Figure 1.
Multiple sequence alignment of homologue small laccases.
Ssl1 from Streptomyces sviceus, SilA from S. ipomoea, EpoA from S. griseus and SLAC from S. coelicolor. The copper binding residues are conserved in all 4 laccases as indicated. All four laccases consist of 2 domains (indicated in Ssl1, black: domain 1, dark grey: domain 2). Signal sequences of the twin-arginine pathway are indicated in light grey. Sequence identity between the four laccases is high and variations are mainly located at the termini.
Figure 2.
SDS-PAGE analysis of recombinant expression and purification process of Ssl1.
Ssl1 migrates at 33 kDa. It was expressed in E coli-CodonPlus (DE3)-RP and purified to homogeneity by heat precipitation and immobilized metal affinity chromatography (IMAC). M: molecular size marker, lane 1: cell extract before induction, lane 2: cell extract after expression, lane 3: soluble fraction from cell disruption, lane 4: soluble fraction from heat precipitation, lane 5: IMAC eluate.
Figure 3.
UV/vis spectrum of Ssl1 in potassium phosphate buffer.
Ssl1 shows a laccase-typical absorption spectrum. The maximum at 592 nm (εSsl1, 592 nm = 2.796±0.191 mM−1 cm−1) corresponds to type I or blue copper and the shoulder around 330 nm is characteristic for type 3 copper centers.
Figure 4.
pH optima and pH stability of Ssl1.
A: Stability of Ssl1 in buffers with different pH values was tested as residual oxidation activity towards 2,6-dimethoxy phenol. Ssl1 was inactivated within 30 min at pH 3 (values shown as diamonds), and within 3 days at pH 4 (circles). Stability at pH 5 (open circles) and pH 7 (squares) was similar with a half-time around 4 to 5 days. At pH 11 (triangles) Ssl1 was most stable with more than 70% residual activity after 7 days. B: Relative activities of Ssl1 at different pH values towards the substrates ABTS (values shown as diamonds), syringaldazine (circles), 2,6-dimethoxy phenol (triangles) and guaiacol (squares). All activities were normalized to the values at optimum pH with the respective substrate. Optimal pH values are 4 for ABTS, 8 for syringaldazine, and 9 for 2,6-dimethoxy phenol and guaiacol.
Table 1.
Figure 5.
Effect of detergents, salts and organic co-solvents on Ssl1.
A: Relative activity of Ssl1 in presence of organic solvents, salts and detergents towards 2,6-dimethoxyphenol oxidation. Water immiscible solvents did not affect Ssl1 activity, with water miscible co-solvents the activity dropped to 20–40%. Ssl1 tolerated the addition of 10 mM sodium azide, with the detergents SDS and Triton-X-100 the activity was reduced to 60 or 80%. B: Residual activity of Ssl1 after 20 h incubation with organic solvents, salts and deteregents. DMSO stabilized Ssl1 whereas acetonitrile lead to a reduction in residual activity. All other studied additives did not lead to major changes of Ssl1 stability.