Study On Biochemistry And Molecular Biology Of Laccase Isozymes From Ligninolytic Fungus Trametes Gallica

White-rot fungus Trametes gallica showed a high laccase-producing ability when it was grown on RB-PDA plate, compared with ligninolytic model fungus—Phanerochate chrysosporium.The laccase production of T. gallica was studied when it was grown in liquid media. The results indicated that 2%(w/v) tryptone used as organic nitrogen source apparently improved the laccae production and the laccase production under high carbon concentration was much higher than that under low ones. High concentration of Cu²⁺ (200μmol/L) strongly increased laccase production. Both TG (Tryptone-Glucose) and PG (Peptone -Glucose) media led to the highest laccase production.Studies of effects of fifty-nine aromatic compounds on the laccase production were carried out. The results indicated that twenty-seven compounds increased the laccase production, among which quinol showed the strongest induction, and other thirty-two inhibited the laccase production in some degree—both 2,6-bichlorophenol and ρ-nitrophenol had the strongest inhibition. Different substituted compounds or isomeric compounds influenced the laccase production at different level.Influences of twelve media on the laccase isozyme patterns under static and shaking conditions were studied by using non-dcnaturated polyacryl amide gel electrophoresis (PAGE)and native staining. The results revealed that different laccase isozymograms were obtained under different media and incubation conditions. T. gallica produced up to 14 laccase isozyme bands in PAG and AAG media under the shaking condition, while only 4 bands in PAG I medium under the static condition. The total number of laccase isozymes reached up to twenty in terms of all incubation conditions.Fourteen laccase isozymes were purified from the extracellular proteins produced by T. gallica (named as Lacl to Lac 14), through ammonium sulfate precipitation, DEAE-Sepharose Fast Flow anion-exchange chromatography, Sephadex G-100 gel penetration chromatography and improved nondenaturated PAGE(IN-PAGE). All of them were shown in one single band on thegelofSDS-PAGE.Physical properties showed that all purified laccases had apparent molecular mass of 60,000 Da (SDS-PAGE), and were glycoproteins containing 10-13.5% glycosylation. The copper contents were determined as 4 coppers per protein by using graphite-furnace atomic absorption spectrometry. The UV-visibale spectrum of the laccase purified with DEAE-Sepharose Fast Flow anion-exchange chromatography and Sephadex G-100 chromatography showed apparent blue absorption at 610nm, which is the typical characteristic of type 1 copper of blue laccase. The pi values of laccase isozymes were between 2.8 to 5.3, and their mobilities were different on the gel of native PAGE.The analysis of biochemical property indicated that laccase was fairly stable at pH6-10, its optimal temperature of reaction was 70^ and the thermal stability was strongly affected by the pH value of solution. Some metal ions inhibited the lacccase activity and Fe2+did most. NaN3 showed the strongest inhibition on the laccase activity compared with several other potential laccase inhibitors such as KF, NaCN, EDTA and so on. The optimal pH was 2.2, 3.0 and 4.0 with ABTS, DMP and guaiacol as substrates, respectively. The results of Km determination displayed that ABTS was the optimal substrate for every laccase isozyme, compared with DMP and guaiacol. The Km values of 14 laccase isozymes were very different and showed correlation with pis. It seemed that the Km values of laccase isozymes with high pi were higher than those with low pi, when ABTS, DMP and guaiacol were used as substrates.Purified laccase isozyme Lac 11 was able to efficiently catalyze oxidation of variousaromatic, non-aromatic compounds, azo dyes and active biological dyes. The optimal pH values for twenty compounds were from 2.2 to 5.4 and oxygen consumption determination indicated that ABTS was still the optimal substrate, and different substituted compounds or isomeric compounds were oxidized at different speeds.The N-terminal amino acid sequences of Lac 10 and Lac 11 were determined as AIGPVADLTISN and S/AIGPVADLTISN, respectively. Homologous analysis demonstrated that the N-terminal amino acid sequences of two laccase isozymes had high consensus with other ones from different Trametes species.Based on the conservation of laccase genes, the primers were designed and three DNA segments were cloned by PCR: lac j\ 47 (147bp), /acg201 (201bp) and /acg450 (450bp) from genomic DNA. By RT-PCR, three cDNA s< nents were cloned: /acc333a (333bp), /acc333b (333bp)and/acA(1488bp).The comparison of nucleic acid and putative amino acid sequences of above six laccase gene segments revealed that /acgl47 was the part of /acg450, and there are high homology up to 99.8% between /acg450 and /acc333a. The sequences of lacA and /acc333b had 99.3% consensus, and /acg201 had less consensus with other five DNA or cDNA sequences. Therefore, it is very possible for the six fragments being grouped into five laccase genes.The lacA encodes a putative mature pepetide of laccase with 496 amino acid residues, containing the ligand sites for three types of copper ions, 4 N-glycosylation sites and 2 disulfide bridge sites. The comparisons of nucleic acid and protein sequences indicated that there was high consensus among lack and other laccase genes, and the difference among them was apparent. Therefore lacA is suggested to be a novel laccase gene.PCR was carried out for tacA cDNA and the product was inserted into E. coli expression vector pET32a(+). The recombinant plasmid named as pET32a-/acA was obtained after transforming into E. Coli BL21. The transformant cells were induced by IPTG and about 30 min later, a 68kDa protein product was observed by SDS-PAGE as predicted. This result was consistent with the prediction. The recombinant protein existed as soluble one and was accounted for 40% of the total proteins. However, there was no laccase activity detected by using ABTS as the substrate.Full-length cDNA fragments were synthesized using Creator? SMART? cDNA LibraryConstruction Kit and a cDNA library with 20,000 independent clones with inserted freagments of 500-4,000 bp were constructed. The recombinant rate was evaluated higher than 60%.