Cloning and sequencing of cel5Z gene from Erwinia chrysanthemi PY35
The phytopathogenic bacterium Erwinia chrysanthemi (Ech) secretes multiple isozymes of plant cell wall disrupting enzymes such as pectate lyase and endoglucanases. We cloned genomic DNA from Ech PY35 digested with Sau3AI and ligated into pBluescript II SK+. One of the E. coli XL1-blue clones had the...
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| Veröffentlicht in: | Molecules and cells 2000-06, Vol.10 (3), p.269-274 |
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| creator | Park, S R Kim, M K Kim, J O Cho, S J Cho, Y U Yun, H D |
| description | The phytopathogenic bacterium Erwinia chrysanthemi (Ech) secretes multiple isozymes of plant cell wall disrupting enzymes such as pectate lyase and endoglucanases. We cloned genomic DNA from Ech PY35 digested with Sau3AI and ligated into pBluescript II SK+. One of the E. coli XL1-blue clones had the ability to hydrolyze carboxymethyl cellulose and polygalacturonic acid. By subsequent subcloning from this 2.9 kb fragment, we obtained a 2.0 kb (pPY401), designated cel5Z, which had the activity of hydrolyzation of carboxymethyl cellulose. The cel5Z gene had an open reading frame (ORF) of 1,281 bp starting with an ATG start codon and followed by a TAA stop codon, encoding 426 amino acids with a signal peptide of 41 amino acids. Since the deduced amino acid sequence of this protein was very similar to that of CelE of Pseudomonas fluorescens, and had the conserved region, VIYEIYNEPL, it belonged to the glycoside hydrolase family 5 of EC 3.2.1.4. The molecular mass of Cel5Z protein from E. coli XL1-blue, as analyzed by CMC-SDS-PAGE, appeared to be 42 kDa. The optimum pH was 6, and the optimum temperature was about 40 degrees C for its enzymatic activity. |
| doi_str_mv | 10.1016/S1016-8478(23)17474-7 |
| format | Article |
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We cloned genomic DNA from Ech PY35 digested with Sau3AI and ligated into pBluescript II SK+. One of the E. coli XL1-blue clones had the ability to hydrolyze carboxymethyl cellulose and polygalacturonic acid. By subsequent subcloning from this 2.9 kb fragment, we obtained a 2.0 kb (pPY401), designated cel5Z, which had the activity of hydrolyzation of carboxymethyl cellulose. The cel5Z gene had an open reading frame (ORF) of 1,281 bp starting with an ATG start codon and followed by a TAA stop codon, encoding 426 amino acids with a signal peptide of 41 amino acids. Since the deduced amino acid sequence of this protein was very similar to that of CelE of Pseudomonas fluorescens, and had the conserved region, VIYEIYNEPL, it belonged to the glycoside hydrolase family 5 of EC 3.2.1.4. The molecular mass of Cel5Z protein from E. coli XL1-blue, as analyzed by CMC-SDS-PAGE, appeared to be 42 kDa. The optimum pH was 6, and the optimum temperature was about 40 degrees C for its enzymatic activity.</description><identifier>ISSN: 1016-8478</identifier><identifier>DOI: 10.1016/S1016-8478(23)17474-7</identifier><identifier>PMID: 10901164</identifier><language>eng</language><publisher>United States</publisher><subject>Amino Acid Sequence ; Bacterial Proteins - chemistry ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Base Sequence ; Carboxymethylcellulose Sodium - metabolism ; Cellulase ; Dickeya chrysanthemi - enzymology ; Dickeya chrysanthemi - genetics ; Electrophoresis, Polyacrylamide Gel ; Genes, Bacterial - genetics ; Glycoside Hydrolases - chemistry ; Glycoside Hydrolases - genetics ; Glycoside Hydrolases - metabolism ; Molecular Sequence Data ; Polysaccharide-Lyases - genetics ; Polysaccharide-Lyases - metabolism ; Sequence