The deletion of amino-terminal domain in Thermoactinomyces vulgaris R-47 alpha-amylases: Effects of domain N on activity, specificity, stability and dimerization
Thermoactinomyces vulgaris R-47 α-amylases, TVA I and TVA II, have a domain N, which is an extra structure in the family 13 enzymes. To investigate the roles of domain N in TVAs, we constructed TVAs-ΔN mutants which are deleted in domain N, and Y14, 16, 68A and Y41, 82, 95A mutants of TVA II. TVAs-Δ...
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| Veröffentlicht in: | Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2001-02, Vol.65 (2), p.401-408 |
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| creator | Yokota, T. (Tokyo Univ. of Agriculture and Technology, Fuchu (Japan)) Tonozuka, T Kamitori, S Sakano, Y |
| description | Thermoactinomyces vulgaris R-47 α-amylases, TVA I and TVA II, have a domain N, which is an extra structure in the family 13 enzymes. To investigate the roles of domain N in TVAs, we constructed TVAs-ΔN mutants which are deleted in domain N, and Y14, 16, 68A and Y41, 82, 95A mutants of TVA II. TVAs-ΔN were unstable under alkaline conditions, and their thermal stabilities were 10°C lower than that of wild-types. The specific activities of TVAs-ΔN for pullulan, starch, cyclodextrins, and oligosaccharides were drastically decreased, being about 1,500- to 10,000-fold smaller than those of wild-types. The k
cat
values of Y14, 16, 68A and Y41, 82, 95A for all tested substrates were markedly decreased, and the K
m
value of Y14, 16, 68A for α-CD and maltotriose were 25- and 3-fold larger, and that of Y41, 82, 92A for starch was 10-fold larger than that of the wild-type. TVA I and TVAs-ΔN in solution are a monomer, while TVA II is a homo-dimer, calculated by their molecular masses. These results suggest domain N in TVAs is an important structure for stabilization of enzymes, recognition and hydrolysis of substartes, and dimerization of TVA II. |
| doi_str_mv | 10.1271/bbb.65.401 |
| format | Article |
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cat
values of Y14, 16, 68A and Y41, 82, 95A for all tested substrates were markedly decreased, and the K
m
value of Y14, 16, 68A for α-CD and maltotriose were 25- and 3-fold larger, and that of Y41, 82, 92A for starch was 10-fold larger than that of the wild-type. TVA I and TVAs-ΔN in solution are a monomer, while TVA II is a homo-dimer, calculated by their molecular masses. These results suggest domain N in TVAs is an important structure for stabilization of enzymes, recognition and hydrolysis of substartes, and dimerization of TVA II.</description><identifier>ISSN: 0916-8451</identifier><identifier>EISSN: 1347-6947</identifier><identifier>DOI: 10.1271/bbb.65.401</identifier><identifier>PMID: 11302176</identifier><language>eng</language><publisher>Tokyo: Japan Society for Bioscience, Biotechnology, and Agrochemistry</publisher><subject>a-Amylase ; ALPHA AMYLASE ; alpha-Amylases - chemistry ; alpha-Amylases - genetics ; alpha-Amylases - metabolism ; Base Sequence ; Biological and medical sciences ; Biotechnology ; CHEMICAL STRUCTURE ; cyclodextrins ; Dimerization ; DNA Primers - genetics ; domain N ; Enzyme Stability ; ENZYMIC ACTIVITY ; family 13 ; Fundamental and applied biological sciences. Psychology ; Isoenzymes - chemistry ; Isoenzymes - genetics ; Isoenzymes - metabolism ; Kinetics ; Methods. Procedures. Technologies ; Micromonosporaceae - enzymology ; Micromonosporaceae - genetics ; Models, Molecular ; Molecular Weight ; oligosaccharides ; Protein engineering ; Protein Structure, Tertiary ; pullulan ; Sequence Deletion ; Substrate Specificity ; THERMOACTINOMYCES ; Thermoactinomyces vulgaris ; TVA ; α-Amylase</subject><ispartof>Bioscience, biotechnology, and biochemistry, 2001-02, Vol.65 (2), p.401-408</ispartof><rights>2001 by Japan Society for Bioscience, Biotechnology, and Agrochemistry 2001</rights><rights>2001 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c513t-38a7e34833eaf081e141234244232555ad0f65f859e3c1210725b196c4c3b42b3</citedby><cites>FETCH-LOGICAL-c513t-38a7e34833eaf081e141234244232555ad0f65f859e3c1210725b196c4c3b42b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=1061880$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11302176$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Yokota, T. (Tokyo Univ. of Agriculture and Technology, Fuchu (Japan))</creatorcontrib><creatorcontrib>Tonozuka, T</creatorcontrib><creatorcontrib>Kamitori, S</creatorcontrib><creatorcontrib>Sakano, Y</creatorcontrib><title>The deletion of amino-terminal