Purification and characterization of invertase from a novel industrial yeast, Schwanniomyces occidentalis
The use of yeast as an expression system for heterologous proteins offers several potential advantages with respect to industrial scale-up and genetics over other expression systems, but suffers from several drawbacks. For example, the secreted proteins of S. cerevisiae, found in the periplasm, are...
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| Veröffentlicht in: | Preparative biochemistry 1989, Vol.19 (4), p.293-319 |
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| creator | Klein, R.D. (The Upjohn Company, Kalamazoo, MI) Deibel, M.R. Jr Sarcich, J.L Zurcher-Neely, H.A Reardon, I.M Heinrikson, R.L |
| description | The use of yeast as an expression system for heterologous proteins offers several potential advantages with respect to industrial scale-up and genetics over other expression systems, but suffers from several drawbacks. For example, the secreted proteins of S. cerevisiae, found in the periplasm, are hyperglycosylated and the organism has a limited range of usable substrates. Other yeasts have similar disadvantages in addition to producing a variety of proteases. We have investigated the use of Schwanniomyces occidentalis as a host for developing a gene expression system in which these and several disadvantages are minimized. The present paper describes the isolation and characterization of an invertase from cell free supernatants of the yeast Schwanniomyces occidentalis grown on lactose. The enzyme is a β-D-fructofuranoside-fructohydrolyase, composed of two identical subunits of 76,000 to 78,000 da. with a native molecular mass of 125,000 +/− 25,000 da. of which approximately 17% can be attributed to N-linked carbohydrate. The enzyme has a Vmax of 0.49 +/− 0.025 units, a Km of 21 +/− 1.5 mM, and temperature and pH optima of 55°C and 3.9-4.5, respectively. The amino acid sequences of the amino terminal region and an internal tryptic peptide support an 81% identity with the invertase from Saccharomyces cerevisiae. The enzyme is induced by low glucose and is catabolite repressed. |
| doi_str_mv | 10.1080/10826068908544919 |
| format | Article |
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(The Upjohn Company, Kalamazoo, MI) ; Deibel, M.R. Jr ; Sarcich, J.L ; Zurcher-Neely, H.A ; Reardon, I.M ; Heinrikson, R.L</creator><creatorcontrib>Klein, R.D. (The Upjohn Company, Kalamazoo, MI) ; Deibel, M.R. Jr ; Sarcich, J.L ; Zurcher-Neely, H.A ; Reardon, I.M ; Heinrikson, R.L</creatorcontrib><description>The use of yeast as an expression system for heterologous proteins offers several potential advantages with respect to industrial scale-up and genetics over other expression systems, but suffers from several drawbacks. For example, the secreted proteins of S. cerevisiae, found in the periplasm, are hyperglycosylated and the organism has a limited range of usable substrates. Other yeasts have similar disadvantages in addition to producing a variety of proteases. We have investigated the use of Schwanniomyces occidentalis as a host for developing a gene expression system in which these and several disadvantages are minimized. The present paper describes the isolation and characterization of an invertase from cell free supernatants of the yeast Schwanniomyces occidentalis grown on lactose. The enzyme is a β-D-fructofuranoside-fructohydrolyase, composed of two identical subunits of 76,000 to 78,000 da. with a native molecular mass of 125,000 +/− 25,000 da. of which approximately 17% can be attributed to N-linked carbohydrate. The enzyme has a Vmax of 0.49 +/− 0.025 units, a Km of 21 +/− 1.5 mM, and temperature and pH optima of 55°C and 3.9-4.5, respectively. The amino acid sequences of the amino terminal region and an internal tryptic peptide support an 81% identity with the invertase from Saccharomyces cerevisiae. The enzyme is induced by low glucose and is catabolite repressed.