Part:BBa_K2588009

Lactate dehydrogenase from E. coli

Lactate dehydrogenase A (ldhA) is an enzyme catalyzing the synthesis of lactate from pyruvate. As lactic acid is known to be an effective attractant for mosquitoes, we used it our project as lure.

Usage and Biology

Usually, under anaerobic conditions, E. coli may grow via two alternative energy-generating modes. If aspiration is not possible, because no electron-acceptors are present, bacteria start fermentation. Hereby, sugars are converted to reduced organic compounds for example lactic acid. The reaction is carried out via the lactate dehydrogenase which converts pyruvate into lactate under consumption of NADH.¹ Therefore, ldhA can be easily used to produce lactic acid.

Characterization

Characterization experiments were carried out with the construct pSB1C3-BBa-K206000-BBa_0034-ldhA. For the generation of cell lysates, DH5α carrying the construct was inoculated with an OD₆₀₀ = 0.1 and induced at OD₆₀₀ = 0.2 with 10 µM arabinose. Six hours after induction, cells were harvested by centrifugation. Cell lysates were either generated through resuspension in RIPA buffer and sonification for SDS-PAGE analysis or by a quick freeze and thaw protocol without detergents to keep the enzyme intact for enzyme activity assays.

First, the production of ldhA was proven by an SDS-PAGE with Coomassie Blue staining (Fig.1).

Fig. 1: SDS-PAGE gel after Coomassie Blue staining.

The SDS PAGE revealed that ldhA was successfully overexpressed in DH5α. The protein has a molecular weight of 36.5 kDa,² which corresponded with the observed band in both transformed DH5α. As a consequence of overexpression, the band of ldhA was much stronger in the induced culture. The negative control, untransformed DH5α did not show any protein band at 36.5 kDa.

Furthermore, the enzyme activity of ldhA was measured through a coupled test system (Fig. 2). It is known, that ldhA catalyzes two reactions: The reduction of pyruvate into lactate and the oxidation of lactate into pyruvate. During oxidation, NAD+ is reduced into NADH. By addition of phenazine methosulfate (PMS), which is directly reduced by NADH, and iodine tetrazoliumchloride (INT), which becomes reduced through PMS, formazan is formed. The formation of formazan can be detected with an absorption at 550 nm.

Fig. 2: Absorbance as a function of time. A linear regression was made. Our results were not significant.

Table 1: Reaction rates determined via activity assay of different cell lysates.

	ΔE /second	ΔE /minute	Coefficient of determination	Reaction rate
DH5α control	0.0002	0.012	0.9515	0.00141176
DH5α with pSB1C3-BBa_ K206000- BBa_0034-ldhA	0.0003	0.018	0.9882	0.00211765

Only a nonsignificant difference in the reaction rates of the control cell lysates from DH5α cells and DH5α transformed with pSB1C3-BBa_ K206000- BBa_0034-ldhA was observed.

Discussion

The presence of ldhA in uninduced transformed cells can be explained using the pBAD-promotor, as it is known to be leaky. Regarding the results enzyme activity assay, a purification of the pure protein may be necessary to further investigate reproducibility of the slightly higher reaction rates for ldhA overexpressing cells compared to not induced cells.

References

1. Bunch, P. K., Mat-Jan, F., Lee, N. & Clark, D. P. The IdhA Gene Encoding the Fermentative Lactate Dehydrogenase of Escherichia Coli. Microbiology 143, 187–195 (1997).
2. Uniprot.org. (2018). ldhA - D-lactate dehydrogenase - Escherichia coli (strain K12) - ldhA gene & protein. [online] Available at: https://www.uniprot.org/uniprot/P52643 [Accessed 17 Oct. 2018].

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 454
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 184