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Heterologous expression of <it>lcc1 </it>gene from <it>Trametes trogii </it>in <it>Pichia pastoris </it>and characterization of the recombinant enzyme

Microbial Cell Factories. 2006;5(1):31 DOI 10.1186/1475-2859-5-31

 

Journal Homepage

Journal Title: Microbial Cell Factories

ISSN: 1475-2859 (Online)

Publisher: BMC

LCC Subject Category: Science: Microbiology

Country of publisher: United Kingdom

Language of fulltext: English

Full-text formats available: PDF, HTML

 

AUTHORS


Buonocore Vincenzo

Garzillo Anna

Lupino Stefania

Colao Maria

Ruzzi Maurizio

EDITORIAL INFORMATION

Blind peer review

Editorial Board

Instructions for authors

Time From Submission to Publication: 10 weeks

 

Abstract | Full Text

<p>Abstract</p> <p>Background</p> <p>Fungal laccases are useful enzymes for industrial applications; they exhibit broad substrate specificity and thus are able to oxidize a variety of xenobiotic compounds including chlorinated phenolics, synthetic dyes, pesticides and polycyclic aromatic hydrocarbons. Unfortunately, the biotechnological exploitation of laccases can be hampered by the difficulties concerning the enzyme production by the native hosts.</p> <p>Results</p> <p>In order to obtain a simple and efficient source of laccase, the <it>lcc1 </it>cDNA isolated from the white-rot fungus <it>Trametes trogii </it>has been successfully expressed in the methylotrophic yeast <it>Pichia pastoris </it>under the control of the methanol induced alcohol oxidase promoter P<sub>AOX1</sub>. The recombinant Lcc1 was produced as a secreted protein with the native N-terminal prepropeptide for signal trafficking, and thus easily recovered from the culture medium. At the 1-liter scale, as calculated on the basis of the specific activity, the recombinant protein was produced at a yield of 17 mg/l. The highest production level obtained in fed-batch culture was 2520 U/l, corresponding to a specific productivity of 31.5 U/g biomass.</p> <p>The purified recombinant laccase exhibited a behaviour similar to the main laccase produced by <it>T. trogii</it>. Lcc1 showed high activity in the presence of organic solvents and a high decolourization capacity towards azo, triarylmethane, indigo carmine and anthraquinonic dyes, that could be significantly enhanced in the presence of the redox mediators 1-hydroxybenzotriazole and violuric acid.</p> <p>Conclusion</p> <p>Heterologous expression of <it>T. trogii </it>laccase <it>lcc1 </it>in the methylotrophic yeast <it>P. pastoris </it>was successfully achieved. The biochemical and kinetic characterization of the recombinant protein suggests potential technological applications for this enzyme.</p>