Braaksma Machtelt, Smilde Age K, van der Werf Mariët J, Punt Peter J
Kluyver Centre for Genomics of Industrial Fermentation, PO Box 5057, 2600 GA Delft, The Netherlands.
TNO Quality of Life, PO Box 360, 3700 AJ Zeist, The Netherlands.
Microbiology (Reading). 2009 Oct;155(Pt 10):3430-3439. doi: 10.1099/mic.0.031062-0. Epub 2009 Jul 23.
Proteolytic degradation by host proteases is one of the key issues in the application of filamentous fungi for non-fungal protein production. In this study the influence of several environmental factors on the production of extracellular proteases of Aspergillus niger was investigated systematically in controlled batch cultures. Of all factors investigated in a series of initial screening experiments, culture pH and nitrogen concentration in particular strongly affected extracellular protease activities. For instance, at a culture pH of 4, protease activity was higher than at pH 5, and protease activity increased with increasing concentrations of ammonium as nitrogen source. Interestingly, an interdependence was observed for several of the factors studied. These possible interaction effects were investigated further using a full factorial experimental design. Amongst others, the results showed a clear interaction effect between nitrogen source and nitrogen concentration. Based on the observed interactions, the selection of environmental factors to reduce protease activity is not straightforward, as unexpected antagonistic or synergistic effects occur. Furthermore, not only were the effects of the process parameters on maximum protease activity investigated, but five other protease-related phenotypes were studied as well, such as maximum specific protease activity and maximum protease productivity. There were significant differences in the effect of the environmental parameters on the various protease-related phenotypes. For instance, pH significantly affected final levels of protease activity, but not protease productivity. The results obtained in this study are important for the optimization of A. niger for protein production.
宿主蛋白酶的蛋白水解降解是丝状真菌用于生产非真菌蛋白应用中的关键问题之一。在本研究中，在受控分批培养中系统地研究了几种环境因素对黑曲霉胞外蛋白酶产生的影响。在一系列初步筛选实验中研究的所有因素中，培养pH值和氮浓度尤其强烈地影响胞外蛋白酶活性。例如，在培养pH值为4时，蛋白酶活性高于pH值为5时，并且蛋白酶活性随着作为氮源的铵浓度增加而增加。有趣的是，观察到所研究的几个因素之间存在相互依赖性。使用全因子实验设计进一步研究了这些可能的相互作用效应。其中，结果表明氮源和氮浓度之间存在明显的相互作用效应。基于观察到的相互作用，选择降低蛋白酶活性的环境因素并非易事，因为会出现意想不到的拮抗或协同效应。此外，不仅研究了工艺参数对最大蛋白酶活性的影响，还研究了其他五个与蛋白酶相关的表型，例如最大比蛋白酶活性和最大蛋白酶生产率。环境参数对各种与蛋白酶相关的表型的影响存在显著差异。例如，pH值显著影响蛋白酶活性的最终水平，但不影响蛋白酶生产率。本研究中获得的结果对于优化黑曲霉用于蛋白质生产非常重要。
