Yuzhi Hong

A new marine bacterial laccase with chloride-enhancing, alkaline-dependent activity and dye decolorization ability

A bacterial laccase gene designated as lac21 was screened from a marine microbial metagenomic library of the South China Sea based on sequence screening strategy. The protein encoded by lac21 shared less than 40% sequence identities with all of the laccases found. Lac21, which was recombinantly expressed in Escherichia coli, showed high activity toward syringaldazine at an optimum pH of 7.5 and temperature of 45°C. Lac21 was stable at pH values ranging from 5.5 to 9.0 and temperatures lower than 40°C. Interestingly, chloride enhanced the laccase activity, with concomitant increase in substrate affinity. Furthermore, Lac21 has high decolorization capability toward azo dyes in the absence of redox mediators, with 80% of Reactive Deep Blue M-2GE (50mg/L) being decolorized by 15U/L enzyme after 24h incubation at 20°C. These unusual properties demonstrate that the new bacterial laccase Lac21 has potentials in specific industrial or environmental applications.

Cloning of a laccase gene from a novel basidiomycete Trametes sp. 420 and its heterologous expression in Pichia pastoris.

The laccase gene lacD, cloned from a novel laccase-producing basidiomycete Trametes sp. 420, contained 2,052 base pairs (bp) interrupted by 8 introns. lacD displayed a relatively high homology with laccase genes from other white rot fungi, whereas the homology between lacD and laccase genes from plants, insects, or bacteria was less than 25%. A 498–amino acid peptide encoded by the lacD cDNA was heterologously expressed in the Pichia pastoris strain GS115, resulting in the highest yield of laccase (8.3 × 104 U/l) as determined with ABTS (2,2′-azinobis [3-ethylbenzothia-zoline-6-sulfonic acid]) as the substrate. Additionally, the enzyme activity of recombinant laccase on decolorization of some industrial dyes was assessed.

Evidence for lignin oxidation by the giant panda fecal microbiome.

The digestion of lignin and lignin-related phenolic compounds from bamboo by giant pandas has puzzled scientists because of the lack of lignin-degrading genes in the genome of the bamboo-feeding animals. We constructed a 16S rRNA gene library from the microorganisms derived from the giant panda feces to identify the possibility for the presence of potential lignin-degrading bacteria. Phylogenetic analysis showed that the phylotypes of the intestinal bacteria were affiliated with the phyla Proteobacteria (53%) and Firmicutes (47%). Two phylotypes were affiliated with the known lignin-degrading bacterium Pseudomonas putida and the mangrove forest bacteria. To test the hypothesis that microbes in the giant panda gut help degrade lignin, a metagenomic library of the intestinal bacteria was constructed and screened for clones that contained genes encoding laccase, a lignin-degrading related enzyme. A multicopper oxidase gene, designated as lac51, was identified from a metagenomic clone. Sequence analysis and copper content determination indicated that Lac51 is a laccase rather than a metallo-oxidase and may work outside its original host cell because it has a TAT-type signal peptide and a transmembrane segment at its N-terminus. Lac51 oxidizes a variety of lignin-related phenolic compounds, including syringaldazine, 2,6-dimethoxyphenol, ferulic acid, veratryl alcohol, guaiacol, and sinapinic acid at conditions that simulate the physiologic environment in giant panda intestines. Furthermore, in the presence of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringic acid, or ferulic acid as mediators, the oxidative ability of Lac51 on lignin was promoted. The absorbance of lignin at 445 nm decreased to 36% for ABTS, 51% for syringic acid, and 51% for ferulic acid after incubation for 10 h. Our findings demonstrate that the intestinal bacteria of giant pandas may facilitate the oxidation of lignin moieties, thereby clarifying the digestion of bamboo lignin by the animal.

Gongronella sp induces overproduction of laccase in Panus rudis.

Laccase is usually produced via chemical induction and is also synthesized by hosts in interaction with the typical bio‐control genus Trichoderma. In this study, we found that a newly isolated non‐laccase‐producing fungus, Gongronella sp. W5, could induce overproduction of laccase in Panus rudis. The enzyme activity, 148,200 U l–1, was 25 times higher than the activity obtained from a chemical induction using copper/o ‐toluidine as inducers. A new laccase isozyme from the interaction of P. rudis and G. W5 was purified and characterized. A further test showed that some pH resistant metabolites secreted by G. W5 acted as signals to induce P. rudis laccase. Laccase is also highly expressed by Trametes sp. AH28‐2 in interaction with Trichoderma sp. ZH1. However, no laccase activity was observed from the cross‐over interactions of P. rudis –Trichoderma sp. ZH1 or Trametes sp. AH28‐2–G. W5. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Structure of native laccase B from Trametes sp. AH28-2.

Fungal laccases are oxidoreductases that belong to the multinuclear copper-containing oxidases. They are able to oxidize a wide range of substrates, preferably phenolic compounds, which makes them suitable for employment in the bioremediation of soil and water as well as in other biotechnological applications. Here, the structural analysis of natural laccase B (LacB) from Trametes sp. AH28-2 is presented. This structure provides the opportunity to study the natural post-translational modifications of the enzyme. The overall fold shows a high homology to those of previously analyzed laccases with known three-dimensional structure. However, LacB contains a new structural element, a protruding loop near the substrate-binding site, compared with the previously reported laccase structures. This unique structural feature may be involved in modulation of the substrate recognition of LacB.