Ma Jinghao, Basit Rana Abdul, Yuan Sihan, Zhao Xuan, Liu Xiaoyan, Fan Guangsen
Key Laboratory of Geriatric Nutrition and Health (Beijing Technology and Business University), Ministry of Education, Beijing, 100048, China.
Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing, 100048, China.
Folia Microbiol (Praha). 2025 Apr;70(2):441-454. doi: 10.1007/s12223-024-01197-6. Epub 2024 Sep 16.
Feruloyl esterases (FAEs) are a crucial component of the hemicellulose-degrading enzyme family that facilitates the degradation of lignocellulose while releasing hydroxycinnamic acids such as ferulic acid with high added value. Currently, the low enzyme yield of FAEs is one of the primary factors limiting its application. Therefore, in this paper, we optimized the fermentation conditions for the expression of FAE BpFae with excellent thermal stability in Escherichia coli by experimental design. Firstly, we explored the effects of 11 factors such as medium type, isopropyl-β-D-thiogalactopyranoside (IPTG) concentration, and inoculum size on BpFae activity separately by the single factor design. Then, the significance of the effects of seven factors, such as post-induction temperature, shaker rotational speed, and inoculum size on BpFae activity, was analyzed by Plackett-Burman design. We identified the main factors affecting the fermentation conditions of E. coli expressing BpFae as post-induction temperature, pre-induction period, and post-induction period. Finally, we used the steepest ascent path design and response surface method to optimize the levels of these three factors further. Under the optimal conditions, the activity of BpFae was 3.58 U/ml, which was a significant 6.6-fold increase compared to the pre-optimization (0.47 U/ml), demonstrating the effectiveness of this optimization process. Moreover, BpFae activity was 1.52 U/ml in a 3-l fermenter under the abovementioned optimal conditions. It was determined that the expression of BpFae in E. coli was predominantly intracellular in the cytoplasm. This study lays the foundation for further research on BpFae in degrading agricultural waste transformation applications.
阿魏酸酯酶（FAEs）是半纤维素降解酶家族的关键组成部分，有助于木质纤维素的降解，同时释放出具有高附加值的羟基肉桂酸，如阿魏酸。目前，FAEs的酶产量低是限制其应用的主要因素之一。因此，在本文中，我们通过实验设计优化了在大肠杆菌中表达具有优异热稳定性的FAE BpFae的发酵条件。首先，我们通过单因素设计分别探究了培养基类型、异丙基-β-D-硫代半乳糖苷（IPTG）浓度和接种量等11个因素对BpFae活性的影响。然后，通过Plackett-Burman设计分析了诱导后温度、摇床转速和接种量等7个因素对BpFae活性影响的显著性。我们确定了影响表达BpFae的大肠杆菌发酵条件的主要因素为诱导后温度、诱导前期和诱导后期。最后，我们使用最速上升路径设计和响应面法进一步优化这三个因素的水平。在最佳条件下，BpFae的活性为3.58 U/ml，与优化前（0.47 U/ml）相比显著提高了6.6倍，证明了该优化过程的有效性。此外，在上述最佳条件下，BpFae在3升发酵罐中的活性为1.52 U/ml。已确定BpFae在大肠杆菌中的表达主要位于细胞质内。本研究为进一步研究BpFae在降解农业废弃物转化应用方面奠定了基础。
