Liu Zhongmei, Wang Xiangyu, Yu Jiayin, Han Laichuang, Zhou Zhemin
School of Biotechnology, Key Laboratory of Industrial Biotechnology (Ministry of Education)，Jiangnan University, Wuxi, Jiangsu, China.
Lab of enzyme technology and bioprocess engineering, Jiangnan University-Rugao Food Biotechnology Institute, Rugao, Jiangsu, China.
Biotechnol Bioeng. 2021 Jul;118(7):2493-2502. doi: 10.1002/bit.27761. Epub 2021 Mar 27.
Enzymatic synthesis of l-alanine has the advantages of less byproducts, strong stereoselectivity, and high catalytic efficiency. Aspartate 4-decarboxylase (ASD) is used industrially in DL-aspartic acid resolution and l-alanine production because it catalyzes the decarboxylation of l-aspartic acid. In this study, the ASD gene from Acinetobacter radioresistens (ArASD) was cloned, and its enzymatic properties were analyzed. ArASD is a dodecamer and has the highest enzyme activity ever reported to date. The optimal conditions for ArASD catalysis are 50°C and pH 4.5. Site-directed mutagenesis was used to improve ArASD stability under acidic conditions to compensate for its weak acid resistance, and the variant N35D with higher catalytic ability was obtained. The conversion by N35 recombinant cells of l-aspartic acid to l-alanine was 92.5% at pH 4.5% and 99.9% at pH 6.0, whereas that of the wild-type recombinant cells was 29.7% and 31.4%, respectively. Aspartase from Escherichia coli (AspA) was employed with ArASD to construct a dual-enzyme system that catalyzes fumaric acid to l-alanine, and the conversion reached 97.1% using recombinant cells harboring the dual-enzyme system. This study explored the enzymatic properties of ArASD and an effective strategy for the acidic resistance modification of ASD. Moreover, the strain expressing the ArASD variant and AspA engineered in this study has great potential application for the l-alanine production industry, especially in the case of high optical purity requirements.
L-丙氨酸的酶法合成具有副产物少、立体选择性强和催化效率高的优点。天冬氨酸4-脱羧酶（ASD）可催化L-天冬氨酸脱羧，因此在工业上用于DL-天冬氨酸拆分和L-丙氨酸生产。本研究克隆了抗辐射不动杆菌的ASD基因（ArASD），并分析了其酶学性质。ArASD是一种十二聚体，具有迄今为止报道的最高酶活性。ArASD催化的最佳条件是50℃和pH 4.5。采用定点诱变提高ArASD在酸性条件下的稳定性，以弥补其耐酸性较弱的问题，并获得了具有更高催化能力的变体N35D。在pH 4.5时，N35重组细胞将L-天冬氨酸转化为L-丙氨酸的转化率为92.5%，在pH 6.0时为99.9%，而野生型重组细胞的转化率分别为29.7%和31.4%。将大肠杆菌天冬氨酸酶（AspA）与ArASD一起用于构建催化富马酸转化为L-丙氨酸的双酶系统，使用含有该双酶系统的重组细胞时转化率达到97.1%。本研究探索了ArASD的酶学性质以及ASD耐酸性修饰的有效策略。此外，本研究中表达ArASD变体和AspA的菌株在L-丙氨酸生产行业具有巨大的潜在应用价值，尤其是在对光学纯度要求较高的情况下。
