High yield expression of novel glutaminase free l-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Bacillus subtilis WB800N
Gene encoding glutaminase-free l -asparaginase II ( ans B2 ) from Pectobacterium carotovorum MTCC 1428 was cloned into pHT43, transformed in Bacillus subtilis WB800N and optimised the expression levels of recombinant enzyme. A three-fold higher enzyme production was observed with an efficient transf...
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| Veröffentlicht in: | Bioprocess and biosystems engineering 2015-11, Vol.38 (11), p.2271-2284 |
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| creator | Chityala, Sushma Venkata Dasu, Veeranki Ahmad, Jamal Prakasham, Reddy Shetty |
| description | Gene encoding glutaminase-free
l
-asparaginase II (
ans B2
) from
Pectobacterium carotovorum
MTCC 1428 was cloned into pHT43, transformed in
Bacillus subtilis
WB800N and optimised the expression levels of recombinant enzyme. A three-fold higher enzyme production was observed with an efficient transformant as compared to native strain. Enzyme localization studies revealed that >90 % of recombinant enzyme is secreted extracellularly, a little fraction is attached to the membrane (>6 %) and localised intracellularly (3 %). The expression of recombinant
l
-asparaginase II was confirmed by SDS-PAGE, IMAC (Immobilised metal ion affinity chromatography) purification followed by Western blotting. Process parameter optimization with OFAT (one factor at a time) revealed that rpm (120), temperature (37 °C), Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration (1 mM) and time of induction (0.8 OD
600nm
) plays a vital role where a maximum of 55 IU/ml was achieved. Further, consecutive induction by IPTG improved the enzyme production up to 105 IU/ml with a specific activity of 101 IU/mg of protein. Molecular modelling analysis depicted that amino acids, GLY60, GLY119 and ALA252 in the active site are responsible for the glutaminase free
l
-asparaginase II activity. This is the first report on enhanced expression of recombinant glutaminase-free
l
-asparaginase II by intermediate addition of IPTG. |
| doi_str_mv | 10.1007/s00449-015-1464-x |
| format | Article |
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l
-asparaginase II (
ans B2
) from
Pectobacterium carotovorum
MTCC 1428 was cloned into pHT43, transformed in
Bacillus subtilis
WB800N and optimised the expression levels of recombinant enzyme. A three-fold higher enzyme production was observed with an efficient transformant as compared to native strain. Enzyme localization studies revealed that >90 % of recombinant enzyme is secreted extracellularly, a little fraction is attached to the membrane (>6 %) and localised intracellularly (3 %). The expression of recombinant
l
-asparaginase II was confirmed by SDS-PAGE, IMAC (Immobilised metal ion affinity chromatography) purification followed by Western blotting. Process parameter optimization with OFAT (one factor at a time) revealed that rpm (120), temperature (37 °C), Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration (1 mM) and time of induction (0.8 OD
600nm
) plays a vital role where a maximum of 55 IU/ml was achieved. Further, consecutive induction by IPTG improved the enzyme production up to 105 IU/ml with a specific activity of 101 IU/mg of protein. Molecular modelling analysis depicted that amino acids, GLY60, GLY119 and ALA252 in the active site are responsible for the glutaminase free
l
-asparaginase II activity. This is the first report on enhanced expression of recombinant glutaminase-free
l
-asparaginase II by intermediate addition of IPTG.</description><identifier>ISSN: 1615-7591</identifier><identifier>EISSN: 1615-7605</identifier><identifier>DOI: 10.1007/s00449-015-1464-x</identifier><identifier>PMID: 26440965</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer Berlin Heidelberg</publisher><subject>Amino acids ; Asparaginase - biosynthesis ; Asparaginase - genetics ; Bacillus subtilis ; Bacillus subtilis - genetics ; Bacillus subtilis - metabolism ; Bacteria ; Bacterial Proteins - biosynthesis ; Bacterial Proteins - genetics ; Biotechnology ; Chemistry ; Chemistry and Materials Science ; Environmental Engineering/Biotechnology ; Food Science ; Gene Expression ; Genes ; Industrial and Production Engineering ; Industrial Chemistry/Chemical Engineering ; Membranes ; Original Paper ; Pectobacterium ; Pectobacterium carotovorum - enzymology ; Pectobacterium carotovorum - genetics ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - genetics</subject><ispartof>Bioprocess and biosystems engineering, 2015-11, Vol.38 (11), p.2271-2284</ispartof><rights>Springer-Verlag Berlin Heidelberg 