Laccases (LACs), belonging to the multicopper oxidase family, are closely associated with various biological functions including lignin synthesis and responses to biotic and abiotic stresses in plants. However, few studies have reported the laccase genes in China rose (Rosa chinensis). Prickles cause difficulties to the management and harvest of R. chinensis and have become a trait concerned in the breeding. To investigate the expression patterns of laccase genes in roses, we cloned a laccase gene from an ancient variety R. chinensis 'Old Blush' and named it RcLAC15. The expression level of RcLAC15 in prickles was significantly higher than those in roots, stems, and leaves. Fifty-eight laccase genes were identified in the genome of R. chinensis, and bioinformatics analysis revealed that RcLAC15 was a homolog of AtLAC15, predicting that RcLAC15 was a stable hydrophilic protein without transmembrane structures. The recombinant expression vector pBI121-proRcLAC15:: GUS was introduced into Arabidopsis, and GUS staining results showed that the RcLAC15 promoter specifically drove GUS gene expression at the edges of Arabidopsis leaves. In summary, RcLAC15 is a gene specifically expressed in the prickles of R. chinensis. This discovery provides a reference for exploring the biological functions of laccase genes in the prickles of R. chinensis.
Laccase/metabolism* ; Rosa/enzymology* ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism* ; Arabidopsis/metabolism* ; Plants, Genetically Modified/metabolism*

We screened for laccase from a marine metagenomic library and obtained a bacterial laccase Lac15 and studied its decolorization ability. Using synthetic azo dyes and anthraquinonic dyes as substrates, we investigated the dye decolorization ability of recombinant Lac15 (rLac15). The purified rLac15 had better decolorization ability towards the azo dyes than the anthraquinonic dyes. When incubated at 45 degrees C and pH 8.5 for 1 h with methylsyringate as the mediator, 20 U/L of rLac15 could decolorize 95% of 100 micromol/L Acid Red 6B (AR-6B), 93% of Reactive Blue 194 (M-2GE), 76% of Reactive Brilliant Orange (K-7R) and 66% of Reactive Blue 171 (KE-R). The decolorization ability of rLac15 decreased with the dye concentration increasing. However, more than 80% of M-2GE and AR-6B were degraded even when the dye concentration was up to 200 micromol/L. At room temperature, rLac51 exhibited significant decolorization ability, with 96% of AR-6B, 86% of M-2GE, 66% of K-7R and 66% of KE-Rdegraded within 24 h at 25 degrees C. rLac15 has the potential of industrial applications.
Anthraquinones ; isolation & purification ; Azo Compounds ; isolation & purification ; Bacteria ; enzymology ; isolation & purification ; Biodegradation, Environmental ; Coloring Agents ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Laccase ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Seawater ; microbiology ; Waste Disposal, Fluid ; methods ; Waste Water ; chemistry

Laccases (benzenediol: oxygen oxidoreductases; EC 1.10.3.2) are copper-containing polyphenol oxidases that can oxidize a wide range of aromatic compounds, concomitantly with the transfer of four electrons and the reduction of molecular oxygen to water. The progress on the research of laccases structure and function is reviewed. Their three-dimensional structures and catalytic mechanism, as well as their applications in different fields are emphasized.
Catalysis ; Hydrocarbons, Aromatic ; isolation & purification ; metabolism ; Laccase ; chemistry ; metabolism ; Oxidation-Reduction

A laccase gene (lacD) from the basidiomycete Trametes sp. 420 was heterologously expressed in Pichia pastoris in two ways, resulting in two recombinant enzymes of rLacDx with native N-terminus and rLacDe with eight additional amino acid residues at N-terminus. The yields of rLacDx and rLacDe in shaken-flask cultures after an 18-day growth were 1.21 x 10(5) u/L and 7.38 x 10(4) u/L, respectively, as determined with 2,2'-azinobis(3-ethylbenzothia-zoline- 6-sulfonic acid) (ABTS) as substrate. The yield of rLacDx was further increased to 2.39 x 10(5) u/L under high-density fermentation while the production process was decreased to 7.5 days. In addition, rLacDx and rLacDe exhibited similar enzymatic characters in oxidizing substrate guaiacol, and were stable at 50 degrees C and at a pH range from 3 to 10. However, the specific activity of rLacDx (1761 u/mg) for ABTS was higher than that of rLacDe (1122 u/mg), and the apparent Km value of rLacDx (427 microM) was less than that of rLacDe (604 microM).
Cloning, Molecular ; Fermentation ; Isoenzymes ; biosynthesis ; genetics ; Laccase ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Trametes ; enzymology ; genetics