</description><identifier>ISSN: 0032-7484</identifier><identifier>EISSN: 2331-0510</identifier><identifier>DOI: 10.1080/10826068908544919</identifier><identifier>PMID: 2622872</identifier><identifier>CODEN: PRBCBQ</identifier><language>eng</language><publisher>New York, NY: Taylor & Francis Group</publisher><subject>ACTIVIDAD ENZIMATICA ; ACTIVITE ENZYMATIQUE ; Amino Acid Sequence ; Amino Acids - analysis ; BETA-FRUCTOFURANOSIDASE ; Biological and medical sciences ; Biotechnology ; Chromatography, High Pressure Liquid ; Electrophoresis, Polyacrylamide Gel ; Enzyme engineering ; ENZYMIC ACTIVITY ; FRUCTOFURANOSIDASA ; FRUCTOFURANOSIDASE ; Fundamental and applied biological sciences. Psychology ; Glycoside Hydrolases - analysis ; Glycoside Hydrolases - isolation & purification ; Glycoside Hydrolases - metabolism ; Glycosylation ; Hydrogen-Ion Concentration ; Improved methods for extraction and purification of enzymes ; LEVADURA ; LEVURE ; Methods. Procedures. Technologies ; Molecular Sequence Data ; PURIFICACION ; PURIFICATION ; Saccharomycetales - enzymology ; Sequence Homology, Nucleic Acid ; Temperature ; YEASTS</subject><ispartof>Preparative biochemistry, 1989, Vol.19 (4), p.293-319</ispartof><rights>Copyright Taylor & Francis Group, LLC 1989</rights><rights>1990 INIST-CNRS</rights><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c494t-42ea13ac1a644a54bfbf10c5c67907a74c1903d84e4fa3818bf05486145718b63</citedby><cites>FETCH-LOGICAL-c494t-42ea13ac1a644a54bfbf10c5c67907a74c1903d84e4fa3818bf05486145718b63</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.tandfonline.com/doi/pdf/10.1080/10826068908544919$$EPDF$$P50$$Ginformaworld$$H</linktopdf><linktohtml>$$Uhttps://www.tandfonline.com/doi/full/10.1080/10826068908544919$$EHTML$$P50$$Ginformaworld$$H</linktohtml><link.rule.ids>314,776,780,4010,27900,27901,27902,59620,60409</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=6927456$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2622872$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Klein, R.D. (The Upjohn Company, Kalamazoo, MI)</creatorcontrib><creatorcontrib>Deibel, M.R. Jr</creatorcontrib><creatorcontrib>Sarcich, J.L</creatorcontrib><creatorcontrib>Zurcher-Neely, H.A</creatorcontrib><creatorcontrib>Reardon, I.M</creatorcontrib><creatorcontrib>Heinrikson, R.L</creatorcontrib><title>Purification and characterization of invertase from a novel industrial yeast, Schwanniomyces occidentalis</title><title>Preparative biochemistry</title><addtitle>Prep Biochem</addtitle><description>The use of yeast as an expression system for heterologous proteins offers several potential advantages with respect to industrial scale-up and genetics over other expression systems, but suffers from several drawbacks. For example, the secreted proteins of S. cerevisiae, found in the periplasm, are hyperglycosylated and the organism has a limited range of usable substrates. Other yeasts have similar disadvantages in addition to producing a variety of proteases. We have investigated the use of Schwanniomyces occidentalis as a host for developing a gene expression system in which these and several disadvantages are minimized. The present paper describes the isolation and characterization of an invertase from cell free supernatants of the yeast Schwanniomyces occidentalis grown on lactose. The enzyme is a β-D-fructofuranoside-fructohydrolyase, composed of two identical subunits of 76,000 to 78,000 da. with a native molecular mass of 125,000 +/− 25,000 da. of which approximately 17% can be attributed to N-linked carbohydrate. The enzyme has a Vmax of 0.49 +/− 0.025 units, a Km of 21 +/− 1.5 mM, and temperature and pH optima of 55°C and 3.9-4.5, respectively. The amino acid sequences of the amino terminal region and an internal tryptic peptide support an 81% identity with the invertase from Saccharomyces cerevisiae. The enzyme is induced by low glucose and is catabolite repressed.