Molecular cloning and production of recombinant Pcal_0672, a family GH57 glycoside hydrolase from Pyrobaculum calidifontis
An open reading frame of putative gene (designated as Pcal_0672), that belongs to family GH57 (UniProt acc # A3MTY1 and GenBank # ABO08098.1), was found in Pyrobaculum calidifontis . It consists of 485 amino acid residues and was amplified, cloned, and produced in Escherichia coli using T7 expressio...
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| Veröffentlicht in: | Biológia 2023-07, Vol.78 (7), p.1861-1874 |
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| creator | Mehboob, Sumaira Ali, Ramzan Bashir, Shahzad Ahmad, Nasir Ahmad, Tuba Butt, Hamama Islam Rashid, Naeem |
| description | An open reading frame of putative gene (designated as Pcal_0672), that belongs to family GH57 (UniProt acc # A3MTY1 and GenBank # ABO08098.1), was found in
Pyrobaculum calidifontis
. It consists of 485 amino acid residues and was amplified, cloned, and produced in
Escherichia coli
using T7 expression vector, pET-22b(+). The BLAST result revealed its low similarity index with characterized amylases of family GH57 i.e., 31–18% with non-specified amylase from thermostable bacterium of De Fuca hydrothermal Vent and maltogenic amylase from
P. furiosus
, respectively. Moreover, two putative catalytic residues, Glu (E
152
) and Asp (D
234
), were found in conserved regions of the enzyme at position 3 and 4, respectively. SDS-PAGE analysis of
E. coli
BL21-CodonPlus (DE3)-RIL cell lysates revealed production of recombinant Pcal_0672 as insoluble aggregates. Despite of its production as insoluble aggregates, the enzyme was able to hydrolyze soluble starch (0.5%), when analyzed by I
2
/KI based enzyme assay (at 80 ºC) followed by zymogram activity on gel. Zymographic activity assay analysis revealed that these insoluble aggregates were highly thermostable and retained their activity even after autoclaving in the presence of 1% Triton X-100 or 5% SDS. It depicts that putative Pcal_0672 is active on starch, highly thermostable archaeal enzyme. |
| doi_str_mv | 10.1007/s11756-023-01338-1 |
| format | Article |
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Pyrobaculum calidifontis
. It consists of 485 amino acid residues and was amplified, cloned, and produced in
Escherichia coli
using T7 expression vector, pET-22b(+). The BLAST result revealed its low similarity index with characterized amylases of family GH57 i.e., 31–18% with non-specified amylase from thermostable bacterium of De Fuca hydrothermal Vent and maltogenic amylase from
P. furiosus
, respectively. Moreover, two putative catalytic residues, Glu (E
152
) and Asp (D
234
), were found in conserved regions of the enzyme at position 3 and 4, respectively. SDS-PAGE analysis of
E. coli
BL21-CodonPlus (DE3)-RIL cell lysates revealed production of recombinant Pcal_0672 as insoluble aggregates. Despite of its production as insoluble aggregates, the enzyme was able to hydrolyze soluble starch (0.5%), when analyzed by I
2
/KI based enzyme assay (at 80 ºC) followed by zymogram activity on gel. Zymographic activity assay analysis revealed that these insoluble aggregates were highly thermostable and retained their activity even after autoclaving in the presence of 1% Triton X-100 or 5% SDS. It depicts that putative Pcal_0672 is active on starch, highly thermostable archaeal enzyme.</description><identifier>ISSN: 1336-9563</identifier><identifier>ISSN: 0006-3088</identifier><identifier>EISSN: 1336-9563</identifier><identifier>DOI: 10.1007/s11756-023-01338-1</identifier><language>eng</language><publisher>Cham: Springer International Publishing</publisher><subject>Aggregates ; Amino acids ; Amylases ; Autoclaving ; Biomedical and Life Sciences ; Cell Biology ; Cloning ; E coli ; Enzymes ; Glucan 1,4-a-maltohydrolase ; Glycosidases ; Glycoside hydrolase ; Hydrothermal vents ; Life Sciences ; Lysates ; Microbiology ; Original Article ; Plant Sciences ; Pyrobaculum calidifontis ; Residues ; Starch ; Zoology</subject><ispartof>Biológia, 2023-07, Vol.78 (7), p.1861-1874</ispartof><rights>The Author(s), under exclusive licence to Plant Science and Biodiversity Centre, Slovak Academy of Sciences (SAS), Institute of Zoology, Slovak Academy of Sciences (SAS), Institute of Molecular Biology, Slovak Academy of Sciences (SAS) 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.