This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Salusjärvi, T.
Right arrow Articles by Miasnikov, A. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Salusjärvi, T.
Right arrow Articles by Miasnikov, A. N.
Agricola
Right arrow Articles by Salusjärvi, T.
Right arrow Articles by Miasnikov, A. N.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, September 2004, p. 5503-5510, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5503-5510.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cloning and Characterization of Gluconolactone Oxidase of Penicillium cyaneo-fulvum ATCC 10431 and Evaluation of Its Use for Production of D-Erythorbic Acid in Recombinant Pichia pastoris

Tuomas Salusjärvi,1* Nisse Kalkkinen,2 and Andrei N. Miasnikov1

Danisco Innovation, Kantvik,1 Institute of Biotechnology, University of Helsinki, Helsinki, Finland2

Received 19 March 2004/ Accepted 8 May 2004

A D-erythorbic acid-forming soluble flavoprotein, gluconolactone oxidase (GLO), was purified from Penicillium cyaneo-fulvum strain ATCC 10431 and partially sequenced. Peptide sequences were used to isolate a cDNA clone encoding the enzyme. The cloned gene (accession no. AY576053) exhibits high levels of similarity with the genes encoding other known eukaryotic lactone oxidases and also with the genes encoding some putative prokaryotic lactone oxidases. Analysis of the coding sequence of the GLO gene indicated the presence of a typical secretion signal sequence at the N terminus of GLO. No other targeting or anchoring signals were found, suggesting that GLO is the first known lactone oxidase that is secreted rather than targeted to the membranes of the endoplasmic reticulum or mitochondria. Experimental evidence, including the N-terminal sequence of mature GLO and data on glycosylation and localization of the enzyme in native and recombinant hosts, supports this analysis. The GLO gene was expressed in Pichia pastoris, and recombinant GLO was produced by using the strong methanol-induced AOX1 promoter. In order to evaluate the suitability of purified GLO for production of D-erythorbic acid, we immobilized it on N-hydroxysuccinimide-activated Sepharose and found that the immobilized GLO retained full activity during immobilization but was rather unstable under reaction conditions. Our results show that both soluble and immobilized forms of GLO can, in principle, be used for production of D-erythorbic acid from D-glucono-{delta}-lactone or (in combination with glucose oxidase and catalase) from glucose. We also demonstrated the feasibility of glucose-D-erythorbic acid fermentation with recombinant strains coexpressing GLO and glucose oxidase genes, and we analyzed problems associated with construction of efficient D-erythorbic acid-producing hosts.

* Corresponding author. Mailing address: Danisco Innovation, Sokeritehtaantie 20, Kantvik 02460, Finland. Phone: 358 405789704. Fax: 358 92982203. E-mail: tuomas.salusjarvi{at}danisco.com.

Applied and Environmental Microbiology, September 2004, p. 5503-5510, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5503-5510.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»