</description><subject>ACTIVIDAD ENZIMATICA</subject><subject>ACTIVITE ENZYMATIQUE</subject><subject>Amino Acid Sequence</subject><subject>Amino Acids - analysis</subject><subject>BETA-FRUCTOFURANOSIDASE</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Enzyme engineering</subject><subject>ENZYMIC ACTIVITY</subject><subject>FRUCTOFURANOSIDASA</subject><subject>FRUCTOFURANOSIDASE</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Glycoside Hydrolases - analysis</subject><subject>Glycoside Hydrolases - isolation & purification</subject><subject>Glycoside Hydrolases - metabolism</subject><subject>Glycosylation</subject><subject>Hydrogen-Ion Concentration</subject><subject>Improved methods for extraction and purification of enzymes</subject><subject>LEVADURA</subject><subject>LEVURE</subject><subject>Methods. Procedures. Technologies</subject><subject>Molecular Sequence Data</subject><subject>PURIFICACION</subject><subject>PURIFICATION</subject><subject>Saccharomycetales - enzymology</subject><subject>Sequence Homology, Nucleic Acid</subject><subject>Temperature</subject><subject>YEASTS</subject><issn>0032-7484</issn><issn>2331-0510</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1989</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkV1rFTEQhoMo9Vj9AQrCXohXruZjkmzAGyl-QaFC7fUyJ5vYyG5Sk2zL8de7hz32ptDezDAzz_smzBDyktH3jHb0wxK4oqoztJMAhplHZMOFYC2VjD4mG0oFbzV08JQ8K-X3UjIp-BE54orzTvMNCT_mHHywWEOKDcahsZeY0VaXw9-1mXwT4rXLFYtrfE5Tg01M125c2sNcag44NjuHpb5rzu3lDcYY0rSzrjTJ2jC4WHEM5Tl54nEs7sUhH5OLL59_nnxrT8--fj_5dNpaMFBb4A6ZQMtQAaCErd96Rq20ShuqUYNlhoqhAwceRce6racSOsVA6qVQ4pi8XX2vcvozu1L7KRTrxhGjS3PptQEt9LKKh0AmQSkq9o5sBW1OpWTn-6scJsy7ntF-f4f-zh0WzeuD-byd3HCrOCx-mb85zLFYHH3GaEO5xZThGuT-6Y8rFqJPecKblMehr7gbU_6vEff94tUq95h6_JUX-uLcMKqBc_EP4t-rSQ</recordid><startdate>1989</startdate><enddate>1989</enddate><creator>Klein, R.D. (The Upjohn Company, Kalamazoo, MI)</creator><creator>Deibel, M.R. Jr</creator><creator>Sarcich, J.L</creator><creator>Zurcher-Neely, H.A</creator><creator>Reardon, I.M</creator><creator>Heinrikson, R.L</creator><general>Taylor & Francis Group</general><general>Dekker</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>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>M7N</scope><scope>M81</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>1989</creationdate><title>Purification and characterization of invertase from a novel industrial yeast, Schwanniomyces occidentalis</title><author>Klein, R.D. (The Upjohn Company, Kalamazoo, MI) ; Deibel, M.R. Jr ; Sarcich, J.L ; Zurcher-Neely, H.A ; Reardon, I.M ; Heinrikson, R.L</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c494t-42ea13ac1a644a54bfbf10c5c67907a74c1903d84e4fa3818bf05486145718b63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1989</creationdate><topic>ACTIVIDAD ENZIMATICA</topic><topic>ACTIVITE ENZYMATIQUE</topic><topic>Amino Acid Sequence</topic><topic>Amino Acids - analysis</topic><topic>BETA-FRUCTOFURANOSIDASE</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Chromatography, High Pressure Liquid</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Enzyme engineering</topic><topic>ENZYMIC ACTIVITY</topic><topic>FRUCTOFURANOSIDASA</topic><topic>FRUCTOFURANOSIDASE</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Glycoside Hydrolases - analysis</topic><topic>Glycoside Hydrolases - isolation & purification</topic><topic>Glycoside Hydrolases - metabolism</topic><topic>Glycosylation</topic><topic>Hydrogen-Ion Concentration</topic><topic>Improved methods for extraction and purification of enzymes</topic><topic>LEVADURA</topic><topic>LEVURE</topic><topic>Methods. Procedures. Technologies</topic><topic>Molecular Sequence Data</topic><topic>PURIFICACION</topic><topic>PURIFICATION</topic><topic>Saccharomycetales - enzymology</topic><topic>Sequence Homology, Nucleic Acid</topic><topic>Temperature</topic><topic>YEASTS</topic><toplevel>online_resources</toplevel><creatorcontrib>Klein, R.D. (The