Alignment</subject><ispartof>Molecules and cells, 2000-06, Vol.10 (3), p.269-274</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c352t-d18738ceed3aa6b3aa96995eb5a4e44295ba166cfaf814687944d4a67524b9463</citedby><cites>FETCH-LOGICAL-c352t-d18738ceed3aa6b3aa96995eb5a4e44295ba166cfaf814687944d4a67524b9463</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10901164$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Park, S R</creatorcontrib><creatorcontrib>Kim, M K</creatorcontrib><creatorcontrib>Kim, J O</creatorcontrib><creatorcontrib>Cho, S J</creatorcontrib><creatorcontrib>Cho, Y U</creatorcontrib><creatorcontrib>Yun, H D</creatorcontrib><title>Cloning and sequencing of cel5Z gene from Erwinia chrysanthemi PY35</title><title>Molecules and cells</title><addtitle>Mol Cells</addtitle><description>The phytopathogenic bacterium Erwinia chrysanthemi (Ech) secretes multiple isozymes of plant cell wall disrupting enzymes such as pectate lyase and endoglucanases. We cloned genomic DNA from Ech PY35 digested with Sau3AI and ligated into pBluescript II SK+. One of the E. coli XL1-blue clones had the ability to hydrolyze carboxymethyl cellulose and polygalacturonic acid. By subsequent subcloning from this 2.9 kb fragment, we obtained a 2.0 kb (pPY401), designated cel5Z, which had the activity of hydrolyzation of carboxymethyl cellulose. The cel5Z gene had an open reading frame (ORF) of 1,281 bp starting with an ATG start codon and followed by a TAA stop codon, encoding 426 amino acids with a signal peptide of 41 amino acids. Since the deduced amino acid sequence of this protein was very similar to that of CelE of Pseudomonas fluorescens, and had the conserved region, VIYEIYNEPL, it belonged to the glycoside hydrolase family 5 of EC 3.2.1.4. The molecular mass of Cel5Z protein from E. coli XL1-blue, as analyzed by CMC-SDS-PAGE, appeared to be 42 kDa. The optimum pH was 6, and the optimum temperature was about 40 degrees C for its enzymatic activity.</description><subject>Amino Acid Sequence</subject><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Base Sequence</subject><subject>Carboxymethylcellulose Sodium - metabolism</subject><subject>Cellulase</subject><subject>Dickeya chrysanthemi - enzymology</subject><subject>Dickeya chrysanthemi - genetics</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Genes, Bacterial - genetics</subject><subject>Glycoside Hydrolases - chemistry</subject><subject>Glycoside Hydrolases - genetics</subject><subject>Glycoside Hydrolases - metabolism</subject><subject>Molecular Sequence Data</subject><subject>Polysaccharide-Lyases - genetics</subject><subject>Polysaccharide-Lyases - metabolism</subject><subject>Sequence Alignment</subject><issn>1016-8478</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpNkE1Lw0AQhveg2Fr9CcqeRA_R_f44SmhVKCioB70sm82mjSSbutsi_fcmbREvM8zwvjMvDwAXGN1ihMXd61AzxaS6JvQGSyZZJo_A-G89AqcpfSGEpSDqBIww0ghjwcYgz5su1GEBbShh8t8bH9wwdhV0vuGfcOGDh1XsWjiNP3WoLXTLuE02rJe-reHLB-Vn4LiyTfLnhz4B77PpW_6YzZ8fnvL7eeYoJ-usxEpS5bwvqbWi6IsWWnNfcMs8Y0TzwmIhXGUrhZlQUjNWMiskJ6zQTNAJuNrfXcWuD5rWpq1Tn7KxwXebZCQmjFI0CPle6GKXUvSVWcW6tXFrMDIDFLMjZgY0hlCzI2Zk77s8PNgUrS__ufa46C_KgGeC</recordid><startdate>20000630</startdate><enddate>20000630</enddate><creator>Park, S R</creator><creator>Kim, M K</creator><creator>Kim, J O</creator><creator>Cho, S J</creator><creator>Cho, Y U</creator><creator>Yun, H D</creator><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>7X8</scope></search><sort><creationdate>20000630</creationdate><title>Cloning and sequencing of cel5Z gene from Erwinia chrysanthemi PY35</title><author>Park, S R ; Kim, M K ; Kim, J O ; Cho, S J ; Cho, Y U ; Yun, H