domain in Thermoactinomyces vulgaris R-47 alpha-amylases: Effects of domain N on activity, specificity, stability and dimerization</title><title>Bioscience, biotechnology, and biochemistry</title><addtitle>Biosci Biotechnol Biochem</addtitle><description>Thermoactinomyces vulgaris R-47 α-amylases, TVA I and TVA II, have a domain N, which is an extra structure in the family 13 enzymes. To investigate the roles of domain N in TVAs, we constructed TVAs-ΔN mutants which are deleted in domain N, and Y14, 16, 68A and Y41, 82, 95A mutants of TVA II. TVAs-ΔN were unstable under alkaline conditions, and their thermal stabilities were 10°C lower than that of wild-types. The specific activities of TVAs-ΔN for pullulan, starch, cyclodextrins, and oligosaccharides were drastically decreased, being about 1,500- to 10,000-fold smaller than those of wild-types. The k
cat
values of Y14, 16, 68A and Y41, 82, 95A for all tested substrates were markedly decreased, and the K
m
value of Y14, 16, 68A for α-CD and maltotriose were 25- and 3-fold larger, and that of Y41, 82, 92A for starch was 10-fold larger than that of the wild-type. TVA I and TVAs-ΔN in solution are a monomer, while TVA II is a homo-dimer, calculated by their molecular masses. These results suggest domain N in TVAs is an important structure for stabilization of enzymes, recognition and hydrolysis of substartes, and dimerization of TVA II.</description><subject>a-Amylase</subject><subject>ALPHA AMYLASE</subject><subject>alpha-Amylases - chemistry</subject><subject>alpha-Amylases - genetics</subject><subject>alpha-Amylases - metabolism</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>CHEMICAL STRUCTURE</subject><subject>cyclodextrins</subject><subject>Dimerization</subject><subject>DNA Primers - genetics</subject><subject>domain N</subject><subject>Enzyme Stability</subject><subject>ENZYMIC ACTIVITY</subject><subject>family 13</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Isoenzymes - chemistry</subject><subject>Isoenzymes - genetics</subject><subject>Isoenzymes - metabolism</subject><subject>Kinetics</subject><subject>Methods. Procedures. Technologies</subject><subject>Micromonosporaceae - enzymology</subject><subject>Micromonosporaceae - genetics</subject><subject>Models, Molecular</subject><subject>Molecular Weight</subject><subject>oligosaccharides</subject><subject>Protein engineering</subject><subject>Protein Structure, Tertiary</subject><subject>pullulan</subject><subject>Sequence Deletion</subject><subject>Substrate Specificity</subject><subject>THERMOACTINOMYCES</subject><subject>Thermoactinomyces vulgaris</subject><subject>TVA</subject><subject>α-Amylase</subject><issn>0916-8451</issn><issn>1347-6947</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU2LFDEQhhtR3HH14l3JQTyIPaby0enxJsv6xaIi6zlUp5PdSLozJj0r7b_xn5pmWvQgCAVVST31VsFbVQ-BboEpeNF13baRW0HhVrUBLlTd7IS6XW3oDpq6FRJOqns5f6W0fEi4W50AcMpANZvq5-W1Jb0NdvJxJNERHPwY68mmkjGQPg7oR1KigGmIaKbSH2ZjM7k5hCtMPpPPtVAEw_4aaxzmgNnml-TcOWumvGiuIh9IWbEI3Phpfk7y3hrvvDk-Jux8KCXBsSe9H2zyP3A56n51x2HI9sGaT6svr88vz97WFx_fvDt7dVEbCXyqeYvKctFybtHRFiwIYFwwIRhnUkrsqWuka-XOcgMMqGKyg11jhOGdYB0_rZ4edfcpfjvYPOnBZ2NDwNHGQ9ZKUUmpov8FoaWqhCrgsyNoUsw5Waf3yQ-YZg1UL87p4pxupC7OFfjxqnroBtv_QVerCvBkBTAbDC7haHz-S7KBtl2uk0fMjy6mAb_HFHo94Rxi-j3D_7n_0XHOYdR4VWzV7z8xSoFSvpOM_wJcBLxs</recordid><startdate>20010201</startdate><enddate>20010201</enddate><creator>Yokota, T. (Tokyo Univ. of Agriculture and Technology, Fuchu (Japan))</creator><creator>Tonozuka, T</creator><creator>Kamitori, S</creator><creator>Sakano, Y</creator><general>Japan Society for Bioscience, Biotechnology, and Agrochemistry</general><general>Japan Society for Bioscience Biotechnology and Agrochemistry</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>C1K</scope><scope>7X8</scope></search><sort><creationdate>20010201</creationdate><title>The deletion of amino-terminal domain in Thermoactinomyces vulgaris R-47 alpha-amylases: Effects of domain N on activity, specificity, stability and dimerization</title><author>Yokota, T. (Tokyo Univ. of Agriculture and Technology, Fuchu (Japan)) ; Tonozuka, T ; Kamitori, S ; Sakano, Y</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c513t-38a7e34833eaf081e141234244232555ad0f65f859e3c1210725b196c4c3b42b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>a-Amylase</topic><topic>ALPHA AMYLASE</topic><topic>alpha-Amylases - chemistry</topic><topic>alpha-Amylases - genetics</topic><topic>alpha-Amylases - metabolism</topic><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>CHEMICAL