DhaA was first conjugated with acetylated pullulan. The anionic conjugate (pullulan-DhaA) interacted with cationic PEI to form PEI-pullulan-DhaA. As a control for PEI-pullulan-DhaA, pullulan-DhaA was used to investigate the effect of PEI on the stability of DhaA.
Characterization of PEI-pullulan-DhaA
As shown in Fig. 1a, DhaA exhibited a single band with an apparent Mw of ∼36.0 kDa in Lane 2. Pullulan-DhaA displayed a wide and disperse electrophoresis band with an Mw range of 40–200 kDa in Lane 3. Thus, DhaA was conjugated with pullulan, and free DhaA was not observed in the conjugate.
The DhaA samples were analyzed by an analytical Superdex 200 column (1 cm × 30 cm). As shown in Fig. 1b, the elution peak of DhaA was single and symmetric at 21.0 mL. Pullulan-DhaA was eluted as a single peak at 16.9 mL, which was left-shifted as compared with DhaA. The elution peak of PEI-pullulan-DhaA (12.1 mL) was in the void volume of the column that exceeded its exclusion limit, which was left-shifted as compared with pullulan-DhaA. This indicated that pullulan conjugation and PEI adsorption could significantly increase the size of DhaA.
As shown in Fig. 1c, the size distributions of the DhaA samples were all found to be unimodal. The centered point was used to obtain the molecular diameters. The molecular diameter of pullulan-DhaA (23.4 ± 5.6 nm) was higher than that of DhaA (8.7 ± 1.8 nm). This indicated that conjugation of pullulan could significantly increase the size of DhaA. In contrast, PEI-pullulan-DhaA showed a molecular diameter of 164.2 ± 23.5 nm with an average PDI of 0.17. This revealed that adsorption with PEI could further increase the size of pullulan-DhaA.
As shown in Fig. 1d, PEI-pullulan-DhaA displayed the appearance of spheres with a size range of 100–150 nm. In addition, PEI-pullulan-DhaA did not show the sign of aggregation with a smooth surface.
The FT-IR spectra of the DhaA samples were measured in the range of 4000 − 400 cm− 1. As shown in Fig. 1e, pullulan exhibited a typical peak at 3420 cm− 1 (O-H stretching). As compared with pullulan, pullulan-DhaA showed a left-shifted peak with stronger absorption at 3436 cm− 1. This was ascribed to O-H stretching and N-H stretching, due to the conjugation of pullulan with DhaA. The peak of PEI-pullulan-DhaA was at 3432 cm− 1, due to the incorporation of amino groups from PEI. The absorption peak appeared at 1000–1100 cm− 1 was observed for pullulan-DhaA and PEI-pullulan-DhaA, due to the stretching vibration of C-C, C-O, and C-H in pullulan. As compared with pullulan and PEI, pullulan-DhaA and PEI-pullulan-DhaA both showed a stronger absorption peak at 1661 cm− 1, the characteristic peak of the amide I band. This was due to the incorporation of N-H (peptide chain) bending vibration from DhaA.
INSERT Fig. 1
Enzymatic activity and kinetic parameters
The enzymatic activity and kinetic parameters of the DhaA samples were summarized in Table 1. The enzymatic activity of DhaA was set as 100%. The relative enzymatic activities of pullulan-DhaA and PEI-pullulan-DhaA were 89.3% and 78.8%, respectively. This suggested that pullulan conjugation and PEI adsorption could slightly decrease the enzymatic activity of DhaA. As compared with DhaA, PEI-pullulan-DhaA exhibited somewhat increased Km, kcat and kcat/Km values, representing slightly increased catalytic efficiency, and weakened enzyme-substrate affinity. The interaction among PEI, pullulan and the substrate rendered the enrichment of the substrate around DhaA. This could increase the catalytic efficiency of DhaA, as reflected by the enhanced kcat and kcat/Km values. In contrast, the bound PEI and pullulan hindered the access of the substrate to the active site of DhaA and decreased the affinity of the substrate to DhaA, as reflected by the enhanced Km value.
