Zhang Xinhong, Wang Chuyan, Ge Yang, Meng Qingnan, Zhang Yi
School of Biology, Food and Environment, Hefei University, Hefei, China.
Institute of Pharmaceutical Biotechnology, University of Science and Technology of China, Hefei, China.
Biotechnol Appl Biochem. 2022 Apr;69(2):587-595. doi: 10.1002/bab.2135. Epub 2021 Mar 18.
Nitrilases can directly hydrolyze nitrile compounds into carboxylic acids and ammonium. To solve the current problems of bioconversions using nitrilases, including the difficult separation of products from the resting cells used as the catalyst and high costs of chemical inducers, a nitrilase from Alcaligenes faecalis was heterologously expressed in Pichia pastoris X33. The stable nitrilase-expressing strain No.39-6-4 was obtained after three rounds of screening based on a combined detection method including dot-blot, SDS-PAGE, and western blot analyses, which confirmed the presence of recombinant nitrilase with a molecular mass of about 50 kDa. The temperature and pH optima of the nitrilase were 45°C and pH 7.5, respectively. Cu , Zn , and Tween 80 strongly inhibited the enzyme activity, but the optical purity of the product R-mandelic acid (R-MA) was stable, with practically 100% enantiomeric excess (ee). The nitrilase-producing P. pastoris strain developed in this study provides a basis for further research on the enzyme.
腈水解酶可将腈类化合物直接水解为羧酸和铵。为解决目前使用腈水解酶进行生物转化存在的问题，包括产物与用作催化剂的静息细胞难以分离以及化学诱导剂成本高，从粪产碱杆菌中获得的一种腈水解酶在毕赤酵母X33中进行了异源表达。基于包括斑点印迹、SDS-PAGE和蛋白质免疫印迹分析在内的联合检测方法，经过三轮筛选，获得了稳定表达腈水解酶的39-6-4菌株，证实存在分子量约为50 kDa的重组腈水解酶。该腈水解酶的最适温度和pH分别为45°C和pH 7.5。铜、锌和吐温80强烈抑制酶活性，但产物R-扁桃酸（R-MA）的光学纯度稳定，对映体过量（ee）几乎达到100%。本研究构建的产腈水解酶毕赤酵母菌株为该酶的进一步研究提供了基础。