2015</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c508t-765b3992c2c1dfa7903bbe46eee5a459d49f78628694d9abb903b6d780e3bced3</citedby><cites>FETCH-LOGICAL-c508t-765b3992c2c1dfa7903bbe46eee5a459d49f78628694d9abb903b6d780e3bced3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00449-015-1464-x$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00449-015-1464-x$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,777,781,27905,27906,41469,42538,51300</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26440965$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Chityala, Sushma</creatorcontrib><creatorcontrib>Venkata Dasu, Veeranki</creatorcontrib><creatorcontrib>Ahmad, Jamal</creatorcontrib><creatorcontrib>Prakasham, Reddy Shetty</creatorcontrib><title>High yield expression of novel glutaminase free l-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Bacillus subtilis WB800N</title><title>Bioprocess and biosystems engineering</title><addtitle>Bioprocess Biosyst Eng</addtitle><addtitle>Bioprocess Biosyst Eng</addtitle><description>Gene encoding glutaminase-free
l
-asparaginase II (
ans B2
) from
Pectobacterium carotovorum
MTCC 1428 was cloned into pHT43, transformed in
Bacillus subtilis
WB800N and optimised the expression levels of recombinant enzyme. A three-fold higher enzyme production was observed with an efficient transformant as compared to native strain. Enzyme localization studies revealed that >90 % of recombinant enzyme is secreted extracellularly, a little fraction is attached to the membrane (>6 %) and localised intracellularly (3 %). The expression of recombinant
l
-asparaginase II was confirmed by SDS-PAGE, IMAC (Immobilised metal ion affinity chromatography) purification followed by Western blotting. Process parameter optimization with OFAT (one factor at a time) revealed that rpm (120), temperature (37 °C), Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration (1 mM) and time of induction (0.8 OD
600nm
) plays a vital role where a maximum of 55 IU/ml was achieved. Further, consecutive induction by IPTG improved the enzyme production up to 105 IU/ml with a specific activity of 101 IU/mg of protein. Molecular modelling analysis depicted that amino acids, GLY60, GLY119 and ALA252 in the active site are responsible for the glutaminase free
l
-asparaginase II activity. This is the first report on enhanced expression of recombinant glutaminase-free
l
-asparaginase II by intermediate addition of IPTG.</description><subject>Amino acids</subject><subject>Asparaginase - biosynthesis</subject><subject>Asparaginase - genetics</subject><subject>Bacillus subtilis</subject><subject>Bacillus subtilis - genetics</subject><subject>Bacillus subtilis - metabolism</subject><subject>Bacteria</subject><subject>Bacterial Proteins - biosynthesis</subject><subject>Bacterial Proteins - genetics</subject><subject>Biotechnology</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Environmental Engineering/Biotechnology</subject><subject>Food Science</subject><subject>Gene Expression</subject><subject>Genes</subject><subject>Industrial and Production Engineering</subject><subject>Industrial Chemistry/Chemical Engineering</subject><subject>Membranes</subject><subject>Original Paper</subject><subject>Pectobacterium</subject><subject>Pectobacterium carotovorum - enzymology</subject><subject>Pectobacterium carotovorum - genetics</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - genetics</subject><issn>1615-7591</issn><issn>1615-7605</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNqNkctu1TAQhi0Eohd4ADbIEhs2AdtxfFnSo9IeqVwWRSwj25kcXDnxwU6q0wfgvXFIixASEiuP7W_-0ehD6AUlbygh8m0mhHNdEdpUlAteHR6hYyrKTQrSPH6oG02P0EnON6SAipGn6IgJzokWzTH6cel33_Cdh9BhOOwT5OzjiGOPx3gLAe_CPJnBjyYD7hMADpXJe5PMbn3bbhf2M7gpWuMmSH4esDMpTvE2plJ_uN5sMOVMYT_iM-N8CHPGebaTDz7jr2eKkI_P0JPehAzP789T9OX9-fXmsrr6dLHdvLuqXEPUVNZqbK01c8zRrjdSk9pa4AIAGsMb3XHdSyWYEpp32li7AKKTikBtHXT1KXq95u5T_D5DntrBZwchmBHinFsqmRSS14z_D8o4Y42iBX31F3oT5zSWRRaKKqnUr0C6Ui7FnBP07T75waS7lpJ20dmuOttiqV10tofS8_I-ebYDdL87HvwVgK1ALl_jDtIfo_-Z-hMtIqpg</recordid><startdate>20151101</startdate><enddate>20151101</enddate><creator>Chityala, Sushma</creator><creator>Venkata Dasu, Veeranki</creator><creator>Ahmad, Jamal</creator><creator>Prakasham, Reddy Shetty</creator><general>Springer Berlin Heidelberg</general><general>Springer Nature B.V</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>3V.</scope><scope>7QL</scope><scope>7T7</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>7X8</scope><scope>7QO</scope></search><sort><creationdate>20151101</creationdate><title>High yield expression of novel glutaminase free l-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Bacillus subtilis WB800N</title><author>Chityala, Sushma ; Venkata Dasu, Veeranki ; Ahmad, Jamal ; Prakasham, Reddy Shetty</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c508t-765b3992c2c1dfa7903bbe46eee5a459d49f78628694d9abb903b6d780e3bced3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Amino acids</topic><topic>Asparaginase - biosynthesis</topic><topic>Asparaginase - genetics</topic><topic>Bacillus subtilis</topic><topic>Bacillus subtilis - genetics</topic><topic>Bacillus