Molecular cloning, Overproduction and characterization of the Bacillus cereus IMP dehydrogenase
The gene of IMP dehydrogenase of Bacillus cereus ts-4, a temperature-sensitive mutant of B. cereus JCM 2152, was subcloned and its sequence was analyzed. A B. cereus ts-4 DNA fragment of 2,065 bp containing the entire impdh gene and flanking regions was sequenced. The fragment contained an open read...
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| Veröffentlicht in: | Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2000-06, Vol.64 (6), p.1210-1216 |
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| creator | Kim, S.I. (Kyushu Univ., Fukuoka (Japan)) Miyamoto, T Honjoh, K Iio, M Hatano, S |
| description | The gene of IMP dehydrogenase of Bacillus cereus ts-4, a temperature-sensitive mutant of B. cereus JCM 2152, was subcloned and its sequence was analyzed. A B. cereus ts-4 DNA fragment of 2,065 bp containing the entire impdh gene and flanking regions was sequenced. The fragment contained an open reading frame of 1,527 bp encoding 509 amino acids with a calculated molecular mass of 55,390 Da. The impdh sequence of JCM 2152 was also analyzed by TA cloning using PCR products amplified with primers from B. cereus ts-4 impdh gene. The gene amplified by PCR was expressed in Escherichia coli using a pET17×b expression plasmid. The N-terminal amino acid sequence of the overproduced enzyme was identified as Met-Trp- Glu-Ser-Lys-Phe-Val-Lys-Glu-Gly-Leu-Thr-Phe-Asp- Asp-Val-Leu-Leu-Val-Pro. The overproduced enzyme was eluted at a molecular mass of about 225 kDa by gel filtration. The molecular mass of the subunit was estimated to be 56 kDa by SDS-PAGE. The overproduced enzyme was active against IMP, IDP, and ITP, and showed the highest activity at pH 9.5. These properties of the recombinant enzyme were almost identical to those of IMP dehydrogenase of B. cereus. |
| doi_str_mv | 10.1271/bbb.64.1210 |
| format | Article |
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(Kyushu Univ., Fukuoka (Japan)) ; Miyamoto, T ; Honjoh, K ; Iio, M ; Hatano, S</creator><creatorcontrib>Kim, S.I. (Kyushu Univ., Fukuoka (Japan)) ; Miyamoto, T ; Honjoh, K ; Iio, M ; Hatano, S</creatorcontrib><description>The gene of IMP dehydrogenase of Bacillus cereus ts-4, a temperature-sensitive mutant of B. cereus JCM 2152, was subcloned and its sequence was analyzed. A B. cereus ts-4 DNA fragment of 2,065 bp containing the entire impdh gene and flanking regions was sequenced. The fragment contained an open reading frame of 1,527 bp encoding 509 amino acids with a calculated molecular mass of 55,390 Da. The impdh sequence of JCM 2152 was also analyzed by TA cloning using PCR products amplified with primers from B. cereus ts-4 impdh gene. The gene amplified by PCR was expressed in Escherichia coli using a pET17×b expression plasmid. The N-terminal amino acid sequence of the overproduced enzyme was identified as Met-Trp- Glu-Ser-Lys-Phe-Val-Lys-Glu-Gly-Leu-Thr-Phe-Asp- Asp-Val-Leu-Leu-Val-Pro. The overproduced enzyme was eluted at a molecular mass of about 225 kDa by gel filtration. The molecular mass of the subunit was estimated to be 56 kDa by SDS-PAGE. The overproduced enzyme was active against IMP, IDP, and ITP, and showed the highest activity at pH 9.5. These properties of the recombinant enzyme were almost identical to those of IMP dehydrogenase of B. cereus.