STRUCTURE</topic><topic>cyclodextrins</topic><topic>Dimerization</topic><topic>DNA Primers - genetics</topic><topic>domain N</topic><topic>Enzyme Stability</topic><topic>ENZYMIC ACTIVITY</topic><topic>family 13</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Isoenzymes - chemistry</topic><topic>Isoenzymes - genetics</topic><topic>Isoenzymes - metabolism</topic><topic>Kinetics</topic><topic>Methods. Procedures. Technologies</topic><topic>Micromonosporaceae - enzymology</topic><topic>Micromonosporaceae - genetics</topic><topic>Models, Molecular</topic><topic>Molecular Weight</topic><topic>oligosaccharides</topic><topic>Protein engineering</topic><topic>Protein Structure, Tertiary</topic><topic>pullulan</topic><topic>Sequence Deletion</topic><topic>Substrate Specificity</topic><topic>THERMOACTINOMYCES</topic><topic>Thermoactinomyces vulgaris</topic><topic>TVA</topic><topic>α-Amylase</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Yokota, T. (Tokyo Univ. of Agriculture and Technology, Fuchu (Japan))</creatorcontrib><creatorcontrib>Tonozuka, T</creatorcontrib><creatorcontrib>Kamitori, S</creatorcontrib><creatorcontrib>Sakano, Y</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>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>Bioscience, biotechnology, and biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Yokota, T. (Tokyo Univ. of Agriculture and Technology, Fuchu (Japan))</au><au>Tonozuka, T</au><au>Kamitori, S</au><au>Sakano, Y</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>The deletion of amino-terminal domain in Thermoactinomyces vulgaris R-47 alpha-amylases: Effects of domain N on activity, specificity, stability and dimerization</atitle><jtitle>Bioscience, biotechnology, and biochemistry</jtitle><addtitle>Biosci Biotechnol Biochem</addtitle><date>2001-02-01</date><risdate>2001</risdate><volume>65</volume><issue>2</issue><spage>401</spage><epage>408</epage><pages>401-408</pages><issn>0916-8451</issn><eissn>1347-6947</eissn><abstract>Thermoactinomyces vulgaris R-47 α-amylases, TVA I and TVA II, have a domain N, which is an extra structure in the family 13 enzymes. To investigate the roles of domain N in TVAs, we constructed TVAs-ΔN mutants which are deleted in domain N, and Y14, 16, 68A and Y41, 82, 95A mutants of TVA II. TVAs-ΔN were unstable under alkaline conditions, and their thermal stabilities were 10°C lower than that of wild-types. The specific activities of TVAs-ΔN for pullulan, starch, cyclodextrins, and oligosaccharides were drastically decreased, being about 1,500- to 10,000-fold smaller than those of wild-types. The k
cat
values of Y14, 16, 68A and Y41, 82, 95A for all tested substrates were markedly decreased, and the K
m
value of Y14, 16, 68A for α-CD and maltotriose were 25- and 3-fold larger, and that of Y41, 82, 92A for starch was 10-fold larger than that of the wild-type. TVA I and TVAs-ΔN in solution are a monomer, while TVA II is a homo-dimer, calculated by their molecular masses. These results suggest domain N in TVAs is an important structure for stabilization of enzymes, recognition and hydrolysis of substartes, and dimerization of TVA II.</abstract><cop>Tokyo</cop><pub>Japan Society for Bioscience, Biotechnology, and Agrochemistry</pub><pmid>11302176</pmid><doi>10.1271/bbb.65.401</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| source | Oxford University Press Journals All Titles (1996-Current); MEDLINE; J-STAGE (Japan Science & Technology Information Aggregator, Electronic) Freely Available Titles - Japanese; Open Access Titles of Japan; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Free Full-Text Journals in Chemistry |
| subjects | a-Amylase ALPHA AMYLASE alpha-Amylases - chemistry alpha-Amylases - genetics alpha-Amylases - metabolism Base Sequence Biological and medical sciences Biotechnology CHEMICAL STRUCTURE cyclodextrins Dimerization DNA Primers - genetics domain N Enzyme Stability ENZYMIC ACTIVITY family 13 Fundamental and applied biological sciences. Psychology Isoenzymes - chemistry Isoenzymes - genetics Isoenzymes - metabolism Kinetics Methods. Procedures. Technologies Micromonosporaceae - enzymology Micromonosporaceae - genetics Models, Molecular Molecular Weight oligosaccharides Protein engineering Protein Structure, Tertiary pullulan Sequence Deletion Substrate Specificity THERMOACTINOMYCES Thermoactinomyces vulgaris TVA α-Amylase |
| title | The deletion of amino-terminal domain in Thermoactinomyces vulgaris R-47 alpha-amylases: Effects of domain N on activity, specificity, stability and dimerization |
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