Characterization of an Unusual Cold-Active β-Glucosidase Belonging to Family 3 of the Glycoside Hydrolases from the Psychrophilic Isolate Paenibacillus sp. Strain C7
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 transform...
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American Society for Microbiology
2005
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ftpubmed:oai:pubmedcentral.nih.gov:1183342 2023-05-15T14:02:34+02:00 Characterization of an Unusual Cold-Active β-Glucosidase Belonging to Family 3 of the Glycoside Hydrolases from the Psychrophilic Isolate Paenibacillus sp. Strain C7 Shipkowski, Stephanie Brenchley, Jean E. 2005-08 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1183342 http://www.ncbi.nlm.nih.gov/pubmed/16085807 https://doi.org/10.1128/AEM.71.8.4225-4232.2005 en eng American Society for Microbiology http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1183342 http://www.ncbi.nlm.nih.gov/pubmed/16085807 http://dx.doi.org/10.1128/AEM.71.8.4225-4232.2005 Copyright © 2005, American Society for Microbiology Enzymology and Protein Engineering Text 2005 ftpubmed https://doi.org/10.1128/AEM.71.8.4225-4232.2005 2013-08-30T12:14:01Z 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-β-d-galactopyranoside) only below 30°C, an indication of cold-active β-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 β-galactosidase activity, belonged to glycoside hydrolase family 3, a family of β-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-β-d-glucopyranoside) than with the galactoside substrate ONPGal (o-nitrophenyl-β-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-β-glucosides such as helicin, MUG (4-methylumbelliferyl-β-d-glucopyranoside), esculin, indoxyl-β-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 β-glucosidases. The hydrolysis of a variety of aryl-β-glucosides suggests that the enzyme may allow the organism to use these substrates in the environment and that its low Km on indoxyl-β-d-glucoside may make it useful for producing indigo. Text Antarc* antarcticus PubMed Central (PMC) Applied and Environmental Microbiology 71 8 4225 4232 |
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English |
topic |
Enzymology and Protein Engineering |
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Enzymology and Protein Engineering Shipkowski, Stephanie Brenchley, Jean E. Characterization of an Unusual Cold-Active β-Glucosidase Belonging to Family 3 of the Glycoside Hydrolases from the Psychrophilic Isolate Paenibacillus sp. Strain C7 |
topic_facet |
Enzymology and Protein Engineering |
description |
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-β-d-galactopyranoside) only below 30°C, an indication of cold-active β-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 β-galactosidase activity, belonged to glycoside hydrolase family 3, a family of β-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-β-d-glucopyranoside) than with the galactoside substrate ONPGal (o-nitrophenyl-β-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-β-glucosides such as helicin, MUG (4-methylumbelliferyl-β-d-glucopyranoside), esculin, indoxyl-β-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 β-glucosidases. The hydrolysis of a variety of aryl-β-glucosides suggests that the enzyme may allow the organism to use these substrates in the environment and that its low Km on indoxyl-β-d-glucoside may make it useful for producing indigo. |
format |
Text |
author |
Shipkowski, Stephanie Brenchley, Jean E. |
author_facet |
Shipkowski, Stephanie Brenchley, Jean E. |
author_sort |
Shipkowski, Stephanie |
title |
Characterization of an Unusual Cold-Active β-Glucosidase Belonging to Family 3 of the Glycoside Hydrolases from the Psychrophilic Isolate Paenibacillus sp. Strain C7 |
title_short |
Characterization of an Unusual Cold-Active β-Glucosidase Belonging to Family 3 of the Glycoside Hydrolases from the Psychrophilic Isolate Paenibacillus sp. Strain C7 |
title_full |
Characterization of an Unusual Cold-Active β-Glucosidase Belonging to Family 3 of the Glycoside Hydrolases from the Psychrophilic Isolate Paenibacillus sp. Strain C7 |
title_fullStr |
Characterization of an Unusual Cold-Active β-Glucosidase Belonging to Family 3 of the Glycoside Hydrolases from the Psychrophilic Isolate Paenibacillus sp. Strain C7 |
title_full_unstemmed |
Characterization of an Unusual Cold-Active β-Glucosidase Belonging to Family 3 of the Glycoside Hydrolases from the Psychrophilic Isolate Paenibacillus sp. Strain C7 |
title_sort |
characterization of an unusual cold-active β-glucosidase belonging to family 3 of the glycoside hydrolases from the psychrophilic isolate paenibacillus sp. strain c7 |
publisher |
American Society for Microbiology |
publishDate |
2005 |
url |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1183342 http://www.ncbi.nlm.nih.gov/pubmed/16085807 https://doi.org/10.1128/AEM.71.8.4225-4232.2005 |
genre |
Antarc* antarcticus |
genre_facet |
Antarc* antarcticus |
op_relation |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1183342 http://www.ncbi.nlm.nih.gov/pubmed/16085807 http://dx.doi.org/10.1128/AEM.71.8.4225-4232.2005 |
op_rights |
Copyright © 2005, American Society for Microbiology |
op_doi |
https://doi.org/10.1128/AEM.71.8.4225-4232.2005 |
container_title |
Applied and Environmental Microbiology |
container_volume |
71 |
container_issue |
8 |
container_start_page |
4225 |
op_container_end_page |
4232 |
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1766272890075873280 |