subtilis - metabolism</topic><topic>Bacteria</topic><topic>Bacterial Proteins - biosynthesis</topic><topic>Bacterial Proteins - genetics</topic><topic>Biotechnology</topic><topic>Chemistry</topic><topic>Chemistry and Materials Science</topic><topic>Environmental Engineering/Biotechnology</topic><topic>Food Science</topic><topic>Gene Expression</topic><topic>Genes</topic><topic>Industrial and Production Engineering</topic><topic>Industrial Chemistry/Chemical Engineering</topic><topic>Membranes</topic><topic>Original Paper</topic><topic>Pectobacterium</topic><topic>Pectobacterium carotovorum - enzymology</topic><topic>Pectobacterium carotovorum - genetics</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Chityala, Sushma</creatorcontrib><creatorcontrib>Venkata Dasu, Veeranki</creatorcontrib><creatorcontrib>Ahmad, Jamal</creatorcontrib><creatorcontrib>Prakasham, Reddy Shetty</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>ProQuest Health and Medical</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>AUTh Library subscriptions: ProQuest Central</collection><collection>ProQuest Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection (Proquest) (PQ_SDU_P3)</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>PML(ProQuest Medical Library)</collection><collection>ProQuest Science Journals</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>ProQuest Biological Science Journals</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>MEDLINE - Academic</collection><collection>Biotechnology Research Abstracts</collection><jtitle>Bioprocess and biosystems engineering</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Chityala, Sushma</au><au>Venkata Dasu, Veeranki</au><au>Ahmad, Jamal</au><au>Prakasham, Reddy Shetty</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>High yield expression of novel glutaminase free l-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Bacillus subtilis WB800N</atitle><jtitle>Bioprocess and biosystems engineering</jtitle><stitle>Bioprocess Biosyst Eng</stitle><addtitle>Bioprocess Biosyst Eng</addtitle><date>2015-11-01</date><risdate>2015</risdate><volume>38</volume><issue>11</issue><spage>2271</spage><epage>2284</epage><pages>2271-2284</pages><issn>1615-7591</issn><eissn>1615-7605</eissn><abstract>Gene encoding glutaminase-free
l
-asparaginase II (
ans B2
) from
Pectobacterium carotovorum
MTCC 1428 was cloned into pHT43, transformed in
Bacillus subtilis
WB800N and optimised the expression levels of recombinant enzyme. A three-fold higher enzyme production was observed with an efficient transformant as compared to native strain. Enzyme localization studies revealed that >90 % of recombinant enzyme is secreted extracellularly, a little fraction is attached to the membrane (>6 %) and localised intracellularly (3 %). The expression of recombinant
l
-asparaginase II was confirmed by SDS-PAGE, IMAC (Immobilised metal ion affinity chromatography) purification followed by Western blotting. Process parameter optimization with OFAT (one factor at a time) revealed that rpm (120), temperature (37 °C), Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration (1 mM) and time of induction (0.8 OD
600nm
) plays a vital role where a maximum of 55 IU/ml was achieved. Further, consecutive induction by IPTG improved the enzyme production up to 105 IU/ml with a specific activity of 101 IU/mg of protein. Molecular modelling analysis depicted that amino acids, GLY60, GLY119 and ALA252 in the active site are responsible for the glutaminase free
l
-asparaginase II activity. This is the first report on enhanced expression of recombinant glutaminase-free
l
-asparaginase II by intermediate addition of IPTG.</abstract><cop>Berlin/Heidelberg</cop><pub>Springer Berlin Heidelberg</pub><pmid>26440965</pmid><doi>10.1007/s00449-015-1464-x</doi><tpages>14</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1615-7591 |
| ispartof | Bioprocess and biosystems engineering, 2015-11, Vol.38 (11), p.2271-2284 |
| issn | 1615-7591 1615-7605 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_1727674324 |
| source | MEDLINE; SpringerLink |
| subjects | Amino acids Asparaginase - biosynthesis Asparaginase - genetics Bacillus subtilis Bacillus subtilis - genetics Bacillus subtilis - metabolism Bacteria Bacterial Proteins - biosynthesis Bacterial Proteins - genetics Biotechnology Chemistry Chemistry and Materials Science Environmental Engineering/Biotechnology Food Science Gene Expression Genes Industrial and Production Engineering Industrial Chemistry/Chemical Engineering Membranes Original Paper Pectobacterium Pectobacterium carotovorum - enzymology Pectobacterium carotovorum - genetics Recombinant Proteins - biosynthesis Recombinant Proteins - genetics |
| title | High yield expression of novel glutaminase free l-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Bacillus subtilis WB800N |
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