F HpaBframes (ORFs) of HpaB, which encodes for the monooxygenase componentopen reading frames (ORFs) and HpaC which encodes for oxidoreductase comThe (GenBank CAD6019151.1), of HpaB, which encodes for the monooxygenase element (GenBank CAD6019161.1), had been HpaC which the expression vectors pETDuet and ponent (GenBank CAD6019151.1), and cloned into encodes for oxidoreductase element pRSFDuet (Novagen, Carlsbad, CA, USA), respectively. The following outcomes pRSFDuet (GenBank CAD6019161.1), were cloned into the expression vectors pETDuet and indicate that HpaB and HpaC had been successfully and recombinantly expressedindicate that HpaB and (Novagen, Carlsbad, CA, USA), respectively. The following benefits in E. coli cells. SDSPAGE evaluation revealed that there have been the presence of important bands SDS-PAGE evaluation HpaC were successfully and recombinantly expressed in E. coli cells. corresponding to HpaB (58.5 that there have been the presence of important bands corresponding to HpaB (58.5 kDa) E. revealed kDa) and HpaC (18.6 kDa) in samples prepared in the soluble fractions of and coli cells(18.six kDa) in samples ready in the soluble fractions of E. coli cells (Figure 1). HpaC (Figure 1).Molecules 2021, 26,Figure 1. SDS-PAGE the proteins HpaB and HpaC. The protein expression of different Figure 1. SDS-PAGE ofof the proteins HpaB and HpaC. The proteinexpression of various plasmids in in BL21 cells. P1: pRSFDuet-HpaBC; P2: pRSFDuet-HpaCB; P3: pETDuet-HpaBC; P4: pETDuet-HpaCB; BL21 cells. P1: pRSFDuet-HpaBC; P2: pRSFDuet-HpaCB; P3: pETDuet-HpaBC; P4: pETDuetHpaCB; P2 3: co-expressionand P3; and P1 four: P1 four: co-expressionandP1 and P4. The places HpaB P2 three: co-expression of P2 of P2 and P3; and co-expression of P1 of P4. The places in the from the HpaB andproteins are indicated by the arrows arrows proper. The molecular weights of your marker and HpaC HpaC proteins are indicated by the on the on the VEGFR1/Flt-1 Purity & Documentation appropriate. The molecular weights on the marker (180 kDa,(180 kDa, 70 kDa, 40 kDa, 35 kDa and 15 kDa) are also shown. shown. proteins proteins one hundred kDa, 100 kDa, 70 kDa, 40 kDa, 35 kDa and 15 kDa) are alsoSeveral HpaBC expression vectors have been constructed, plus a a two-step PKCθ list fermentation Several HpaBC expression vectors have been constructed, and two-step fermentation was performed working with NN as a substrate (final concentration of 200 mg -1N N and E had been L was performed applying as a substrate (final concentration of 200 mg-1); ); and E have been detected by HPLC [18]. The HPLC final results showed that the strains had remarkably unique differdetected by HPLC [18]. The HPLC benefits showed that the strains had ent ortho-hydroxylation activities (Figure two). The enzyme activities on the strains carrying ortho-hydroxylation activities (Figure 2). The enzyme activities of the strains carrying the the P1 and P3(containing HpaB in MCS-1 and HpaC in MCS-2) plasmids had been significantly P1 and P3 (containing HpaB and HpaC in MCS-2) plasmids were substantially reduce than these of of strains carrying the P2 and P4 (containing HpaC in MCS-1 and HpaB reduce than those strains carrying the P2 and P4 (containing HpaC in MCS-1 and HpaB in in MCS-2) plasmids. instance, the conversion efficiency on the on the P2-carrying(7.47 MCS-2) plasmids. For As an example, the conversion efficiency P2-carrying strain strain (7.47 0.41 ,15.81 0.86 was three.81-fold larger than that of that from the P1-carrying and 0.41 ,15.81 0.86 mg-1) mg -1 ) was three.81-fold higher thanthe P1-carrying strain, strain, L and that from the P4-car.