</description><identifier>ISSN: 0916-8451</identifier><identifier>EISSN: 1347-6947</identifier><identifier>DOI: 10.1271/bbb.64.1210</identifier><identifier>PMID: 10923792</identifier><language>eng</language><publisher>Tokyo: Japan Society for Bioscience, Biotechnology, and Agrochemistry</publisher><subject>Amino Acid Sequence ; BACILLUS CEREUS ; Bacillus cereus - enzymology ; Bacillus cereus - genetics ; Bacteriology ; Base Sequence ; Biological and medical sciences ; cloning ; Cloning, Molecular ; DNA Primers - genetics ; DNA, Bacterial - genetics ; Escherichia coli ; Escherichia coli - genetics ; expression ; Fundamental and applied biological sciences. Psychology ; Genes, Bacterial ; IMP dehydrogenase ; IMP Dehydrogenase - biosynthesis ; IMP Dehydrogenase - chemistry ; IMP Dehydrogenase - genetics ; impdh gene ; Metabolism. Enzymes ; Microbiology ; MOLECULAR CLONING ; Molecular Sequence Data ; Molecular Weight ; Mutation ; NUCLEOTIDE SEQUENCE ; Open Reading Frames ; OXIDOREDUCTASES ; PURINES ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics ; sporulation ; Substrate Specificity ; Temperature</subject><ispartof>Bioscience, biotechnology, and biochemistry, 2000-06, Vol.64 (6), p.1210-1216</ispartof><rights>2000 by Japan Society for Bioscience, Biotechnology, and Agrochemistry 2000</rights><rights>2000 INIST-CNRS</rights><rights>Copyright Japan Science and Technology Agency 2000</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,777,781,27905,27906</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=1467474$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10923792$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kim, S.I. (Kyushu Univ., Fukuoka (Japan))</creatorcontrib><creatorcontrib>Miyamoto, T</creatorcontrib><creatorcontrib>Honjoh, K</creatorcontrib><creatorcontrib>Iio, M</creatorcontrib><creatorcontrib>Hatano, S</creatorcontrib><title>Molecular cloning, Overproduction and characterization of the Bacillus cereus IMP dehydrogenase</title><title>Bioscience, biotechnology, and biochemistry</title><addtitle>Biosci Biotechnol Biochem</addtitle><description>The gene of IMP dehydrogenase of Bacillus cereus ts-4, a temperature-sensitive mutant of B. cereus JCM 2152, was subcloned and its sequence was analyzed. A B. cereus ts-4 DNA fragment of 2,065 bp containing the entire impdh gene and flanking regions was sequenced. The fragment contained an open reading frame of 1,527 bp encoding 509 amino acids with a calculated molecular mass of 55,390 Da. The impdh sequence of JCM 2152 was also analyzed by TA cloning using PCR products amplified with primers from B. cereus ts-4 impdh gene. The gene amplified by PCR was expressed in Escherichia coli using a pET17×b expression plasmid. The N-terminal amino acid sequence of the overproduced enzyme was identified as Met-Trp- Glu-Ser-Lys-Phe-Val-Lys-Glu-Gly-Leu-Thr-Phe-Asp- Asp-Val-Leu-Leu-Val-Pro. The overproduced enzyme was eluted at a molecular mass of about 225 kDa by gel filtration. The molecular mass of the subunit was estimated to be 56 kDa by SDS-PAGE. The overproduced enzyme was active against IMP, IDP, and ITP, and showed the highest activity at pH 9.5. These properties of the recombinant enzyme were almost identical to those of IMP dehydrogenase of B. cereus.