Upjohn Company, Kalamazoo, MI)</creatorcontrib><creatorcontrib>Deibel, M.R. Jr</creatorcontrib><creatorcontrib>Sarcich, J.L</creatorcontrib><creatorcontrib>Zurcher-Neely, H.A</creatorcontrib><creatorcontrib>Reardon, I.M</creatorcontrib><creatorcontrib>Heinrikson, R.L</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>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biochemistry Abstracts 3</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Preparative biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Klein, R.D. (The Upjohn Company, Kalamazoo, MI)</au><au>Deibel, M.R. Jr</au><au>Sarcich, J.L</au><au>Zurcher-Neely, H.A</au><au>Reardon, I.M</au><au>Heinrikson, R.L</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Purification and characterization of invertase from a novel industrial yeast, Schwanniomyces occidentalis</atitle><jtitle>Preparative biochemistry</jtitle><addtitle>Prep Biochem</addtitle><date>1989</date><risdate>1989</risdate><volume>19</volume><issue>4</issue><spage>293</spage><epage>319</epage><pages>293-319</pages><issn>0032-7484</issn><eissn>2331-0510</eissn><coden>PRBCBQ</coden><abstract>The use of yeast as an expression system for heterologous proteins offers several potential advantages with respect to industrial scale-up and genetics over other expression systems, but suffers from several drawbacks. For example, the secreted proteins of S. cerevisiae, found in the periplasm, are hyperglycosylated and the organism has a limited range of usable substrates. Other yeasts have similar disadvantages in addition to producing a variety of proteases. We have investigated the use of Schwanniomyces occidentalis as a host for developing a gene expression system in which these and several disadvantages are minimized. The present paper describes the isolation and characterization of an invertase from cell free supernatants of the yeast Schwanniomyces occidentalis grown on lactose. The enzyme is a β-D-fructofuranoside-fructohydrolyase, composed of two identical subunits of 76,000 to 78,000 da. with a native molecular mass of 125,000 +/− 25,000 da. of which approximately 17% can be attributed to N-linked carbohydrate. The enzyme has a Vmax of 0.49 +/− 0.025 units, a Km of 21 +/− 1.5 mM, and temperature and pH optima of 55°C and 3.9-4.5, respectively. The amino acid sequences of the amino terminal region and an internal tryptic peptide support an 81% identity with the invertase from Saccharomyces cerevisiae. The enzyme is induced by low glucose and is catabolite repressed.</abstract><cop>New York, NY</cop><pub>Taylor & Francis Group</pub><pmid>2622872</pmid><doi>10.1080/10826068908544919</doi><tpages>27</tpages></addata></record> |
| fulltext | fulltext |
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| ispartof | Preparative biochemistry, 1989, Vol.19 (4), p.293-319 |
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| language | eng |
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| source | Taylor & Francis; MEDLINE |
| subjects | ACTIVIDAD ENZIMATICA ACTIVITE ENZYMATIQUE Amino Acid Sequence Amino Acids - analysis BETA-FRUCTOFURANOSIDASE Biological and medical sciences Biotechnology Chromatography, High Pressure Liquid Electrophoresis, Polyacrylamide Gel Enzyme engineering ENZYMIC ACTIVITY FRUCTOFURANOSIDASA FRUCTOFURANOSIDASE Fundamental and applied biological sciences. Psychology Glycoside Hydrolases - analysis Glycoside Hydrolases - isolation & purification Glycoside Hydrolases - metabolism Glycosylation Hydrogen-Ion Concentration Improved methods for extraction and purification of enzymes LEVADURA LEVURE Methods. Procedures. Technologies Molecular Sequence Data PURIFICACION PURIFICATION Saccharomycetales - enzymology Sequence Homology, Nucleic Acid Temperature YEASTS |
| title | Purification and characterization of invertase from a novel industrial yeast, Schwanniomyces occidentalis |
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