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 Shipkowski, S.
Right arrow Articles by Brenchley, J. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shipkowski, S.
Right arrow Articles by Brenchley, J. E.
Agricola
Right arrow Articles by Shipkowski, S.
Right arrow Articles by Brenchley, J. E.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, August 2005, p. 4225-4232, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4225-4232.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characterization of an Unusual Cold-Active ß-Glucosidase Belonging to Family 3 of the Glycoside Hydrolases from the Psychrophilic Isolate Paenibacillus sp. Strain C7

Stephanie Shipkowski* and Jean E. Brenchley

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802

Received 30 November 2004/ Accepted 26 February 2005

We selected for spore-forming psychrophilic bacteria able to use lactose as a carbon source and one isolate, designated Paenibacillus sp. strain C7, that was phylogenetically related to, but distinct from both Paenibacillus macquariensis and Paenibacillus antarcticus. Some Escherichia coli transformants obtained with genomic DNA from this isolate hydrolyzed X-Gal (5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside) only below 30°C, an indication of cold-active ß-galactosidase activity. Sequencing of the cloned insert revealed an open reading frame encoding a 756-amino acid protein that, rather than belonging to a family typically known for ß-galactosidase activity, belonged to glycoside hydrolase family 3, a family of ß-glucosidases. Because of this unusual placement, the recombinant enzyme (BglY) was purified and characterized. Consistent with its classification, the enzyme had seven times greater activity with the glucoside substrate ONPGlu (o-nitrophenyl-ß-D-glucopyranoside) than with the galactoside substrate ONPGal (o-nitrophenyl-ß-D-galactopyranoside). In addition, the enzyme had, with ONPGlu, a thermal optimum around 30 to 35°C, activity over a broad pH range (5.5 to 10.9), and an especially low Km (<0.003 mM). Further examination of substrate preference showed that the BglY enzyme also hydrolyzed other aryl-ß-glucosides such as helicin, MUG (4-methylumbelliferyl-ß-D-glucopyranoside), esculin, indoxyl-ß-D-glucoside (a natural indigo precursor), and salicin, but had no activity with glucosidic disaccharides or lactose. These characteristics and substrate preferences make the BglY enzyme unique among the family 3 ß-glucosidases. The hydrolysis of a variety of aryl-ß-glucosides suggests that the enzyme may allow the organism to use these substrates in the environment and that its low Km on indoxyl-ß-D-glucoside may make it useful for producing indigo.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 211 South Frear, University Park, PA 16802. Phone: (814) 865-3330. Fax: (814) 865-3330. E-mail: sar242{at}psu.edu.

Applied and Environmental Microbiology, August 2005, p. 4225-4232, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4225-4232.2005
Copyright © 2005, 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»