Abstract：
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 Km and Vmax of NAP-Das2.3 towards N-acetyl-glufosinate were 25.32 mM and 19.23 μmol mg-1 min-1, 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.P above 99.9%) was obtained. Therefore, the recombinant NAP-Das2.3 might be an alternative for L-glufosinate biosynthesis.
摘要：
具有强大的除草活性，L-草铵膦的生物催化合成已引起人们的关注。在目前的研究中，NAP-Das2.3，一种脱乙酰酶，能够将N-乙酰基-L-草铵膦立体选择性地解析为从麦芽分藻中开采的L-草铵膦，异源表达和表征。在大肠杆菌中，由于包含体的形成,NAP-Das2.3活性仅达到0.25U/L。在巴斯德毕赤酵母中实现了NAP-Das2.3的有效可溶性表达。在摇瓶和5升生物反应器发酵中，重组巴斯德毕赤酵母的NAP-Das2.3活性达到107.39U/L和1287.52U/L,分别。NAP-Das2.3水解N-乙酰基-草铵膦的最佳温度和pH分别为45°C和pH8.0。NAP-Das2.3对N-乙酰基-草铵膦的Km和Vmax分别为25.32mM和19.23μmolmg-1min-1。90分钟内,通过NAP-Das2.3转化了100mM外消旋N-乙酰基-草铵膦中的92.71％的L-对映体。获得具有高光学纯度(e.e.P高于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品。
