Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, No. 18, Chaowang Road, Hangzhou, 310014, Zhejiang, People's Republic of China.
Engineering Research Centre of Bioconversion and Biopurification, Ministry of Education, Zhejiang University of Technology, No. 18,Chaowang Road, Hangzhou, 310014, Zhejiang, People's Republic of China.
Bioprocess Biosyst Eng. 2023 Nov;46(11):1639-1650. doi: 10.1007/s00449-023-02925-x. Epub 2023 Sep 21.
With potent herbicidal activity, biocatalysis synthesis of L-glufosinate has drawn attention. In present research, NAP-Das2.3, a deacetylase capable of stereoselectively resolving N-acetyl-L-glufosinate to L-glufosinate mined from Arenimonas malthae, was heterologously expressed and characterized. In Escherichia coli, NAP-Das2.3 activity only reached 0.25 U/L due to the formation of inclusive bodies. Efficient soluble expression of NAP-Das2.3 was achieved in Pichia pastoris. In shake flask and 5 L bioreactor fermentation, NAP-Das2.3 activity by recombinant P. pastoris reached 107.39 U/L and 1287.52 U/L, respectively. The optimum temperature and pH for N-acetyl-glufosinate hydrolysis by NAP-Das2.3 were 45 °C and pH 8.0, respectively. The K and V of NAP-Das2.3 towards N-acetyl-glufosinate were 25.32 mM and 19.23 μmol mg min, respectively. Within 90 min, 92.71% of L-enantiomer in 100 mM racemic N-acetyl-glufosinate was converted by NAP-Das2.3. L-glufosinate with high optical purity (e.e. above 99.9%) was obtained. Therefore, the recombinant NAP-Das2.3 might be an alternative for L-glufosinate biosynthesis.
具有强大除草活性的 L-草铵膦可以通过生物催化合成，这引起了人们的关注。在本研究中，从 Arenimonas malthae 中挖掘出一种能够立体选择性地拆分 N-乙酰-L-草铵膦为 L-草铵膦的脱乙酰酶 NAP-Das2.3，并对其进行了异源表达和特性分析。由于包涵体的形成，NAP-Das2.3 在大肠杆菌中的活性仅达到 0.25 U/L。在巴斯德毕赤酵母中实现了 NAP-Das2.3 的高效可溶性表达。在摇瓶和 5 L 生物反应器发酵中，重组毕赤酵母的 NAP-Das2.3 活性分别达到 107.39 U/L 和 1287.52 U/L。NAP-Das2.3 水解 N-乙酰草铵膦的最适温度和 pH 分别为 45°C 和 pH 8.0。NAP-Das2.3 对 N-乙酰草铵膦的 K 和 V 分别为 25.32 mM 和 19.23 μmol·mg·min。在 90 分钟内，100 mM 外消旋 N-乙酰草铵膦中 92.71%的 L-对映体被 NAP-Das2.3 转化。获得了光学纯度高（ee 值大于 99.9%）的 L-草铵膦。因此，重组 NAP-Das2.3 可能是 L-草铵膦生物合成的替代方法。
