TABLE 2

Characterization of purified hAOX1-WT, the codon-optimized co-hAOX1 protein, and hAOX1 variants C44W, S1271L, and G1269R after expression in E. coli TP1000 cells

hAOX1-WTco-hAOX1hAOX1- S1271LhAOX1- C44WhAOX1- G1269R
Total yield of protein 
(mg/l of culture)0.09 ± 0.021.1 ± 0.30.9 ± 0.1≥0.00051.2 ± 0.3
Specific activitya (mU/mg) of purified enzyme w/o in vitro chemical sulfuration113.6 ± 34.1141.4 ± 29.8126.7 ± 21
Specific activitya (mU/mg) of purified enzyme after in vitro chemical sulfuration1516.7 ± 182.71459.1 ± 170.51132.1 ± 155.3
Mo content (%)b55.2 ± 8.4166.4 ± 6.5841.3 ± 3.5431.2 ± 3.43
Fe content (%)b71.2 ± 10.3776.7 ± 5.6358.6 ± 4.2172.3 ± 4.66
  • a Specific activity was determined using 40 μM phenanthridine as substrate and molecular oxygen as electron acceptor.

  • b Molybdenum and iron content were determined by inductively coupled plasma optical emission spectrometry (ICP-OES). Values are related to the theoretical 100% full complement of Moco and the 2× [2Fe2S] clusters.