Promising approaches for the assembly of the catalytically active, recombinant Desulfomicrobium baculatum hydrogenase with substitutions at the active site

Table 1 Comparison of the recombinant Dmb hydrogenase variant activities in the spectrophotometric methyl viologen assay

Construct (sample tested)	average hydrogenase activity [µmoles H₂/min/mg of protein]	standard deviation
LH U493C_STOP499_pMCSG53 + SH_pMCSG53 (in vitro assembly)	81.368	± 0.15
SH_rbs517_LH U493C_STOP499_pMCSG53 (in vivo assembly)	85.344	± 0.11
LH U493M_STOP499_pMCSG53 + SH_pMCSG53 (in vitro assembly)	28.644	± 0.11
SH_rbs517_LH U493M_STOP499_pMCSG53 (in vivo assembly)	38.278	± 0.01
LH U493M_STOP499_pMCSG53	0	0
LH U493C_STOP499_pMCSG53	0	0
SH_pMCSG53	0	0
assay buffer	0	0
E. coli BL21-Gold(DE3) strain (aerobic conditions)	0.0016	0
E. coli BL21-Gold(DE3) strain	0.0017	0

The recombinant Dmb H2ase preparations, containing 0.3 mg of the purified protein variant, were obtained via in vivo or in vitro assembly. The small H2ase subunit (SH) was subjected to a solubilization procedure prior to in vitro assembly. Single H2ase subunits: SH, LH U493C_STOP499 (truncated), LH U493M_STOP499 (truncated), prepared in a similar manner to the operon derived enzyme, showed no activity. A crude lysate from the E. coli BL21(DE3)Gold strain culture, which was grown in parallel with the IPTG induced recombinant Dmb H2ase producing cultures, was used as a negative control. An additional control sample of the strain, grown overnight in standard aerobic conditions, was also included. A sample of the assay buffer contained all of the components of the assay except the enzyme preparation/lysate. All the samples were prepared in triplicate and measurements were undertaken with shaking during 6 min kinetic runs with a measurement interval of 1 min.

ISSN: 1475-2859