Pullulanase is an important starch-debranching enzyme mostly used in starch processing-related food industries. However, the levels of pullulanase produced from recombinant Bacillus subtilis, a Generally Recognized as Safe (GRAS) host, are generally limited. To enhance the activity of pullulanase, batch fermentation and fed-batch fermentation were systematically investigated. The overall purpose is to improve the fermentation yield by optimizing the feeding strategy in the fermentation process, thereby increasing the enzyme activity of pullulanase. Therefore, in this study, the feeding methods, the feeding ingredients, the feeding concentration, and pH values were studied in detail. The optimized fermentation conditions for pullulanase production from recombinant B. subtilis were determined as following: inoculum volume 7%, pH 6.5, the dissolved oxygen level 30%, and constant-rate feeding of 100 mL glucose solution (400 g L-1) in late logarithmic growth. The OD600 of recombinant B. subtilis and enzyme activity were 84.54 and 102.75 U mL-1, which were respectively 141% and 144% higher than that before optimization. These findings provided a prerequisite for further amplification of the fermentation system to obtain higher enzyme activity.

中文翻译：

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通过优化饲喂策略和发酵条件，可增强重组枯草芽孢杆菌产生支链淀粉酶的能力。

支链淀粉酶是一种重要的淀粉脱支酶，主要用于与淀粉加工相关的食品工业中。然而，通常限制了由重组枯草芽孢杆菌（通常被认为是安全的（GRAS）宿主）产生的支链淀粉酶的水平。为了提高支链淀粉酶的活性，系统地研究了分批发酵和补料分批发酵。总体目的是通过优化发酵过程中的进料策略来提高发酵产量，从而增加支链淀粉酶的酶活性。因此，在这项研究中，详细研究了喂养方法，喂养成分，喂养浓度和pH值。确定从重组枯草芽孢杆菌生产支链淀粉酶的最佳发酵条件如下：接种量7％，pH 6.5，溶氧水平为30％，并在对数生长后期恒速补料100 mL葡萄糖溶液（400 g L-1）。重组枯草芽孢杆菌的OD600和酶活性分别为84.54和102.75 U mL-1，分别比优化前提高了141％和144％。这些发现为进一步扩增发酵系统以获得更高的酶活性提供了前提。