</rights><rights>The Author(s), under exclusive licence to Plant Science and Biodiversity Centre, Slovak Academy of Sciences (SAS), Institute of Zoology, Slovak Academy of Sciences (SAS), Institute of Molecular Biology, Slovak Academy of Sciences (SAS) 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c319t-cd73c6c09bb8f4e271a5c4fc5aa719eb8e50080a9c33315c5e48cc4d1fd734603</citedby><cites>FETCH-LOGICAL-c319t-cd73c6c09bb8f4e271a5c4fc5aa719eb8e50080a9c33315c5e48cc4d1fd734603</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/s11756-023-01338-1$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s11756-023-01338-1$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27924,27925,41488,42557,51319</link.rule.ids></links><search><creatorcontrib>Mehboob, Sumaira</creatorcontrib><creatorcontrib>Ali, Ramzan</creatorcontrib><creatorcontrib>Bashir, Shahzad</creatorcontrib><creatorcontrib>Ahmad, Nasir</creatorcontrib><creatorcontrib>Ahmad, Tuba</creatorcontrib><creatorcontrib>Butt, Hamama Islam</creatorcontrib><creatorcontrib>Rashid, Naeem</creatorcontrib><title>Molecular cloning and production of recombinant Pcal_0672, a family GH57 glycoside hydrolase from Pyrobaculum calidifontis</title><title>Biológia</title><addtitle>Biologia</addtitle><description>An open reading frame of putative gene (designated as Pcal_0672), that belongs to family GH57 (UniProt acc # A3MTY1 and GenBank # ABO08098.1), was found in
Pyrobaculum calidifontis
. It consists of 485 amino acid residues and was amplified, cloned, and produced in
Escherichia coli
using T7 expression vector, pET-22b(+). The BLAST result revealed its low similarity index with characterized amylases of family GH57 i.e., 31–18% with non-specified amylase from thermostable bacterium of De Fuca hydrothermal Vent and maltogenic amylase from
P. furiosus
, respectively. Moreover, two putative catalytic residues, Glu (E
152
) and Asp (D
234
), were found in conserved regions of the enzyme at position 3 and 4, respectively. SDS-PAGE analysis of
E. coli
BL21-CodonPlus (DE3)-RIL cell lysates revealed production of recombinant Pcal_0672 as insoluble aggregates. Despite of its production as insoluble aggregates, the enzyme was able to hydrolyze soluble starch (0.5%), when analyzed by I
2
/KI based enzyme assay (at 80 ºC) followed by zymogram activity on gel. Zymographic activity assay analysis revealed that these insoluble aggregates were highly thermostable and retained their activity even after autoclaving in the presence of 1% Triton X-100 or 5% SDS. It depicts that putative Pcal_0672 is active on starch, highly thermostable archaeal enzyme.</description><subject>Aggregates</subject><subject>Amino acids</subject><subject>Amylases</subject><subject>Autoclaving</subject><subject>Biomedical and Life Sciences</subject><subject>Cell Biology</subject><subject>Cloning</subject><subject>E coli</subject><subject>Enzymes</subject><subject>Glucan 1,4-a-maltohydrolase</subject><subject>Glycosidases</subject><subject>Glycoside hydrolase</subject><subject>Hydrothermal vents</subject><subject>Life Sciences</subject><subject>Lysates</subject><subject>Microbiology</subject><subject>Original Article</subject><subject>Plant Sciences</subject><subject>Pyrobaculum calidifontis</subject><subject>Residues</subject><subject>Starch</subject><subject>Zoology</subject><issn>1336-9563</issn><issn>0006-3088</issn><issn>1336-9563</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2023</creationdate><recordtype>article</recordtype><recordid>eNp9kDFPwzAUhC0EEqXwB5gssRKw4zhORlRBi1REB5gt58UurhK72MkQfj2GIsHE9G64707vELqk5IYSIm4jpYKXGclZRihjVUaP0CyJMqt5yY7_6FN0FuOOkEJwQmfo48l3GsZOBQydd9ZtsXIt3gffjjBY77A3OGjwfWOdcgPegOokKUV-jRU2qrfdhJcrLvC2m8BH22r8NrXBdypqbILv8WYKvlGpY-xxgm1rjXeDjefoxKgu6oufO0evD_cvi1W2fl4-Lu7WGTBaDxm0gkEJpG6ayhQ6F1RxKAxwpQStdVNpTkhFVA2MMcqB66ICKFpqEliUhM3R1SE3PfU-6jjInR-DS5Uyr_K6ojkRIrnygwuCjzFoI_fB9ipMkhL5tbE8bCzTxvJ7Y0kTxA5QTGa31eE3-h_qE9vLgCQ</recordid><startdate>20230701</startdate><enddate>20230701</enddate><creator>Mehboob, Sumaira</creator><creator>Ali, Ramzan</creator><creator>Bashir, Shahzad</creator><creator>Ahmad, Nasir</creator><creator>Ahmad, Tuba</creator><creator>Butt, Hamama Islam</creator><creator>Rashid, Naeem</creator><general>Springer International Publishing</general><general>Springer Nature