Optimization of the production of Chondrus crispus hexose oxidase in Pichia pastoris.
Optimization of the production of Chondrus crispus hexose oxidase in Pichia pastoris.
复制标题
毕赤酵母中蒺藜角叉菜己糖氧化酶的生产优化。
DOI：
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发表时间：
2001
影响因子：
1.6
通讯作者：
P. Stougaard
中科院分区：
文献类型：
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作者：
A. Wolff;O. C. Hansen;U. Poulsen;S. Madrid;P. Stougaard
已糖氧化酶（D-hexose：O（2）-oxidoreductase，EC 1.1.3.5，HOX）在不同的寄主系统中异源产生，该酶在红角叉菜中发现。在大肠杆菌中产生了全长HOX多肽，但没有检测到HOX活性。相比之下，活性HOX可以在甲醇营养型酵母毕赤酵母中产生。研究了几种优化己糖氧化酶生产的生长、生理和遗传途径。我们的研究结果表明，特定的生长条件是必不可少的，以产生具有正确构象的活性HOX。此外，HOX似乎通过全长多肽链的蛋白水解切割成两个片段而被激活，这两个片段保持物理缔合。使用广泛使用的来自酿酒酵母转化酶或α-交配因子的分泌信号将HOX引导至细胞外区室的尝试失败了。然而，我们在这项研究中表明，HOX是通过一种迄今为止未知的机制运输出巴斯德毕赤酵母，并且有可能通过诱变将这种分泌从低于检测限提高到至少250 mg胞外酶/升。
Hexose oxidase (D-hexose:O(2)-oxidoreductase, EC 1.1.3.5, HOX) normally found in the red alga Chondrus crispus was produced heterologously in different host systems. Full-length HOX polypeptide was produced in Escherichia coli, but no HOX activity could be detected. In contrast, active HOX could be produced in the methylotrophic yeast Pichia pastoris. Several growth physiological and genetic approaches for optimization of hexose oxidase production in P. pastoris were investigated. Our results indicate that specific growth conditions are essential in order to produce active HOX with the correct conformation. Furthermore, HOX seems to be activated by proteolytic cleavage of the full-length polypeptide chain into two fragments, which remain physically associated. Attempts to direct HOX to the extracellular compartment using the widely used secretion signals from Saccharomyces cerevisiae invertase or alpha-mating factor failed. However, we show in this study that HOX is transported out of P. pastoris via a hitherto unknown mechanism and that it is possible to enhance this secretion by mutagenesis from below the detection limit to at least 250 mg extracellular enzyme per liter.
影响因子：
3.2
作者：
Monosov, EZ;Wenzel, TJ;Subramani, S
通讯作者：
Subramani, S