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