高密度发酵产酪氨酸酚裂解酶及催化合成L-DOPA

High-density fermentation for preparing tyrosine phenol lyase and application in L-DOPA synthesis

为了提高酪氨酸酚裂解酶(tyrosine phenol-lyase,TPL)的表达量和酶活,使之更高效地催化合成左旋多巴(3,4-2-dihydroxylphenylalanine,L-DOPA),在摇瓶优化的基础上,研究了溶氧控制策略、补料策略、诱导温度和诱导策略对菌体生长、TPL酶活和L-DOPA产量的影响。结果表明,在5 L发酵罐中25℃诱导条件下,采用DO-stat补料策略控制罐内溶氧的体积分数为20%,菌体浓度、TPL酶活和催化合成L-DOPA产量较分批培养分别提高了17.9%、57.7%和27.8%。诱导后控制溶氧体积分数在40%相比于20%更利于TPL的表达,酶活相比于20%的DO的提高了33.8%。通过优化起始诱导时间,在菌体生长的对数前期(OD 600=7)诱导,细胞浓度提高到51.2 g/L,酶活提高到2.43 U/mL,L-DOPA产量为56.58 g/L,较分批培养分别提高了64.1%、170%和209.2%。初步实现了重组大肠杆菌高密度培养生产TPL,为L-DOPA产业化研究奠定了基础。

In order to improve the expression level of tyrosine phenol lyase(TPL)and to make it more efficient to catalyze L-DOPA synthesis,influences of dissolved oxygen control strategy,feeding strategy and induction strategy on cell growth,TPL activity and L-DOPA production were studied.By using DO-stat feeding strategy to maintain the volume fraction of dissolved oxygen at 20%in a 5 L fermenter at 25℃,the cell density,TPL activity and the titer of L-DOPA increased by 17.9%,57.7%,and 27.8%,respectively.The enzyme activity further increased by 33.8%with the volume fraction of the dissolved oxygen changed from 20%to 40%after induction.When the cells were inducted during the logarithmic phase of cell growth(OD 60 0=7),the maximal cell density,TPL activity and L-DOPA titer raised to 51.2 g/L,2.43 U/mL,and 56.58 g/L,respectively,which was 64.1%,170%,and 209.2%,respectively,higher than that in batch cultures.This study preliminarily realized high density culture of recombinant Escherichia coli to produce TPL,which provides technical supports for the industrialization of L-DOPA.