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), were 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 benefits pRSFDuet (GenBank CAD6019161.1), have been cloned into the expression vectors pETDuet and PKCη supplier indicate that HpaB and HpaC were effectively and recombinantly expressedindicate that HpaB and (Novagen, Carlsbad, CA, USA), respectively. The following benefits in E. coli cells. SDSPAGE analysis revealed that there were the presence of significant bands SDS-PAGE analysis HpaC have been successfully and recombinantly expressed in E. coli cells. corresponding to HpaB (58.5 that there were the presence of main bands corresponding to HpaB (58.five kDa) E. revealed kDa) and HpaC (18.six kDa) in samples prepared from the soluble fractions of and coli cells(18.6 kDa) in samples prepared 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 diverse Figure 1. SDS-PAGE ofof the proteins HpaB and HpaC. The proteinexpression of distinct 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 4: P1 4: co-expressionandP1 and P4. The locations HpaB P2 3: co-expression of P2 of P2 and P3; and co-expression of P1 of P4. The areas of your of the HpaB andproteins are indicated by the arrows arrows appropriate. The molecular weights of your marker and HpaC HpaC proteins are indicated by the around the on the correct. The molecular weights from the marker (180 kDa,(180 kDa, 70 kDa, 40 kDa, 35 kDa and 15 kDa) are also shown. shown. proteins proteins 100 kDa, one hundred kDa, 70 kDa, 40 kDa, 35 kDa and 15 kDa) are alsoSeveral HpaBC expression vectors have been Nav1.5 Compound constructed, and also a a two-step fermentation Numerous HpaBC expression vectors were constructed, and two-step fermentation was performed using NN as a substrate (final concentration of 200 mg -1N N and E had been L was performed using as a substrate (final concentration of 200 mg-1); ); and E were detected by HPLC [18]. The HPLC outcomes showed that the strains had remarkably distinct differdetected by HPLC [18]. The HPLC results showed that the strains had ent ortho-hydroxylation activities (Figure 2). The enzyme activities with the strains carrying ortho-hydroxylation activities (Figure two). The enzyme activities from the strains carrying the the P1 and P3(containing HpaB in MCS-1 and HpaC in MCS-2) plasmids had been substantially P1 and P3 (containing HpaB and HpaC in MCS-2) plasmids were substantially lower than these of of strains carrying the P2 and P4 (containing HpaC in MCS-1 and HpaB lower than these strains carrying the P2 and P4 (containing HpaC in MCS-1 and HpaB in in MCS-2) plasmids. example, the conversion efficiency from the of 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 3.81-fold greater than that of that from the P1-carrying and 0.41 ,15.81 0.86 mg-1) mg -1 ) was three.81-fold greater thanthe P1-carrying strain, strain, L and that on the P4-car.