Part:BBa_K2936003

S- glutathione hydrolase(Regulated by flhA gene)

This is the coding sequence of the S- glutathione hydrolase. The S- glutathione hydrolase oxidizes GSCHO to formic acid.

Usage and Biology

Since the accumulation of formic acid would affect the growth of the strain, in order to further optimize the degradation efficiency of FDS-2 for formaldehyde, we found that LBFDH existing in L. buchneri could degrade formate into CO2. By introducing the gene controlling LBFDH expression, hoping to reduce the accumulation of formic acid in bacteria and improve the degradation efficiency of formate. ( Degradation principle is shown in Figure 1, and the plasmid map is shown in Figure2)

Characterize

In order to find an appropriate enzyme activity test method, after searching for literature and patents, we found a high-throughput screening method: first, we used the developer for preliminary screening, the formulation and concentration of the developer are shown in Figure 3，and the color rendering effect is shown in Figure 4.

Then we used different concentrations of NADPH to measure the OD340 value, and a linear relationship was determined by plotting, so as to determine that the value of OD340 was independent of ammonium formate and other concentrations. (the result is shown in Figure 6)
Next, we added the substrate ammonium formate, NADP+, and crude enzyme solution into the VP tube, measured the OD340, and determined the appropriate concentration of the reaction system. (The result is shown in Figure 7)
The concentration of different substrates (ammonium formate, NADP+) was used to measure the OD340 of Lbfdh, and the Km and Kcat values were calculated respectively. (The result is shown in Figure 8)
Finally, we did an enzyme assay. We calculated the values of Kcat and Km, and tested the enzyme activity of lbfdh gene, and set BL21 strain as the control group. Meanwhile, according to the defined enzyme activity formula, the enzyme activity of the experimental group and the control group was calculated and compared. Enzyme activity calculation formula = (OD340*50" dilution factor ")/(6.22" absorption coefficient per μmol NADH at 340 nm "*OD600" concentration of bacteria solution before crushing "*time" reaction time). The result is shown in Figure 9.

Experimental results

In order to get the lbfdh fragment, we conducted plasmid PCR experiments, and the nucleic acid map is shown in Figure 5.

There was a linear relationship between OD340 and NADPH, and the influence of ammonium formate and other substances on OD340 was basically ignored.
The appropriate results for each component are shown Figure 7. And the definition of IU：The amount of enzyme required to produce 1μmol NADPH in 1min.


The above data proved that the LBFDH gene we imported had enzyme activity in host bacteria.