Functional and structural characterization of family 6 carbohydrate-binding module (CtCBM6A) of Clostridium thermocellum α-L-arabinofuranosidase
The gene encoding the family 6 carbohydrate-binding module ( Ct CBM6A) from Clostridium thermocellum , cloned in pET-21a(+) expression vector, was overexpressed using Escherichia coli BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of the recombinant...
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| Veröffentlicht in: | Biochemistry (Moscow) 2013-11, Vol.78 (11), p.1272-1279 |
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| creator | Ahmed, S. Luís, A. S. Brás, J. L. A. Fontes, C. M. G. A. Goyal, A. |
| description | The gene encoding the family 6 carbohydrate-binding module (
Ct
CBM6A) from
Clostridium thermocellum
, cloned in pET-21a(+) expression vector, was overexpressed using
Escherichia coli
BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of the recombinant
Ct
CBM6A showed molecular size of approximately 15 kDa. Ligand-binding analysis of
Ct
CBM6A with rye arabinoxylan and oat spelt xylan by affinity gel electrophoresis showed low affinity for these ligands (
K
a
of 40 and 26 liter/g, respectively), and analysis by fluorescence spectroscopy (
K
a
of 33 and 15 liter/g, respectively) corroborated lower binding affinity with the above soluble ligands. However,
Ct
CBM6A displayed significantly higher ligand-binding affinity with insoluble wheat arabinoxylan with equilibrium association constant
K
a
of 230 M
−1
and binding capacity (
N
0
) of 11 μmole/g. The protein melting curve of
Ct
CBM6A displayed a peak shift from 53 to 58°C in the presence of Ca
2+
, indicating that Ca
2+
imparts thermal stability to the
Ct
CBM6A structure. Homology modeling of
Ct
CBM6A revealed a characteristic β-sandwich core structure. The Ramachandran plot of
Ct
CBM6A showed 89% of the residues in the most favorable region, 10% in additionally favored region, and 1% in generously allowed region, indicating that
Ct
CBM6A has a stable conformation. |
| doi_str_mv | 10.1134/S0006297913110072 |
| format | Article |
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Ct
CBM6A) from
Clostridium thermocellum
, cloned in pET-21a(+) expression vector, was overexpressed using
Escherichia coli
BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of the recombinant
Ct
CBM6A showed molecular size of approximately 15 kDa. Ligand-binding analysis of
Ct
CBM6A with rye arabinoxylan and oat spelt xylan by affinity gel electrophoresis showed low affinity for these ligands (
K
a
of 40 and 26 liter/g, respectively), and analysis by fluorescence spectroscopy (
K
a
of 33 and 15 liter/g, respectively) corroborated lower binding affinity with the above soluble ligands. However,
Ct
CBM6A displayed significantly higher ligand-binding affinity with insoluble wheat arabinoxylan with equilibrium association constant
K
a
of 230 M
−1
and binding capacity (
N
0
) of 11 μmole/g. The protein melting curve of
Ct
CBM6A displayed a peak shift from 53 to 58°C in the presence of Ca
2+
, indicating that Ca
2+
imparts thermal stability to the
Ct
CBM6A structure. Homology modeling of
Ct
CBM6A revealed a characteristic β-sandwich core structure. The Ramachandran plot of
Ct
CBM6A showed 89% of the residues in the most favorable region, 10% in additionally favored region, and 1% in generously allowed region, indicating that
Ct
CBM6A has a stable conformation.</description><identifier>ISSN: 0006-2979</identifier><identifier>EISSN: 1608-3040</identifier><identifier>DOI: 10.1134/S0006297913110072</identifier><identifier>PMID: 24460941</identifier><language>eng</language><publisher>Boston: Springer US</publisher><subject>Amino Acid Sequence ; Bacterial Proteins - chemistry ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Binding proteins ; Biochemistry ; Biomedical and Life Sciences ; Biomedicine ; Bioorganic Chemistry ; Clostridium ; Clostridium thermocellum ; Clostridium thermocellum - enzymology ; Escherichia coli ; Escherichia coli - metabolism ; Genetic aspects ; Glycoside Hydrolases - chemistry ; Glycoside Hydrolases - genetics ; Glycoside Hydrolases - metabolism ; Life Sciences ; Ligands ; Microbiology ; Molecular Sequence Data ; Physiological aspects ; Protein Binding ; Protein Denaturation ; Protein Stability ; Protein Structure, Tertiary ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - chemistry ; Recombinant Proteins - isolation & purification ; Sequence Alignment ; Triticum - metabolism ; Triticum aestivum ; Xylans - chemistry ; Xylans - metabolism</subject><ispartof>Biochemistry (Moscow), 2013-11, Vol.78 (11), p.1272-1279</ispartof><rights>Pleiades Publishing, Ltd. 2013</rights><rights>COPYRIGHT 2013 Springer</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c444t-291fcd3f9a6c5bcbf1662aecc090bd6afb29309c1912033be5a17feeb4302ec93</citedby><cites>FETCH-LOGICAL-c444t-291fcd3f9a6c5bcbf1662aecc090bd6afb29309c1912033be5a17feeb4302ec93</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1134/S0006297913110072$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1134/S0006297913110072$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24460941$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ahmed, S.