Characterization of the Alkaline Laccase Ssl1 from Streptomyces sviceus with Unusual Properties Discovered by Genome Mining

Matthias Gunne,Giovanni Maga,Vlada B. Urlacher

doi:10.1371/journal.pone.0052360

Abstract

Fungal laccases are well investigated enzymes with high potential in diverse applications like bleaching of waste waters and textiles, cellulose delignification, and organic synthesis. However, they are limited to acidic reaction conditions and require eukaryotic expression systems. This raises a demand for novel laccases without these constraints. We have taken advantage of the laccase engineering database LccED derived from genome mining to identify and clone the laccase Ssl1 from Streptomyces sviceus which can circumvent the limitations of fungal laccases. Ssl1 belongs to the family of small laccases that contains only few characterized enzymes. After removal of the twin-arginine signal peptide Ssl1 was readily expressed in E. coli. Ssl1 is a small laccase with 32.5 kDa, consists of only two cupredoxin-like domains, and forms trimers in solution. Ssl1 oxidizes 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and phenolic substrates like 2,6-dimethoxy phenol, guaiacol, and syringaldazine. The kcat value for ABTS oxidation was at least 20 times higher than for other substrates. The optimal pH for oxidation reactions is substrate dependent: for phenolic substrates the highest activities were detected at alkaline conditions (pH 9.0 for 2,6-dimethoxy phenol and guaiacol and pH 8.0 for syringaldazine), while the highest reaction rates with ABTS were observed at pH 4.0. Though originating from a mesophilic organism, Ssl demonstrates remarkable stability at elevated temperatures (T1/2,60°C = 88 min) and in a wide pH range (pH 5.0 to 11.0). Notably, the enzyme retained 80% residual activity after 5 days of incubation at pH 11. Detergents and organic co-solvents do not affect Ssl1 stability. The described robustness makes Ssl1 a potential candidate for industrial applications, preferably in processes that require alkaline reaction conditions.

Highlights

Significant progress has been achieved in enzyme engineering, the discovery and characterization of novel enzymes from diverseorganisms still plays an essential role for the development of biocatalytic processes
The electrons are channeled through highly conserved copper binding residues from the substrate oxidizing T1 copper site to the T2/T3 trinuclear copper cluster where oxygen is bound and reduced to water by four electrons [2]
The characteristics and biotechnological potential of these enzymes are poorly investigated and still few reports on bacterial laccases have been published. These reports demonstrate the thermal robustness and more alkaline activity profiles of bacterial laccases compared to fungal enzymes

Summary

Introduction

Significant progress has been achieved in enzyme engineering, the discovery and characterization of novel enzymes from diverse (micro)organisms still plays an essential role for the development of biocatalytic processes. These reports demonstrate the thermal robustness and more alkaline activity profiles of bacterial laccases compared to fungal enzymes. We describe the cloning, expression and characterization of the small two-domain Ssl1 laccase from Streptomyces sviceus.

Results

Conclusion