Copper ion was necessary for the transcription of all laccase isozyme genes from Trametes sp. AH28-2, with higher concentrations of Cu2+ (1-2 mmol/L) being more favorable to the synthesis of laccase. In the glucose media containing 0.5 mmol/L Cu2+, the laccase activity of the supernate was rather low (44.3 u/L) and had an increase of 60.3% (71.0 u/L) when 4.0 mmol/L o-toluidine was added. Moreover, the activity reached up to 2584 u/L as the glucose was replaced by cellobiose. And Native-PAGE showed that LacA was the main laccase component if fungus was induced by o-toluidine or copper ions. It had been demonstrated by quantitative RT-PCR that the regulation of lacA expression, induced by o-toluidine, occurred at the transcriptional level, with the accumulation of mRNA transcripts being accompanied by the increase of laccase activity of the culture fluid. In addition, the structural gene of lacA interrupted by 10 introns was 2110 bp in length and the corresponding cDNA sequence was 1560 bp encoding a 520 aa protein, which had high similarities with other laccases from basidiomycetes. Furthermore, a length of 1881 bp of 5'-terminal sequence of transcription control of lacA, amplified by the improved inverse PCR, contained a TATA box, seven CAAT boxes as well as a number of putative cis-acting elements important for its expression, including five MREs, nine CreA-binding sites, four XREs, two STREs and seven nitrogen factor binding sites. The existence of these elements was well in agreement with the data obtained from Trametes sp. AH28-2 shaken-flask cultures.
5' Untranslated Regions ; genetics ; Base Sequence ; Cloning, Molecular ; Copper ; pharmacology ; Fungal Proteins ; genetics ; metabolism ; Gene Expression Regulation, Enzymologic ; Isoenzymes ; Laccase ; genetics ; metabolism ; Molecular Sequence Data ; Trametes ; enzymology ; genetics ; Transcription, Genetic

The production conditions of extracellular laccase from Armilliria mellea and the characteristics of the enzyme were studied. The experiment proved that initial pH5.5 of the culture medium and temperature at 25 degrees C were favorable for laccase synthesis. As carbon resources, cellobiose and raffinose were better in terms of productivity than maltose, sorbose and galactose. Organic nitrogen source was more suitable for Armilliria mellea to synthesize laccase than inorganic nitrogen source. Peat extract (PE) enhanced notably the yield of laccase; the maximal yield was 7 times as much as that of the control when PE concentration was 50%. Three isozymes were detected in culture supernatant named A, B and C respectively after their mobility on PAGE. After concentrated by (NH4)2SO4 precipitation, laccase A was further purified to homogeneity by preparative native PAGE and anion exchange column chromatography. The native enzyme was a single polypeptide with a molecular mass of approximately 59 kD estimated by SDS-PAGE, while 58 kD by gel filtration chromatography under non-denaturing conditions. Determined by IEF its isoelectric point was 4.0. The optimal pH value and temperature were 5.6 and 60 degrees C respectively in catalytic reaction of oxidizing guaiacol. At 60 degrees C and 65 degrees C, half-lives of laccase A were 45 min and 36.8 min, respectively. Enzyme activity was inhibited with 100 mmol/L Cl-, but was activated with 1 mmol/L SO4(2-). However, if the concentration of NaN3 was only 1 mmol/L, laccase A lost its activity completely. 10 mmol/L EDTA had no effect on laccase A, while 1 mmol/L Cu2+ could enhance its activity. Laccase A showed a good stability when the pH of the buffer varied from 5.2 to 7.2. Using guaiacol as the substrate, the Km was 1.026 mmol/L and the Vmax was 5 mumol/(min.mg); using ABTS instead, the Km was 0.22 mmol/L and Vmax was 69 mumol/(min.mg).
Agaricales ; drug effects ; enzymology ; growth & development ; Carbohydrates ; pharmacology ; Culture Media, Conditioned ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Enzyme Stability ; Hydrogen-Ion Concentration ; Laccase ; Oxidoreductases ; isolation & purification ; metabolism