Heterologous expression of lcc1 from Lentinula edodes in tobacco BY-2 cells results in the production an active, secreted form of fungal laccase.
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.
Sakamoto
Yuichi
Y
Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan. sakamoto@ibrc.or.jp
Nakade
Keiko
K
Yano
Akira
A
Nakagawa
Yuko
Y
Hirano
Tatsuya
T
Irie
Toshikazu
T
Watanabe
Hisayuki
H
Nagai
Masaru
M
Sato
Toshitsugu
T
D002460:Cell Line; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D005818:Genetic Engineering; D042845:Laccase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D021381:Protein Transport; D020942:Shiitake Mushrooms / Q000201:enzymology*; D013379:Substrate Specificity; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D005656:Fungal Proteins; D042845:Laccase