苯基乳酸(phenyllactic acid, PLA)是一种重要的有机酸,广泛应用于食品、医药、化妆品等领域。通过构建D-乳酸脱氢酶重组大肠杆菌BL21(DE3)/pET28a-ldh D利用全细胞催化合成D-苯基乳酸。对重组大肠杆菌的诱导及转化条件进行了优化,最终确定了最佳的诱导条件:OD600=0.8时,添加IPTG至终浓度为0.2 mmol/L,在25 ℃下诱导4 h;最佳的转化条件:pH 7.0磷酸缓冲液,13 g/L苯丙酮酸钠,5 g/L葡萄糖,30 g/L菌体(干重),37 ℃,200 r/min,转化2 h。在最优的条件下,苯基乳酸的产物浓度达到5.2 g/L,产率为40%。该文还探索了苯丙酮酸钠与葡萄糖分批添加对苯基乳酸合成的影响,并初步得到了最佳工艺,经3 h转化,苯基乳酸产物质量浓度和产率分别提高到7.38 g/L和52.7%,显示了较好的应用前景,也为后续酶的改造奠定了基础。
Phenyllactic acid is an important organic acid that has been widely used in foods, pharmaceuticals, cosmetics and other fields. D-phenyllactic acid was catalytically synthesized by whole cells through constructing recombinant Escherichia coli BL21 (DE3)/pET28a-ldh D. The induction and transformation conditions were optimized and determined as follows: IPTG was added to a final concentration of 0.2 mmol/L when OD600 reached 0.8 and cells were induced at 25 ℃ for 4 h. The optimal transformation conditions were as follows: using pH=7.0 phosphate buffer, 13 g/L sodium phenylpyruvate, 5 g/L glucose, 30 g/L cells (dry weight), at 37 ℃ and 200 r/min for 2 h. Under the optimal conditions, phenyllactic acid accumulated up to 5.2 g/L and the yield was 40%. Effects of fed-batch process using sodium pyruvate and glucoase on phenyllactic acid synthesis were explored, and the best processing scheme was obtained. After 3 h of conversion, the concentration of D-phenyllactic and its yield increased to 7.38 g/L and 52.7%, respectively. This study showed that the process of whole cell transformation for D-phenyllactic acid synthesis had good application prospects, which laid a foundation for subsequent enzymatic modification.
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