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Bioengineered. 2012 Jul-Aug;3(4):232-5. doi: 10.4161/bioe.20037. Epub 2012 Jun 18.

Constitutive expression of Botrytis aclada laccase in Pichia pastoris.

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1
Food Biotechnology Laboratory, Department of Food Sciences and Technology, University of Natural Resources and Life Sciences, Vienna, Austria.

Abstract

The heterologous expression of laccases is important for their large-scale production and genetic engineering--a prerequisite for industrial application. Pichia pastoris is the preferred expression host for fungal laccases. The recently cloned laccase from the ascomycete Botrytis aclada (BaLac) has been efficiently expressed in P. pastoris under the control of the inducible alcohol oxidase (AOX1) promoter. In this study, we compare these results to the constitutive expression in the same organism using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The results show that the amounts of BaLac produced with the GAP system (517 mgL(-1)) and the AOX1 system (495 mgL(-1)) are comparable. The constitutive expression is, however, faster, and the specific activity of BaLac in the culture supernatant is higher (41.3 Umg(-1) GAP, 14.2 Umg(-1) AOX1). In microtiter plates, the constitutive expression provides a clear advantage due to easy manipulation (simple medium, no methanol feeding) and fast enzyme production (high-throughput screening assays can already be performed after 48 h).

PMID:
22705842
PMCID:
PMC3476866
DOI:
10.4161/bioe.20037
[Indexed for MEDLINE]
Free PMC Article
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