</description><subject>Amino Acid Sequence</subject><subject>BACILLUS CEREUS</subject><subject>Bacillus cereus - enzymology</subject><subject>Bacillus cereus - genetics</subject><subject>Bacteriology</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>cloning</subject><subject>Cloning, Molecular</subject><subject>DNA Primers - genetics</subject><subject>DNA, Bacterial - genetics</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>expression</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Genes, Bacterial</subject><subject>IMP dehydrogenase</subject><subject>IMP Dehydrogenase - biosynthesis</subject><subject>IMP Dehydrogenase - chemistry</subject><subject>IMP Dehydrogenase - genetics</subject><subject>impdh gene</subject><subject>Metabolism. Enzymes</subject><subject>Microbiology</subject><subject>MOLECULAR CLONING</subject><subject>Molecular Sequence Data</subject><subject>Molecular Weight</subject><subject>Mutation</subject><subject>NUCLEOTIDE SEQUENCE</subject><subject>Open Reading Frames</subject><subject>OXIDOREDUCTASES</subject><subject>PURINES</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><subject>sporulation</subject><subject>Substrate Specificity</subject><subject>Temperature</subject><issn>0916-8451</issn><issn>1347-6947</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0Utv1DAQAGALgehSOHEGRaLiAikex7HjI614FLVqD3C2Jn7spvLGrZ2All-PSxaBEBKnsUbfjGY8hDwFegxMwpu-748FL2-g98gKGi5robi8T1ZUgag73sIBeZTzNaUl0cJDcgBUsUYqtiL6IgZn5oCpMiGOw7h-XV1-dekmRTubaYhjhaOtzAYTmsml4Tv-TEZfTRtXnaAZQphzZVxyJZxdXFXWbXY2xbUbMbvH5IHHkN2TfTwkX96_-3z6sT6__HB2-va8Ni2FqQZQYHsmW4nQWWOhw1aAs0YpyrqeU8GbzrS9ElRYaL1vpDVAy67O-bJKc0heLn3L4Lezy5PeDtm4EHB0cc5alq-SksF_IciuYaoVBb74C17HOY1lCQ2cK94IxlhRrxZlUsw5Oa9v0rDFtNNA9d15dDmPFlzfnafo5_uec7919g-73KOAoz3AbDD4hKMZ8m_HheSSFyYWNow-pi1-iylYPeEuxPSrpvn3AM-WQo9R4zoV9-mKUQqUMlqW_gHO7bG2</recordid><startdate>20000601</startdate><enddate>20000601</enddate><creator>Kim, S.I. (Kyushu Univ., Fukuoka (Japan))</creator><creator>Miyamoto, T</creator><creator>Honjoh, K</creator><creator>Iio, M</creator><creator>Hatano, S</creator><general>Japan Society for Bioscience, Biotechnology, and Agrochemistry</general><general>Japan Society for Bioscience Biotechnology and Agrochemistry</general><general>Oxford University Press</general><scope>FBQ</scope><scope>IQODW</scope><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>7TM</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>20000601</creationdate><title>Molecular cloning, Overproduction and characterization of the Bacillus cereus IMP dehydrogenase</title><author>Kim, S.I. (Kyushu Univ., Fukuoka (Japan)) ; Miyamoto, T ; Honjoh, K ; Iio, M ; Hatano, S</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c501t-1191db2757a18dcd18a561edc99028b406438c5b9606d15ff37dc10347eef2373</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>Amino Acid Sequence</topic><topic>BACILLUS CEREUS</topic><topic>Bacillus cereus - enzymology</topic><topic>Bacillus cereus - genetics</topic><topic>Bacteriology</topic><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>cloning</topic><topic>Cloning, Molecular</topic><topic>DNA Primers - genetics</topic><topic>DNA, Bacterial - genetics</topic><topic>Escherichia coli</topic><topic>Escherichia coli - genetics</topic><topic>expression</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Genes, Bacterial</topic><topic>IMP dehydrogenase</topic><topic>IMP Dehydrogenase - biosynthesis</topic><topic>IMP Dehydrogenase - chemistry</topic><topic>IMP Dehydrogenase - genetics</topic><topic>impdh gene</topic><topic>Metabolism. Enzymes</topic><topic>Microbiology</topic><topic>MOLECULAR CLONING</topic><topic>Molecular Sequence Data</topic><topic>Molecular Weight</topic><topic>Mutation</topic><topic>NUCLEOTIDE SEQUENCE</topic><topic>Open Reading Frames</topic><topic>OXIDOREDUCTASES</topic><topic>PURINES</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - genetics</topic><topic>sporulation</topic><topic>Substrate Specificity</topic><topic>Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kim, S.I. (Kyushu Univ., Fukuoka (Japan))</creatorcontrib><creatorcontrib>Miyamoto, T</creatorcontrib><creatorcontrib>Honjoh, K</creatorcontrib><creatorcontrib>Iio, M</creatorcontrib><creatorcontrib>Hatano, S</creatorcontrib><collection>AGRIS</collection><collection>Pascal-Francis</collection><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>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Bioscience, biotechnology, and biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Kim, S.I. (Kyushu Univ., Fukuoka (Japan))</au><au>Miyamoto, T</au><au>Honjoh, K</au><au>Iio, M</au><au>Hatano, S</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Molecular cloning, Overproduction and characterization of the Bacillus cereus IMP dehydrogenase</atitle><jtitle>Bioscience, biotechnology, and biochemistry</jtitle><addtitle>Biosci Biotechnol Biochem</addtitle><date>2000-06-01</date><risdate>2000</risdate><volume>64</volume><issue>6</issue><spage>1210</spage><epage>1216</epage><pages>1210-1216</pages><issn>0916-8451</issn><eissn>1347-6947</eissn><abstract>The gene of IMP dehydrogenase of Bacillus cereus ts-4, a temperature-sensitive mutant of B. cereus JCM 2152, was subcloned and its sequence was analyzed. A B. cereus ts-4 DNA fragment of 2,065 bp containing the entire impdh gene and flanking regions was sequenced. The fragment contained an open reading frame of 1,527 bp encoding 509 amino acids with a calculated molecular mass of 55,390 Da. The impdh sequence of JCM 2152 was also analyzed by TA cloning using PCR products amplified with primers from B. cereus ts-4 impdh gene. The gene amplified by PCR was expressed in Escherichia coli using a pET17×b expression plasmid. The N-terminal amino acid sequence of the overproduced enzyme was identified as Met-Trp- Glu-Ser-Lys-Phe-Val-Lys-Glu-Gly-Leu-Thr-Phe-Asp- Asp-Val-Leu-Leu-Val-Pro. The overproduced enzyme was eluted at a molecular mass of about 225 kDa by gel filtration. The molecular mass of the subunit was estimated to be 56 kDa by SDS-PAGE. The overproduced enzyme was active against IMP, IDP, and ITP, and showed the highest activity at pH 9.5. These properties of the recombinant enzyme were almost identical to those of IMP dehydrogenase of B. cereus.</abstract><cop>Tokyo</cop><pub>Japan Society for Bioscience, Biotechnology, and Agrochemistry</pub><pmid>10923792</pmid><doi>10.1271/bbb.64.1210</doi><tpages>7</tpages></addata></record> |
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| source | J-STAGE Free; MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Oxford University Press Journals All Titles (1996-Current); Freely Accessible Japanese Titles; Free Full-Text Journals in Chemistry |
| subjects | Amino Acid Sequence BACILLUS CEREUS Bacillus cereus - enzymology Bacillus cereus - genetics Bacteriology Base Sequence Biological and medical sciences cloning Cloning, Molecular DNA Primers - genetics DNA, Bacterial - genetics Escherichia coli Escherichia coli - genetics expression Fundamental and applied biological sciences. Psychology Genes, Bacterial IMP dehydrogenase IMP Dehydrogenase - biosynthesis IMP Dehydrogenase - chemistry IMP Dehydrogenase - genetics impdh gene Metabolism. Enzymes Microbiology MOLECULAR CLONING Molecular Sequence Data Molecular Weight Mutation NUCLEOTIDE SEQUENCE Open Reading Frames OXIDOREDUCTASES PURINES Recombinant Proteins - biosynthesis Recombinant Proteins - chemistry Recombinant Proteins - genetics sporulation Substrate Specificity Temperature |
| title | Molecular cloning, Overproduction and characterization of the Bacillus cereus IMP dehydrogenase |
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