Details
| Original language | English |
|---|---|
| Pages (from-to) | 34-42 |
| Number of pages | 9 |
| Journal | Protein Expression and Purification |
| Volume | 137 |
| Publication status | Published - 23 Jun 2017 |
Abstract
A laccase of the basidiomycete Pleurotus pulmonarius (PpuLcc) possessed strong decolorizing abilities towards artificial and natural dyes. The PpuLcc was purified from the culture supernatant via FPLC, and the corresponding gene cloned and expressed in Pichia pastoris GS115. To examine the impact of the C-terminal tail region and the signal peptide on the recombinant expression of PpuLcc, a non-modified version or different truncations (−2, −5, −13 AA) of the target protein were combined with different secretion signals. Heterologous expression of codon optimized constructs resulted in extracellular activities of the PpuLcc variants of up to 7000 U L−1 (substrate ABTS) which was six times higher than non-codon optimized constructs. In contrast to previous works, altering the C-terminal end of the protein did not influence kinetic parameters or the rate of expression. The His-Tag purified enzymes showed high temperature optima (50–70 °C) and thermo stability. All of the recombinant variants degraded triarylmethane and azo dyes. Rapid bleaching of β-carotene (E 160a) and the polyene acid norbixin (E 160b) using a laccase was found for the first time. Thus, the enzyme may be useful in decolorizing unwanted polyene pigments, for example from the processing of cheese, bakery, desserts, ice cream or coloured casings.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
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In: Protein Expression and Purification, Vol. 137, 23.06.2017, p. 34-42.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Variants of PpuLcc, a multi-dye decolorizing laccase from Pleurotus pulmonarius expressed in Pichia pastoris
AU - Behrens, Christoph J.
AU - Linke, Diana
AU - Allister, Aldrige B.
AU - Zelena, Katerina
AU - Berger, Ralf G.
PY - 2017/6/23
Y1 - 2017/6/23
N2 - A laccase of the basidiomycete Pleurotus pulmonarius (PpuLcc) possessed strong decolorizing abilities towards artificial and natural dyes. The PpuLcc was purified from the culture supernatant via FPLC, and the corresponding gene cloned and expressed in Pichia pastoris GS115. To examine the impact of the C-terminal tail region and the signal peptide on the recombinant expression of PpuLcc, a non-modified version or different truncations (−2, −5, −13 AA) of the target protein were combined with different secretion signals. Heterologous expression of codon optimized constructs resulted in extracellular activities of the PpuLcc variants of up to 7000 U L−1 (substrate ABTS) which was six times higher than non-codon optimized constructs. In contrast to previous works, altering the C-terminal end of the protein did not influence kinetic parameters or the rate of expression. The His-Tag purified enzymes showed high temperature optima (50–70 °C) and thermo stability. All of the recombinant variants degraded triarylmethane and azo dyes. Rapid bleaching of β-carotene (E 160a) and the polyene acid norbixin (E 160b) using a laccase was found for the first time. Thus, the enzyme may be useful in decolorizing unwanted polyene pigments, for example from the processing of cheese, bakery, desserts, ice cream or coloured casings.
AB - A laccase of the basidiomycete Pleurotus pulmonarius (PpuLcc) possessed strong decolorizing abilities towards artificial and natural dyes. The PpuLcc was purified from the culture supernatant via FPLC, and the corresponding gene cloned and expressed in Pichia pastoris GS115. To examine the impact of the C-terminal tail region and the signal peptide on the recombinant expression of PpuLcc, a non-modified version or different truncations (−2, −5, −13 AA) of the target protein were combined with different secretion signals. Heterologous expression of codon optimized constructs resulted in extracellular activities of the PpuLcc variants of up to 7000 U L−1 (substrate ABTS) which was six times higher than non-codon optimized constructs. In contrast to previous works, altering the C-terminal end of the protein did not influence kinetic parameters or the rate of expression. The His-Tag purified enzymes showed high temperature optima (50–70 °C) and thermo stability. All of the recombinant variants degraded triarylmethane and azo dyes. Rapid bleaching of β-carotene (E 160a) and the polyene acid norbixin (E 160b) using a laccase was found for the first time. Thus, the enzyme may be useful in decolorizing unwanted polyene pigments, for example from the processing of cheese, bakery, desserts, ice cream or coloured casings.
UR - http://www.scopus.com/inward/record.url?scp=85021422728&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2017.06.014
DO - 10.1016/j.pep.2017.06.014
M3 - Article
C2 - 28651974
AN - SCOPUS:85021422728
VL - 137
SP - 34
EP - 42
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
ER -