INSERT Table 1
Thermal stability and structural analysis
The DhaA samples were treated at 50 ℃ to evaluate the heat stability of DhaA. As shown in Fig. 2a, the enzymatic activity of DhaA decreased quickly at 50 ℃. The enzymatic activity of pullulan-DhaA rapidly decreased and its decrease rate was slightly slower than that of DhaA. In contrast, the enzymatic activity of PEI-pullulan-DhaA decreased slowly. In particular, the relative activities of PEI-pullulan-DhaA were 49.8% at 20 hours and 10.1% at 60 hours, whereas those of pullulan-DhaA and DhaA were almost abolished at 20 hours. Moreover, the half-life time of PEI-pullulan-DhaA (15.9 h− 1) was higher than those of DhaA (1.3 h− 1) and pullulan-DhaA (1.9 h− 1). This indicated that the heat stability of DhaA was slightly increased by conjugation of pullulan and significantly enhanced by adsorption with PEI.
The secondary structures of the DhaA samples were analyzed by far-UV CD spectroscopy. As shown in Fig. 2b, DhaA exhibited double negative maximum ellipticity around 208 nm and 222 nm, characteristic of α-helical content. In contrast, the CD spectra of pullulan-DhaA and PEI-pullulan-DhaA showed slightly changes in the double negative maximum ellipticity. This suggested that the secondary structure of DhaA remained preserved upon pullulan conjugation and PEI adsorption. The ellipticity values of the three samples all decreased upon heat treatment at 50 oC for 1 hour, indicating the structural disorder of DhaA. However, PEI-pullulan-DhaA showed larger ellipticity than DhaA and pullulan-DhaA upon heat treatment. This indicated that conjugation with pullulan and adsorption with PEI increased the resistance of DhaA to heat treatment by maintaining the secondary structure of DhaA.
As shown in Fig. 2c, DhaA showed a single peak with the maximum intrinsic fluorescence intensity at 335 nm when excited at 280 nm. Pullulan-DhaA and PEI-pullulan-DhaA both exhibited slightly lower fluorescence intensity than DhaA, along with the maximum fluorescence emission wavelength (λmax) of 336 nm. The fluorescence intensities of the three DhaA samples all reduced upon heat treatment. However, PEI-pullulan-DhaA showed higher intensity than DhaA and pullulan-DhaA upon heat treatment. Moreover, the λmax values of the heat-treated DhaA and pullulan-DhaA were red-shifted to 342 nm, whereas the λmax value of the heat-treated PEI-pullulan-DhaA was still at 336 nm. This indicated that conjugation with AG and adsorption with PEI could maintain the tertiary structure of DhaA and prevent the enzyme conformation from being disordered upon heat treatment.
ANS acted as a fluorescent probe to study the hydrophobicity of the DhaA samples. As shown in Fig. 2d, the extrinsic fluorescence (EF) intensity of pullulan-DhaA was slightly higher than that of DhaA and lower than that of PEI-pullulan-DhaA, along with λmax of 491 nm. This was due to the binding of ANS with the amphiphilic PEI. The EF intensities of the three samples all increased upon heat treatment. However, PEI-pullulan-DhaA showed lower intensity than DhaA and pullulan-DhaA upon heat treatment. Moreover, the λmax value of the heat-treated DhaA was red-shifted to 478 nm, whereas the λmax values of the heat-treated pullulan-DhaA and PEI-pullulan-DhaA were both at 485 nm. This suggested that pullulan conjugation and PEI adsorption could additively stabilize the conformation of DhaA upon heat treatment.
INSERT Fig. 2
pH stability and structural analysis
As shown in Fig. 3a, the enzymatic activities of the DhaA samples were relatively stable in the pH range of 7.0–9.0. In contrast, the enzymatic activities of the DhaA samples all quickly decreased below pH 6.0. Interestingly, the relative activities of PEI-pullulan-DhaA were higher than those of DhaA and pullulan-DhaA in the pH range of 3.0–6.0. In particular, the enzymatic activity of DhaA almost abolished at pH 3.0 and pH 4.0. Pullulan-DhaA showed low activity at pH 3.0 (5.1%) and pH 4.0 (8.6%). In contrast, PEI-pullulan-DhaA exhibited higher relative activity at pH 3.0 (19.1%) and pH 4.0 (44.4%). This indicated that adsorption with PEI played a predominant role to maintain the pH stability of DhaA.
