Heterologous expression of a Streptomyces cyaneus laccase for biomass modification applications

Abstract

Laccases are used for the conversion of biomass into fermentable sugars but it is difficult to produce high yields of active laccases in heterologous expression systems. We overcame this challenge by expressing Streptomyces cyaneus CECT 3335 laccase in Escherichia coli (ScLac) and we achieved a yield of up to 104 mg L−1 following purification by one-step affinity chromatography. Stability and activity assays using simple lignin model substrates showed that the purified enzyme preparation was active over a broad pH range and at high temperatures, suggesting it would be suitable for biomass degradation. The reusability of ScLac was also demonstrated by immobilizing the enzyme on agarose beads with a binding yield of 33%, and by the synthesis of cross-linked enzyme aggregates with an initial activity recovery of 72%.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0387-0) contains supplementary material, which is available to authorized users.

Identification and Characterization of New Laccase Biocatalysts from Pseudomonas Species Suitable for Degradation of Synthetic Textile Dyes

“…Obtained results are in accordance with available literature data: CopA from P. putida KT2440, laccase from Bacillus coagulans, and B. clausii laccase [26] exhibited maximum activity on ABTS as a substrate on pH 4 [5]. Furthermore, laccase from B. licheniformis showed maximal activity at pH 3 [3] while laccase from Streptomyces cyaneus had the maximum activity at pH 5 [4]. maximum activity at pH 5 [4].…”

“…All tested enzymes exhibited high temperature stability at 60 °C, since their activities were slightly elevated in comparison to control (enzyme activity measured before incubation at 60 °C and 80 °C) that was arbitrarily set to 100%, even after 2h of incubation ( Figure 4C). Temperature activation of enzyme is also previously shown by Ece and coworkers [4]. McoCA3 enzyme exhibited the best temperature stability overall, as it retained 55% of its initial activity after 120 min at 80 °C, while Cbp, CopA, and McoKT retained 16%, 29%, and 20% of activity, respectively ( Figure 4D).…”

“…Furthermore, laccase from B. licheniformis showed maximal activity at pH 3 [3] while laccase from Streptomyces cyaneus had the maximum activity at pH 5 [4]. maximum activity at pH 5 [4].…”

“…Proteins of interest were purified with Ni-NTA affinity chromatography, and their activity was confirmed using zymography assay ( Figure 3). Although most of the laccases studied thus far are of fungal origin, they have limitations including difficulties in heterologous protein expression due to the need for posttranslational modifications, as well as narrow temperature and pH range [4,14]. During the last decade bacterial laccases gained more attention [31,45] due to the number of advantages over fungal laccases, one of which is being more amenable for recombinant expression and directed evolution studies in an E. coli host.…”

“…Laccases are blue multicopper enzymes (EC 1.10.3.2) that oxidize a broad range of both phenolic and non-phenolic substrates, via a four-electron reduction of molecular oxygen to water [1,2]. These enzymes are widely distributed in nature and have been isolated from bacteria [3][4][5], fungi [6,7] and plants [8,9], as well as from lichens [10], and sponges [11]. Although laccases are heterogeneous in different species, implying diversity in their function, four copper-binding motifs are conserved in the…”