</creatorcontrib><creatorcontrib>Luís, A. S.</creatorcontrib><creatorcontrib>Brás, J. L. A.</creatorcontrib><creatorcontrib>Fontes, C. M. G. A.</creatorcontrib><creatorcontrib>Goyal, A.</creatorcontrib><title>Functional and structural characterization of family 6 carbohydrate-binding module (CtCBM6A) of Clostridium thermocellum α-L-arabinofuranosidase</title><title>Biochemistry (Moscow)</title><addtitle>Biochemistry Moscow</addtitle><addtitle>Biochemistry (Mosc)</addtitle><description>The gene encoding the family 6 carbohydrate-binding module (
Ct
CBM6A) from
Clostridium thermocellum
, cloned in pET-21a(+) expression vector, was overexpressed using
Escherichia coli
BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of the recombinant
Ct
CBM6A showed molecular size of approximately 15 kDa. Ligand-binding analysis of
Ct
CBM6A with rye arabinoxylan and oat spelt xylan by affinity gel electrophoresis showed low affinity for these ligands (
K
a
of 40 and 26 liter/g, respectively), and analysis by fluorescence spectroscopy (
K
a
of 33 and 15 liter/g, respectively) corroborated lower binding affinity with the above soluble ligands. However,
Ct
CBM6A displayed significantly higher ligand-binding affinity with insoluble wheat arabinoxylan with equilibrium association constant
K
a
of 230 M
−1
and binding capacity (
N
0
) of 11 μmole/g. The protein melting curve of
Ct
CBM6A displayed a peak shift from 53 to 58°C in the presence of Ca
2+
, indicating that Ca
2+
imparts thermal stability to the
Ct
CBM6A structure. Homology modeling of
Ct
CBM6A revealed a characteristic β-sandwich core structure. The Ramachandran plot of
Ct
CBM6A showed 89% of the residues in the most favorable region, 10% in additionally favored region, and 1% in generously allowed region, indicating that
Ct
CBM6A has a stable conformation.</description><subject>Amino Acid Sequence</subject><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Binding proteins</subject><subject>Biochemistry</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedicine</subject><subject>Bioorganic Chemistry</subject><subject>Clostridium</subject><subject>Clostridium thermocellum</subject><subject>Clostridium thermocellum - enzymology</subject><subject>Escherichia coli</subject><subject>Escherichia coli - metabolism</subject><subject>Genetic aspects</subject><subject>Glycoside Hydrolases - chemistry</subject><subject>Glycoside Hydrolases - genetics</subject><subject>Glycoside Hydrolases - metabolism</subject><subject>Life Sciences</subject><subject>Ligands</subject><subject>Microbiology</subject><subject>Molecular Sequence Data</subject><subject>Physiological aspects</subject><subject>Protein Binding</subject><subject>Protein Denaturation</subject><subject>Protein Stability</subject><subject>Protein Structure, Tertiary</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Sequence Alignment</subject><subject>Triticum - metabolism</subject><subject>Triticum aestivum</subject><subject>Xylans - chemistry</subject><subject>Xylans - metabolism</subject><issn>0006-2979</issn><issn>1608-3040</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNks1u1DAQxy1ERZfCA3BBkbiUQ4q_4tTHbUQBaasegHPk2ONdV4ldbOewvAWPwov0mepoCxIqQsgHa2Z-_5nxeBB6RfAZIYy_-4wxFlS2kjBCMG7pE7QiAp_XDHP8FK2WcL3Ej9HzlG6KSbFkz9Ax5VxgyckK_bicvc4ueDVWypsq5TjrPMdi6p2KSmeI7rtaiCrYyqrJjftKVFrFIez2JqoM9eC8cX5bTcHMI1SnXe4ursT67aLoxlByOuPmqco7iFPQMI7FuPtZb-pSoYiDLQV9SM6oBC_QkVVjgpcP9wn6evn-S_ex3lx_-NStN7XmnOfyLGK1YVYqoZtBD5YIQRVojSUejFB2oJJhqYkkFDM2QKNIawEGzjAFLdkJOj3kvY3h2wwp95NLS2_KQ5hTT7ikbRlZg_8LFeSctaSgbw7oVo3QO29DLkNc8H7NGto2DIumUGd_ocoxMDkdPFhX_H8IyEGgY0gpgu1vo5tU3PcE98su9I92oWheP3Q9DxOY34pfn18AegBSCfktxP4mzLFsQvpH1nsaMr81</recordid><startdate>20131101</startdate><enddate>20131101</enddate><creator>Ahmed, S.</creator><creator>Luís, A. S.</creator><creator>Brás, J. L. A.</creator><creator>Fontes, C. M. G. A.</creator><creator>Goyal, A.</creator><general>Springer US</general><general>Springer</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>C1K</scope><scope>7X8</scope></search><sort><creationdate>20131101</creationdate><title>Functional and structural characterization of family 6 carbohydrate-binding module (CtCBM6A) of Clostridium thermocellum α-L-arabinofuranosidase</title><author>Ahmed, S. ; Luís, A. S. ; Brás, J. L. A. ; Fontes, C. M. G. A. ; Goyal, A.