D</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c352t-d18738ceed3aa6b3aa96995eb5a4e44295ba166cfaf814687944d4a67524b9463</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>Amino Acid Sequence</topic><topic>Bacterial Proteins - chemistry</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Base Sequence</topic><topic>Carboxymethylcellulose Sodium - metabolism</topic><topic>Cellulase</topic><topic>Dickeya chrysanthemi - enzymology</topic><topic>Dickeya chrysanthemi - genetics</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Genes, Bacterial - genetics</topic><topic>Glycoside Hydrolases - chemistry</topic><topic>Glycoside Hydrolases - genetics</topic><topic>Glycoside Hydrolases - metabolism</topic><topic>Molecular Sequence Data</topic><topic>Polysaccharide-Lyases - genetics</topic><topic>Polysaccharide-Lyases - metabolism</topic><topic>Sequence Alignment</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Park, S R</creatorcontrib><creatorcontrib>Kim, M K</creatorcontrib><creatorcontrib>Kim, J O</creatorcontrib><creatorcontrib>Cho, S J</creatorcontrib><creatorcontrib>Cho, Y U</creatorcontrib><creatorcontrib>Yun, H D</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Molecules and cells</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Park, S R</au><au>Kim, M K</au><au>Kim, J O</au><au>Cho, S J</au><au>Cho, Y U</au><au>Yun, H D</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Cloning and sequencing of cel5Z gene from Erwinia chrysanthemi PY35</atitle><jtitle>Molecules and cells</jtitle><addtitle>Mol Cells</addtitle><date>2000-06-30</date><risdate>2000</risdate><volume>10</volume><issue>3</issue><spage>269</spage><epage>274</epage><pages>269-274</pages><issn>1016-8478</issn><abstract>The phytopathogenic bacterium Erwinia chrysanthemi (Ech) secretes multiple isozymes of plant cell wall disrupting enzymes such as pectate lyase and endoglucanases. We cloned genomic DNA from Ech PY35 digested with Sau3AI and ligated into pBluescript II SK+. One of the E. coli XL1-blue clones had the ability to hydrolyze carboxymethyl cellulose and polygalacturonic acid. By subsequent subcloning from this 2.9 kb fragment, we obtained a 2.0 kb (pPY401), designated cel5Z, which had the activity of hydrolyzation of carboxymethyl cellulose. The cel5Z gene had an open reading frame (ORF) of 1,281 bp starting with an ATG start codon and followed by a TAA stop codon, encoding 426 amino acids with a signal peptide of 41 amino acids. Since the deduced amino acid sequence of this protein was very similar to that of CelE of Pseudomonas fluorescens, and had the conserved region, VIYEIYNEPL, it belonged to the glycoside hydrolase family 5 of EC 3.2.1.4. The molecular mass of Cel5Z protein from E. coli XL1-blue, as analyzed by CMC-SDS-PAGE, appeared to be 42 kDa. The optimum pH was 6, and the optimum temperature was about 40 degrees C for its enzymatic activity.</abstract><cop>United States</cop><pmid>10901164</pmid><doi>10.1016/S1016-8478(23)17474-7</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Amino Acid Sequence Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacterial Proteins - metabolism Base Sequence Carboxymethylcellulose Sodium - metabolism Cellulase Dickeya chrysanthemi - enzymology Dickeya chrysanthemi - genetics Electrophoresis, Polyacrylamide Gel Genes, Bacterial - genetics Glycoside Hydrolases - chemistry Glycoside Hydrolases - genetics Glycoside Hydrolases - metabolism Molecular Sequence Data Polysaccharide-Lyases - genetics Polysaccharide-Lyases - metabolism Sequence Alignment |
| title | Cloning and sequencing of cel5Z gene from Erwinia chrysanthemi PY35 |
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