Fig.1. SDS-PAGE analysis of AE5 mutant strain CotA . SDS-PAGE was used to analyze the expression of CotA. Recombinant vectors pHJ5 transformed into BL21 (DE3) competent cells and induced by 0.1 mM IPTG in LB medium for 20 h at 16 ℃ , respectively. All the samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie Blue in the gel. Lane M, protein marker. Lane 1-2, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-cotA which was induced. . Lane 5-6, whole bacterial lysate of the E.coli BL21 (DE3) containing empty pET28a. S: Supernant; P: Pellet.

We changed the temperature and the concentration of the inducer. The figure above clearly shows that the target protein induced in LB medium is present in the supernatant (Fig. 1).

Fig.2. SDS-PAGE analysis of AE5 mutant strain CotA.SDS-PAGE was used to analyze the expression of CotA . Recombinant vectors pHJ5 transformed into BL21 (DE3) competent cells and induced by 0.1 mM IPTG in LB medium for 20 h at 16 ℃ , respectively. The pellet was then dissolved in MSM medium without IPTG for 2 d at 20 ℃. All the samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie Blue in the gel. Lane M, protein marker. Lane 1-2, whole bacterial lysate of the E.coli . Lane 5-6, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-cotA and pET28a-lipH8 which were induced. Lane 7-8, whole bacterial lysate of the E.coli BL21 (DE3) containing empty pET28a. S: Supernant; P: Pellet.

After 2 d of induction in MSM medium without IPTG, the target proteins CotA can be clearly visualized in supernatant fraction of the whole bacterial lysate, which proves that the engineering iteration is effective (Fig. 2).

Fig.3. Oxidation of phenanthrene with the whole bacteria of CotA and LipH8 at 20 ℃ for 1 d, 3d, and 5d. The experiment was carried out in MSM medium without IPTG, and the oxidation was determined using noncellular components as the control. The differences in the PAH oxidation were determined by comparing the controls based on one-way ANOVA followed by Dunnett’s test (* P < 0.05).

SDS-PAGE results showed that the constructed expression system was successful. In order to verify whether the protein had biological activity, the concentration of phenanthrene was detected by HPLC. Both CotA and LipH8 could degrade phenanthrene, and coexpression of CotA and LipH8 was more effective (Fig. 3).

Fig.1. SDS-PAGE analysis of AE6 mutant strain LipH8. SDS-PAGE was used to analyze the expression of LipH8. Recombinant vectors pHJ6 transformed into BL21 (DE3) competent cells and induced by 0.1 mM IPTG in LB medium for 20 h at 20 ℃, respectively. All the samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie Blue in the gel. Lane M, protein marker. Lane 3-4, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-lipH8 which was induced. Lane 5-6, whole bacterial lysate of the E.coli BL21 (DE3) containing empty pET28a. S: Supernant; P: Pellet.

We changed the temperature and the concentration of the inducer. The figure above clearly shows that the target protein induced in LB medium is present in the supernatant (Fig. 1).

Fig.2. SDS-PAGE analysis of AE5 mutant strain CotA and AE6 mutant strain LipH8. SDS-PAGE was used to analyze the expression of CotA and LipH8. Recombinant vectors pHJ5 and pHJ6 transformed into BL21 (DE3) competent cells and induced by 0.1 mM IPTG in LB medium for 20 h at 16 ℃ and 20 ℃, respectively. The pellet was then dissolved in MSM medium without IPTG for 2 d at 20 ℃. All the samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie Blue in the gel. Lane M, protein marker. Lane 3-4, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-lipH8 which was induced. Lane 5-6, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-cotA and pET28a-lipH8 which were induced. Lane 7-8, whole bacterial lysate of the E.coli BL21 (DE3) containing empty pET28a. S: Supernant; P: Pellet.

After 2 d of induction in MSM medium without IPTG, the target proteins LipH8 can be clearly visualized in supernatant fraction of the whole bacterial lysate, which proves that the engineering iteration is effective (Fig. 2).

Fig.3. Oxidation of phenanthrene with the whole bacteria of CotA and LipH8 at 20 ℃ for 1 d, 3d, and 5d. The experiment was carried out in MSM medium without IPTG, and the oxidation was determined using noncellular components as the control. The differences in the PAH oxidation were determined by comparing the controls based on one-way ANOVA followed by Dunnett’s test (* P < 0.05).

SDS-PAGE results showed that the constructed expression system was successful. In order to verify whether the protein had biological activity, the concentration of phenanthrene was detected by HPLC. Both CotA and LipH8 could degrade phenanthrene, and coexpression of CotA and LipH8 was more effective (Fig. 3).