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c444t-291fcd3f9a6c5bcbf1662aecc090bd6afb29309c1912033be5a17feeb4302ec93</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Amino Acid Sequence</topic><topic>Bacterial Proteins - chemistry</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Binding proteins</topic><topic>Biochemistry</topic><topic>Biomedical and Life Sciences</topic><topic>Biomedicine</topic><topic>Bioorganic Chemistry</topic><topic>Clostridium</topic><topic>Clostridium thermocellum</topic><topic>Clostridium thermocellum - enzymology</topic><topic>Escherichia coli</topic><topic>Escherichia coli - metabolism</topic><topic>Genetic aspects</topic><topic>Glycoside Hydrolases - chemistry</topic><topic>Glycoside Hydrolases - genetics</topic><topic>Glycoside Hydrolases - metabolism</topic><topic>Life Sciences</topic><topic>Ligands</topic><topic>Microbiology</topic><topic>Molecular Sequence Data</topic><topic>Physiological aspects</topic><topic>Protein Binding</topic><topic>Protein Denaturation</topic><topic>Protein Stability</topic><topic>Protein Structure, Tertiary</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Sequence Alignment</topic><topic>Triticum - metabolism</topic><topic>Triticum aestivum</topic><topic>Xylans - chemistry</topic><topic>Xylans - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ahmed, S.</creatorcontrib><creatorcontrib>Luís, A. S.</creatorcontrib><creatorcontrib>Brás, J. L. A.</creatorcontrib><creatorcontrib>Fontes, C. M. G. A.</creatorcontrib><creatorcontrib>Goyal, A.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemistry (Moscow)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ahmed, S.</au><au>Luís, A. S.</au><au>Brás, J. L. A.</au><au>Fontes, C. M. G. A.</au><au>Goyal, A.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Functional and structural characterization of family 6 carbohydrate-binding module (CtCBM6A) of Clostridium thermocellum α-L-arabinofuranosidase</atitle><jtitle>Biochemistry (Moscow)</jtitle><stitle>Biochemistry Moscow</stitle><addtitle>Biochemistry (Mosc)</addtitle><date>2013-11-01</date><risdate>2013</risdate><volume>78</volume><issue>11</issue><spage>1272</spage><epage>1279</epage><pages>1272-1279</pages><issn>0006-2979</issn><eissn>1608-3040</eissn><abstract>The gene encoding the family 6 carbohydrate-binding module (
Ct
CBM6A) from
Clostridium thermocellum
, cloned in pET-21a(+) expression vector, was overexpressed using
Escherichia coli
BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of the recombinant
Ct
CBM6A showed molecular size of approximately 15 kDa. Ligand-binding analysis of
Ct
CBM6A with rye arabinoxylan and oat spelt xylan by affinity gel electrophoresis showed low affinity for these ligands (
K
a
of 40 and 26 liter/g, respectively), and analysis by fluorescence spectroscopy (
K
a
of 33 and 15 liter/g, respectively) corroborated lower binding affinity with the above soluble ligands. However,
Ct
CBM6A displayed significantly higher ligand-binding affinity with insoluble wheat arabinoxylan with equilibrium association constant
K
a
of 230 M
−1
and binding capacity (
N
0
) of 11 μmole/g. The protein melting curve of
Ct
CBM6A displayed a peak shift from 53 to 58°C in the presence of Ca
2+
, indicating that Ca
2+
imparts thermal stability to the
Ct
CBM6A structure. Homology modeling of
Ct
CBM6A revealed a characteristic β-sandwich core structure. The Ramachandran plot of
Ct
CBM6A showed 89% of the residues in the most favorable region, 10% in additionally favored region, and 1% in generously allowed region, indicating that
Ct
CBM6A has a stable conformation.</abstract><cop>Boston</cop><pub>Springer US</pub><pmid>24460941</pmid><doi>10.1134/S0006297913110072</doi><tpages>8</tpages></addata></record> |
| fulltext | fulltext |
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| issn | 0006-2979 1608-3040 |
| language | eng |
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| source | MEDLINE; SpringerNature Journals |
| subjects | Amino Acid Sequence Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacterial Proteins - metabolism Binding proteins Biochemistry Biomedical and Life Sciences Biomedicine Bioorganic Chemistry Clostridium Clostridium thermocellum Clostridium thermocellum - enzymology Escherichia coli Escherichia coli - metabolism Genetic aspects Glycoside Hydrolases - chemistry Glycoside Hydrolases - genetics Glycoside Hydrolases - metabolism Life Sciences Ligands Microbiology Molecular Sequence Data Physiological aspects Protein Binding Protein Denaturation Protein Stability Protein Structure, Tertiary Recombinant Proteins - biosynthesis Recombinant Proteins - chemistry Recombinant Proteins - isolation & purification Sequence Alignment Triticum - metabolism Triticum aestivum Xylans - chemistry Xylans - metabolism |
| title | Functional and structural characterization of family 6 carbohydrate-binding module (CtCBM6A) of Clostridium thermocellum α-L-arabinofuranosidase |
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