全细胞催化合成L-苯基乳酸重组大肠杆菌的构建

Production of L-phenyllactic acid from whole-cell recombinant Escherichia coli

苯基乳酸是一种新型天然广谱抑菌物质,L-乳酸脱氢酶是微生物转化苯丙酮酸合成L-苯基乳酸的关键酶,通过构建过量表达L-乳酸脱氢酶的重组大肠杆菌,提高大肠杆菌合成L-苯基乳酸(苯基乳酸,phenyllactic acid,PLA)的能力。以Bacillus megaterium Z2013513基因组为模板,经PCR扩增得到L-乳酸脱氢酶基因,连接表达载体p ET-28a(+)并导入到大肠杆菌BL21(DE3),获得重组大肠杆菌BL21(DE3)/p ET-28a-ldh L。SDS-PAGE电泳和酶活分析表明,约在40 ku处出现显著特异性条带,粗酶液酶活力达3.4 U/mg。在37℃、200 r/min条件下,25 g/L(干重)重组大肠杆菌经60 min将70.32 mmol/L苯丙酮酸全细胞转化合成50.59 mmol/L L-苯基乳酸。由于具有较高的产物光学纯度(96.98%e.e.)和底物摩尔转化率(71.94%),表明一步生物转化法能高效地合成L-苯基乳酸。

Phenyllactic acid has recently been discovered to be a potential antiseptic agent with broad spectrum activity. L-lactate dehydrogenase is a key enzyme for microbial synthesis of L-phenyllactic acid from phenylpyruvic acid. Recombinant E. coli BL21（ DE3） / p ET28a-ldh L overexpressing L-lactate dehydrogenase from Bacillus megaterium Z2013513 was constructed in this study to enhance the production of L-PLA. The L-lactate dehydrogenase gene ldh L of B. megaterium was first cloned into vector p ET-28a（ ＋）. The recombinant vector was then transfered into E.coli BL21（ DE3）. SDS-PAGE analysis showed that ldh L was successfully over expressed in the recombinant strain,which was about 40 ku. Enzyme activity analysis showed the specific activity of the crude cell-free extract was 3. 4 U /mg. Using 25 g / L（ dry cell weight） recombinant E. coli whole-cells,50. 59 mmol / L L-phenyllactic acid was obtained from 70. 32 mmol / L phenylpyruvic acid at 37 ℃,200 r / min after 60 min transformation. Due to an excellent product enantiomeric excess of 96. 98% and a high molar conversions ratio of 71. 94%,L-phenyllactic acid was efficiently produced by the one-pot biotransformation system.