B.V</general><scope>AAYXX</scope><scope>CITATION</scope><scope>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7T7</scope><scope>7TK</scope><scope>7TM</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope></search><sort><creationdate>20230701</creationdate><title>Molecular cloning and production of recombinant Pcal_0672, a family GH57 glycoside hydrolase from Pyrobaculum calidifontis</title><author>Mehboob, Sumaira ; Ali, Ramzan ; Bashir, Shahzad ; Ahmad, Nasir ; Ahmad, Tuba ; Butt, Hamama Islam ; Rashid, Naeem</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c319t-cd73c6c09bb8f4e271a5c4fc5aa719eb8e50080a9c33315c5e48cc4d1fd734603</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2023</creationdate><topic>Aggregates</topic><topic>Amino acids</topic><topic>Amylases</topic><topic>Autoclaving</topic><topic>Biomedical and Life Sciences</topic><topic>Cell Biology</topic><topic>Cloning</topic><topic>E coli</topic><topic>Enzymes</topic><topic>Glucan 1,4-a-maltohydrolase</topic><topic>Glycosidases</topic><topic>Glycoside hydrolase</topic><topic>Hydrothermal vents</topic><topic>Life Sciences</topic><topic>Lysates</topic><topic>Microbiology</topic><topic>Original Article</topic><topic>Plant Sciences</topic><topic>Pyrobaculum calidifontis</topic><topic>Residues</topic><topic>Starch</topic><topic>Zoology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Mehboob, Sumaira</creatorcontrib><creatorcontrib>Ali, Ramzan</creatorcontrib><creatorcontrib>Bashir, Shahzad</creatorcontrib><creatorcontrib>Ahmad, Nasir</creatorcontrib><creatorcontrib>Ahmad, Tuba</creatorcontrib><creatorcontrib>Butt, Hamama Islam</creatorcontrib><creatorcontrib>Rashid, Naeem</creatorcontrib><collection>CrossRef</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><jtitle>Biológia</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Mehboob, Sumaira</au><au>Ali, Ramzan</au><au>Bashir, Shahzad</au><au>Ahmad, Nasir</au><au>Ahmad, Tuba</au><au>Butt, Hamama Islam</au><au>Rashid, Naeem</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Molecular cloning and production of recombinant Pcal_0672, a family GH57 glycoside hydrolase from Pyrobaculum calidifontis</atitle><jtitle>Biológia</jtitle><stitle>Biologia</stitle><date>2023-07-01</date><risdate>2023</risdate><volume>78</volume><issue>7</issue><spage>1861</spage><epage>1874</epage><pages>1861-1874</pages><issn>1336-9563</issn><issn>0006-3088</issn><eissn>1336-9563</eissn><abstract>An open reading frame of putative gene (designated as Pcal_0672), that belongs to family GH57 (UniProt acc # A3MTY1 and GenBank # ABO08098.1), was found in
Pyrobaculum calidifontis
. It consists of 485 amino acid residues and was amplified, cloned, and produced in
Escherichia coli
using T7 expression vector, pET-22b(+). The BLAST result revealed its low similarity index with characterized amylases of family GH57 i.e., 31–18% with non-specified amylase from thermostable bacterium of De Fuca hydrothermal Vent and maltogenic amylase from
P. furiosus
, respectively. Moreover, two putative catalytic residues, Glu (E
152
) and Asp (D
234
), were found in conserved regions of the enzyme at position 3 and 4, respectively. SDS-PAGE analysis of
E. coli
BL21-CodonPlus (DE3)-RIL cell lysates revealed production of recombinant Pcal_0672 as insoluble aggregates. Despite of its production as insoluble aggregates, the enzyme was able to hydrolyze soluble starch (0.5%), when analyzed by I
2
/KI based enzyme assay (at 80 ºC) followed by zymogram activity on gel. Zymographic activity assay analysis revealed that these insoluble aggregates were highly thermostable and retained their activity even after autoclaving in the presence of 1% Triton X-100 or 5% SDS. It depicts that putative Pcal_0672 is active on starch, highly thermostable archaeal enzyme.</abstract><cop>Cham</cop><pub>Springer International Publishing</pub><doi>10.1007/s11756-023-01338-1</doi><tpages>14</tpages></addata></record> |
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| subjects | Aggregates Amino acids Amylases Autoclaving Biomedical and Life Sciences Cell Biology Cloning E coli Enzymes Glucan 1,4-a-maltohydrolase Glycosidases Glycoside hydrolase Hydrothermal vents Life Sciences Lysates Microbiology Original Article Plant Sciences Pyrobaculum calidifontis Residues Starch Zoology |
| title | Molecular cloning and production of recombinant Pcal_0672, a family GH57 glycoside hydrolase from Pyrobaculum calidifontis |
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