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Heterologous expression of lcc1 from Lentinula edodes in tobacco BY-2 cells results in the production an active, secreted form of fungal laccase

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Abstract

Laccase (Lcc) is a lignin-degrading enzyme produced by white-rot fungi and has been the subject of much interest in the field of bioremediation due to its ability to oxidize phenolic compounds. In this report, we describe the isolation and characterization of lcc1, a novel gene of Lentinula edodes that encodes Lcc1, and demonstrate that recombinant Lcc1 is expressed in an active, secreted form in tobacco BY-2 cells in culture. The open reading frame of lcc1 was 1,557 base pairs in length and encoded a putative protein of 518 amino acids. We introduced a chimeric form of lcc1 (CaMV35Sp:clcc1) into tobacco BY-2 cells and obtained several stable clcc1 transformants that expressed active Lcc1. Lcc1 activity in BY-2 culture media was higher than in cellular extracts, which indicated that recombinant Lcc1 was produced in a secreted form. Recombinant Lcc1 had a smaller apparent molecular weight and exhibited a different pattern of posttranslational modification than Lcc1 purified from L. edodes. The substrate specificity of purified recombinant Lcc1 was similar to L. edodes Lcc1, and both enzymes were able to decolorize the same set of dyes. These results suggest that heterologous expression of fungal Lcc1 in BY-2 cells will be a valuable tool for the production of sufficient quantities of active laccase for bioremediation.

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Author notes

  1. Tatsuya Hirano

    Present address: Faculty of Agriculture, Meijo University, 1-501 Shiogamaguchi, Tenpaku, Nagoya, Aichi, 468-2502, Japan

  2. Toshikazu Irie

    Present address: Department of Biological Resources, School of Environmental Science, The University of Shiga Prefecture, 2500 Hassaka-cho, Hikone, Shiga, 522-8533, Japan

  3. Hisayuki Watanabe

    Present address: Nichiro Corporation, Maikon City, Kuriki Area, 2-6-14 Kuriki, Asao-ku, Kawasaki, Kanagawa, 215-0033, Japan

  4. Masaru Nagai

    Present address: Institute for Environmental Science, 1-7 Ienomae, Obuchi, Rokkasho-mura, Kamikita, Aomori, 039-3212, Japan

Authors and Affiliations

  1. Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate, 024-0003, Japan

    Yuichi Sakamoto, Keiko Nakade, Akira Yano, Yuko Nakagawa, Tatsuya Hirano, Toshikazu Irie, Hisayuki Watanabe, Masaru Nagai & Toshitsugu Sato

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  1. Yuichi Sakamoto
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Corresponding author

Correspondence to Yuichi Sakamoto.

Electronic supplementary material

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Supplementary Figure 1.

Nucleotide sequence and deduced amino acid sequence of L. edodes lccl gene. Red letters: signal peptide, blue letters: cupper binding site, green letters: N-glycosylation site (JPEG 1.41 MB)

Supplementary Figure 2.

Southern blot analysis of L. edodes lccl gene. Full length of lccl cDNA was used for the probe. In the probe, there are no recognition sequences of all restriction enzymes used for southern bolt analysis. (JPEG 308 KB)

Supplementary Figure 3.

Northern blot analysis of L. edodes lccl gene. Northern blot analysis was carried out by the method of Hirano et al. (1999). 1; vegetative mycelia, 2; primordia, 3; stipe of mature fruit body, 4; gill of mature fruit body, 5; pileus of mature fruit body. gpd: glyceraldehyde-3-phosphate dehydrogenase gene. ras: ras gene (JPEG 503 KB)

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Sakamoto, Y., Nakade, K., Yano, A. et al. Heterologous expression of lcc1 from Lentinula edodes in tobacco BY-2 cells results in the production an active, secreted form of fungal laccase. Appl Microbiol Biotechnol 79, 971–980 (2008). https://doi.org/10.1007/s00253-008-1507-1

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  • DOI: https://doi.org/10.1007/s00253-008-1507-1

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