme into the culture fluid was most likely delayed due to retention by the cell wall.
利用酶活性测定以及凝胶电泳技术，研究了重组黑曲霉NRRL-3（GOD3-18）分泌葡萄糖氧化酶（GOD）的动力学。大部分GOD是在培养第一阶段快速生长期间产生的。此阶段较高的分泌速率并未阻止GOD在细胞内积累至总可溶性细胞蛋白的40%，这表明该酶的产生速率超过了其分泌到培养基中的速率。在培养的第二阶段，GOD的分泌速率较慢，尽管第一阶段产生的大部分GOD在第二阶段被分泌到培养基中。最后，从培养基中回收了约90%的总产生的GOD。二维凝胶电泳表明，细胞内和细胞外的GOD无法区分，揭示了相同的翻译后修饰（例如，信号序列切割、糖基化模式）。结果表明，分泌途径的初始步骤很快，并且酶分泌到培养液中很可能由于被细胞壁滞留而延迟。
