Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa

137

Pycnoporus cinnabarinus laccase lac1 gene was overexpressed in Aspergillus niger, a well-known fungal host producing a large amount of homologous or heterologous enzymes for industrial applications. The corresponding cDNA was placed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter as a strong and constitutive promoter. The laccase signal peptide or the glucoamylase preprosequence of A. niger was used to target the secretion. Both signal peptides directed the secretion of laccase into the culture medium as an active protein, but the A. niger preprosequence allowed an 80-fold increase in laccase production. The identity of the recombinant protein was further confirmed by immunodetection using Western blot analysis and N-terminal sequencing. The molecular mass of the mature laccase was 70 kDa as expected, similar to that of the native form, suggesting no hyperglycosylation. The recombinant laccase was purified in a three-step procedure including a fractionated precipitation using ammonium sulfate, and a concentration by ultrafiltration followed by a Mono Q column. All the characteristics of the recombinant laccase are in agreement with those of the native laccase. This is the first report of the production of a white-rot laccase in A. niger.

Section: Discussionmentioning

confidence: 84%

“…Laccase activity was then demonstrated in fungi, plants and more recently in bacteria [3]. Laccases are glycoproteins, usually monomeric, although some multimeric structures were described in Podospora anserina [4], Agaricus bisporus [5] and Trametes villosa [6]. Laccases are heterogeneous in their biochemical properties and molecular structures.…”

mentioning

confidence: 99%

Expression of the Pycnoporus cinnabarinus laccase gene in Aspergillus niger and characterization of the recombinant enzyme

Record¹,

Punt

Chamkha

et al. 2002

137

“…However, wide variations are seen with respect to the induction of laccase gene transcription by aromatic compounds with both inducible and noninducible forms described [46], suggesting that only certain isoenzymes serve in a protective capacity. Transcription of one laccase gene, lcc1, from Trametes villosa was induced 17-fold by the addition of XYL but a second gene (lcc2) was constituitive under the conditions tested [29]. Amounts of laccase mRNA and laccase activity in 10-day-old cultures of T. versicolor were a direct function of concentrations of the laccase inducers, 1-hydroxybenzotriazole (HBT) and XYL but no induction was observed after the addition of FA and veratric acid (VA) [2].…”

Section: Discussionmentioning

confidence: 93%

“…Basidiomycetes typically produce multiple laccase isoforms [5,[23][24][25][26][27][28][29] and V. volvacea produces at least two protein bands with laccase activity when grown in submerged culture under different conditions [1]. Previous physiological studies have shown that, as in other basidiomycetes, laccase production by V. volvacea is induced by copper and by various aromatic compounds.…”

Section: Discussionmentioning

confidence: 99%

Biochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariella volvacea

Chen

Buswell

2003

We have isolated a laccase (lac1) from culture fluid of Volvariella volvacea, grown in a defined medium containing 150 µm CuSO4, by ion‐exchange and gel filtration chromatography. Lac1 has a molecular mass of 58 kDa as determined by SDS/PAGE and an isoelectric point of 3.7. Degenerate primers based on the N‐terminal sequence of purified lac1 and a conserved copper‐binding domain were used to generate cDNA fragments encoding a portion of the lac1 protein and RACE was used to obtain full‐length cDNA clones. The cDNA of lac1 contained an ORF of 1557 bp encoding 519 amino acids. The amino acid sequence from Ala25 to Asp41 corresponded to the N‐terminal sequence of the purified protein. The first 24 amino acids are presumed to be a signal peptide. The expression of lac1 is regulated at the transcription level by copper and various aromatic compounds. RT‐PCR analysis of gene transcription in fungal mycelia grown on rice‐straw revealed that, apart from during the early stages of substrate colonization, lac1 was expressed at every stage of the mushroom developmental cycle defined in this study, although the levels of transcription varied considerably depending upon the developmental phase. Transcription of lac1 increased sharply during the latter phase of substrate colonization and reached maximum levels during the very early stages (primordium formation, pinhead stage) of fruit body morphogenesis. Gene expression then declined to ≈ 20–30% of peak levels throughout the subsequent stages of sporophore development.

“…Ligninolytic enzymes are generally difficult to overexpress in heterologous organisms in an active form. However, the expression of active recombinant laccases has been reported in the filamentous fungus Aspergillus oryzae [10], and the yeasts Saccharomyces cerevisiae [11] and Pichia pastoris [12].…”

mentioning

confidence: 99%

Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I‐937 and expression in Pichia pastoris

Otterbein¹,

Record²,

Longhi

et al. 2000

112

Laccases are multicopper-containing enzymes which catalyse the oxidation of phenolic and nonphenolic compounds with the concomitant reduction of molecular oxygen. In this study, a full-length cDNA coding for laccase (lac1) from Pycnoporus cinnabarinus I-937 was isolated and characterized. The corresponding open reading frame is 1557 nucleotides long and encodes a protein of 518 amino acids. The cDNA encodes a precursor protein containing a 21 amino-acid signal sequence corresponding to a putative signal peptide. The deduced amino-acid sequence of the encoded protein was similar to that of other laccase proteins, with the residues involved in copper coordination sharing the greatest extent of similarity. The cDNA encoding for laccase was placed under the control of the alcohol oxidase (Aox 1) promoter and expressed in the methylotropic yeast Pichia pastoris. The laccase leader peptide, as well as the Saccharomyces cerevisiae a-factor signal peptide, efficiently directed the secretion into the culture medium of laccase in an active form. Moreover, the laccase activity was directly detected in plates. The identity of the recombinant product was further confirmed by protein immunoblotting. The expected molecular mass of the mature protein is 81 kDa. However, the apparent molecular mass of the recombinant protein is 110 k Da, thus suggesting that the protein expressed in P. pastoris may be hyperglycosylated.