厌氧表达乳酸脱氢酶以提高大肠杆菌产D-乳酸光学纯度

Anaerobic Expression of Lactate Dehydrogenase to Improve the D-lactic Acid Optical Purity Procluced by Escherichia coli

【目的】为解决在D-乳酸工业发酵生产中使用农业粗加工或废弃物等廉价原料(含有少量L-乳酸)而导致终产物D-乳酸光纯降低的问题。【方法】构建了带有不同启动子的L-乳酸脱氢酶基因lldD的表达质粒:pUC19-PLD(PpflBp6)、pUC19-NLD(PnirB)及pUC19-PNLD(PpflBp6-PnirB),并将它们分别转化入大肠杆菌D-乳酸工程菌HBUT-D中,得到菌株HBUT-D3、HBUT-D5和HBUT-D7。通过LldD的酶活检测以及对NBS培养基(添加1 g/L L-乳酸)发酵结果的TOPSIS多元评估,优选出可快速去除L-乳酸且不影响D-乳酸发酵的菌株,并使用农业廉价原料进行发酵。【结果】HBUT-D7的LldD比酶活为64 U/g,L-乳酸的消耗速率为34 mg/(L·h),D-乳酸的生产强度为4.09 g/(L·h),综合评估为最优菌株。以玉米浆为原料进行发酵时,HBUT-D及HBUT-D7的L-乳酸消耗速率分别为10.41 mg/(L·h)及34.75 mg/(L·h);D-乳酸生产强度分别为4.24 g/(L·h)及3.87 g/(L·h);D-乳酸光学纯度分别为99.07%及99.92%。以糖蜜为原料进行发酵时,HBUT-D及HBUT-D7的L-乳酸消耗速率为6.87 mg/(L·h)和17.18 mg/(L·h);D-乳酸生产强度分别为1.93 g/(L·h)及1.88 g/(L·h);D-乳酸光学纯度分别为99.22%及99.99%。【结论】厌氧诱导启动子的表达质粒可提高菌株HBUT-D7的L-乳酸脱氢酶酶活,使其在使用廉价原料发酵时能有效消除L-乳酸,提高发酵终产物D-乳酸的光学纯度。

【Objective】This work aims to solve the problem that the light purity of the final product D-lactic acid is reduced while using cheap raw materials(containing a small amount of L-lactic acid)such as agricultural rough processing or waste in the industrial fermentation production of D-lactic acid.【Method】The expressing plasmids of L-lactate dehydrogenase gene lldD,pUC19-PLD(PpflBp6),pUC19-NLD(PnirB)and pUC19-PNLD(PpflBp6-PnirB)with different promoters,were constructed and transformed into Escherichia coli D-lactic acid engineering strain HBUT-D,and strain HBUT-D3,HBUT-D5 and HBUT-D7 was acquired respectively.Through LldD enzymatic activity test and TOPSIS multivariate evaluation on NBS fermentative experiments(supplemented with 1 g/L L-lactic acid),one strain was screened by its capability for fast elimination of L-lactic and high fermentative efficiency of D-lactic acid,for following fermentative experiments with cheap raw materials.【Result】HBUT-D7 was evaluated as the optimal strain based on the LldD specific enzyme activity of 64 U/g,L-lactate consumption rate of 34 mg/(L·h),and D-lactic acid productivity of 4.09 g/(L·h).When fermented with corn syrup,the L-lactic acid consumption rates of HBUT-D and HBUT-D7 were 10.41 mg/(L·h)and 34.75 mg/(L·h),respectively.The productivities of D-lactic acid were 4.24 g/(L·h)and 3.87 g/(L·h),respectively.The optical purities of D-lactic acid were 99.07%and 99.92%,respectively.When fermented with molasses,the L-lactic acid consumption rates of HBUT-D and HBUT-D7 were 6.87 mg/(L·h)and 17.18 mg/(L·h),respectively.The productivities of D-lactic acid were 1.93 g/(L·h)and 1.88 g/(L·h),respectively.The optical purities of D-lactic acid were 99.22%and 99.99%,respectively.【Conclusion】The expressing plasmid of the anaerobic inducible promoter may increase the L-lactate dehydrogenase enzyme activity in strain HBUT-D7,thus it can eliminate the L-lactic acid to increase the optical purity of D-lactic acid while using cheap raw materials for fermentation.