As shown in Fig. 3b, the ellipticity values of the DhaA samples decreased at pH 4.0 as compared with those at pH 7.4. Moreover, the CD spectrum of DhaA at pH 4.0 was almost abolished. Pullulan-DhaA at pH 4.0 showed larger ellipticity than DhaA at pH 4.0 and lower ellipticity than PEI-pullulan-DhaA at pH 4.0. Thus, conjugation with pullulan and adsorption with PEI increased the pH stability of DhaA by maintaining the secondary structure of DhaA.
As shown in Fig. 3c, the fluorescence intensities of the three DhaA samples at pH 4.0 were lower than those at pH 7.4, indicating that these samples were in disordered conformation at pH 4.0. Moreover, pullulan-DhaA at pH 4.0 showed larger intensity than DhaA at pH 4.0 and lower intensity than PEI-pullulan-DhaA at pH 4.0. Thus, conjugation with pullulan and adsorption with PEI could enhance the resistance of DhaA to low pH by maintaining the tertiary structure of DhaA.
As shown in Fig. 3d, the EF intensities of the DhaA samples at pH 7.4 were comparable with each other, along with the maximum at 488 nm. The EF intensities of the DhaA samples at pH 4.0 were higher than those at pH 7.4. Moreover, DhaA at pH 4.0 showed the highest intensity among the samples, along with the maximum blue-shifted to 470 nm. In contrast, PEI-pullulan-DhaA at pH 4.0 showed the lowest intensity among the three samples, along with the maximum blue-shifted to 477 nm. Thus, conjugation with pullulan and adsorption with PEI increased the pH stability of DhaA.
INSERT Fig. 3
Hypersaline stability
The hypersaline stability of the DhaA samples was investigated in the presence of 1.5 M NaCl and 2 M urea, respectively. As shown in Fig. 4a, the remaining activity of pullulan-DhaA (33.1%) was slightly higher than that of DhaA (27.3%) in 1.5 M NaCl (P < 0.05). In contrast, the remaining activity of PEI-pullulan-DhaA (61.1%) was significantly higher than that of pullulan-DhaA (33.1%) in 1.5 M NaCl. As shown in Fig. 4b, the remaining activity of pullulan-DhaA (54.9%) did not show significant difference to that of DhaA (51.9%) in 2 M urea. In contrast, the remaining activity of PEI-pullulan-DhaA (87.1%) was significantly higher than that of pullulan-DhaA (54.9%) in 2 M urea (P < 0.01). This suggested that adsorption with PEI played a predominant role in protection of DhaA against hypersaline.
Organic solvent stability
Halogenated compounds are byproducts commonly produced in organic solvent-based industries. Thus, the organic solvent stability of the DhaA samples was investigated in the presence of 40% (v/v) DMSO solution. As shown in Fig. 4c, the remaining activity of DhaA was 17.4% in 40% (v/v) DMSO solution at 25°C. The remaining activity of pullulan-DhaA (57.3%), was higher than that of DhaA (17.4%, P < 0.01) and lower than that of pullulan-DhaA (87.1%, P < 0.01). This suggested that pullulan conjugation and adsorption with PEI both additively increased the organic solvent stability of DhaA in 40% (v/v) DMSO solution.
Storage stability
The DhaA samples were all incubated at 20 oC. As shown in Fig. 4d, the enzymatic activity of DhaA decreased significantly during the range of 0–9 days, and then lost activity. In contrast, the enzymatic activity of pullulan-DhaA was higher than that of DhaA during the entire incubation time, along with the relative activity of 6.3% on 40 days. The enzymatic activity of PEI-pullulan-DhaA slightly decreased during the range of 0–40 days. Particularly, the activity of PEI-pullulan-DhaA was 64.2% on 40 days. Thus, PEI adsorption could significantly improve the long-term storage stability of DhaA.
INSERT Fig. 4