Enzymatic Production of Ascorbic Acid-2-phosphate by Recombinant Acid Phosphatase
Enzymatic Production of Ascorbic Acid-2-phosphate by Recombinant Acid Phosphatase
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重组酸性磷酸酶酶法生产抗坏血酸-2-磷酸
DOI：
10.1021/acs.jafc.7b00612
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发表时间：
2017
影响因子：
6.1
通讯作者：
Wu Jing
中科院分区：
文献类型：
--
作者：
Zheng Kai;Song Wei;Sun Anran;Chen Xiulai;Liu Jia;Luo Qiuling;Wu Jing
本研究以L-抗坏血酸（阿萨）为原料，建立了一种环境友好、高效的酶法合成L-抗坏血酸-2-磷酸酯（阿萨-2 P）的方法。铜绿假单胞菌酸性磷酸酶（PaAPase）在大肠杆菌BL 21中表达。重组PaAP酶的最适温度、最适pH、Km、kcat和催化效率分别为50 °C、5.0、93 mM、4.2 s-1和2.7 mM-1 min-1。最大菌体干重和PaAP酶磷酸化活性分别达到8.5g/L和1127.7U/L。最高阿萨-2 P浓度（50.0 g/L）和最大转化率在最佳条件下，75 g/L完整细胞与88 g/L阿萨和160 g/L焦磷酸钠共同孵育，可获得39.2（0.1 mM Ca 2+，pH 4.0，30 °C）10小时;平均阿萨-2 P生产速率为5.0 g/L/h，并且阿萨-2 P生产系统成功地扩大到7.5 L发酵罐。因此，酶法生产阿萨-2 P具有很大的工业化潜力。
In this study, an environmentally friendly and efficient enzymatic method for the synthesis ofl-ascorbic acid-2-phosphate (AsA-2P) froml-ascorbic acid (AsA) was developed. ThePseudomonas aeruginosaacid phosphatase (PaAPase) was expressed inEscherichia coliBL21. The optimal temperature, optimal pH,Km,kcat, and catalytic efficiency of recombinantPaAPase were 50 °C, 5.0, 93 mM, 4.2 s–1, and 2.7 mM–1min–1, respectively. The maximal dry cell weight andPaAPase phosphorylating activity reached 8.5 g/L and 1127.7 U/L, respectively. The highest AsA-2P concentration (50.0 g/L) and the maximal conversion (39.2%) were obtained by incubating 75 g/L intact cells with 88 g/L AsA and 160 g/L sodium pyrophosphate under optimal conditions (0.1 mM Ca2+, pH 4.0, 30 °C) for 10 h; the average AsA-2P production rate was 5.0 g/L/h, and the AsA-2P production system was successfully scaled up to a 7.5 L fermenter. Therefore, the enzymatic process showed great potential for production of AsA-2P in industry.