Sequence Engineering of an Aspergillus niger Tannase to Produce in Pichia pastoris a Single-Chain Enzyme with High Specific Activity
Tannin acyl hydrolases or tannases (E.C.3.1.1.20) are enzymes that hydrolyze the ester bond of tannins to produce gallic acid and glucose. We engineered the Aspergillus niger GH1 tannase sequence and Pichia pastoris strains to produce and secrete the enzyme as a single-chain protein. The recombinant...
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| Veröffentlicht in: | Molecular biotechnology 2022-04, Vol.64 (4), p.388-400 |
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| creator | Ordaz-Pérez, Daniela Fuentes-Garibay, José Antonio Guerrero-Olazarán, Martha Viader-Salvadó, José María |
| description | Tannin acyl hydrolases or tannases (E.C.3.1.1.20) are enzymes that hydrolyze the ester bond of tannins to produce gallic acid and glucose. We engineered the
Aspergillus niger
GH1 tannase sequence and
Pichia pastoris
strains to produce and secrete the enzyme as a single-chain protein. The recombinant tannase was N-glycosylated, had a molecular mass after N-deglycosylation of 65.4 kDa, and showed activity over broad pH and temperature ranges, with optimum pH and temperature of 5.0 and 20 °C. Furthermore, the single-chain tannase had an 11-fold increased specific activity in comparison to the double-chain
A. niger
GH1 tannase, which was also produced in
P. pastoris
. Structural analysis suggested that the high specific activity may be due to the presence of a flexible loop in the lid domain, which can control and drive the substrate to the active site. In contrast, the low specific activity of the double-chain tannase may be due to the presence of a disordered and flexible loop that could hinder the substrate’s access to the binding site. Based on its biochemical properties, high specific activity, and the possibility of its production in
P. pastoris
, the tannase described could be used in food and beverage processing at low and medium temperatures. |
| doi_str_mv | 10.1007/s12033-021-00416-6 |
| format | Article |
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Aspergillus niger
GH1 tannase sequence and
Pichia pastoris
strains to produce and secrete the enzyme as a single-chain protein. The recombinant tannase was N-glycosylated, had a molecular mass after N-deglycosylation of 65.4 kDa, and showed activity over broad pH and temperature ranges, with optimum pH and temperature of 5.0 and 20 °C. Furthermore, the single-chain tannase had an 11-fold increased specific activity in comparison to the double-chain
A. niger
GH1 tannase, which was also produced in
P. pastoris
. Structural analysis suggested that the high specific activity may be due to the presence of a flexible loop in the lid domain, which can control and drive the substrate to the active site. In contrast, the low specific activity of the double-chain tannase may be due to the presence of a disordered and flexible loop that could hinder the substrate’s access to the binding site. Based on its biochemical properties, high specific activity, and the possibility of its production in
P. pastoris
, the tannase described could be used in food and beverage processing at low and medium temperatures.</description><identifier>ISSN: 1073-6085</identifier><identifier>EISSN: 1559-0305</identifier><identifier>DOI: 10.1007/s12033-021-00416-6</identifier><identifier>PMID: 34655039</identifier><language>eng</language><publisher>New York: Springer US</publisher><subject>Aspergillus niger ; Binding sites ; Biochemistry ; Biological Techniques ; Biotechnology ; Cell Biology ; Chains ; Chemistry ; Chemistry and Materials Science ; Deglycosylation ; Enzymes ; Food processing ; Gallic acid ; Human Genetics ; Original Paper ; pH effects ; Pichia pastoris ; Protein Science ; Structural analysis ; Substrates ; Tannase ; Tannins</subject><ispartof>Molecular biotechnology, 2022-04, Vol.64 (4), p.388-400</ispartof><rights>The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021</rights><rights>2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.</rights><rights>The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c375t-72c1dc3d31ef43461e2353877eecc437c5ed68bf24880d63a2c0e48fd3272f363</citedby><cites>FETCH-LOGICAL-c375t-72c1dc3d31ef43461e2353877eecc437c5ed68bf24880d63a2c0e48fd3272f363</cites><orcidid>0000-0003-4801-9192</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s12033-021-00416-6$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s12033-021-00416-6$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,776,780,27901,27902,41464,42533,51294</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/34655039$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ordaz-Pérez, Daniela</creatorcontrib><creatorcontrib>Fuentes-Garibay, José Antonio</creatorcontrib><creatorcontrib>Guerrero-Olazarán, Martha</creatorcontrib><creatorcontrib>Viader-Salvadó, José María</creatorcontrib><title>Sequence Engineering of an Aspergillus niger Tannase to Produce in Pichia pastoris a Single-Chain Enzyme with High Specific Activity</title><title>Molecular biotechnology</title><addtitle>Mol Biotechnol</addtitle><addtitle>Mol Biotechnol</addtitle><description>Tannin acyl hydrolases or tannases (E.C.3.1.1.20) are enzymes that hydrolyze the ester bond of tannins to produce gallic acid and glucose. We engineered the
Aspergillus niger
GH1 tannase sequence and
Pichia pastoris
strains to produce and secrete the enzyme as a single-chain protein. The recombinant tannase was N-glycosylated, had a molecular mass after N-deglycosylation of 65.4 kDa, and showed activity over broad pH and temperature ranges, with optimum pH and temperature of 5.0 and 20 °C. Furthermore, the single-chain tannase had an 11-fold increased specific activity in comparison to the double-chain
A. niger
GH1 tannase, which was also produced in
P. pastoris
. Structural analysis suggested that the high specific activity may be due to the presence of a flexible loop in the lid domain, which can control and drive the substrate to the active site. In contrast, the low specific activity of the double-chain tannase may be due to the presence of a disordered and flexible loop that could hinder the substrate’s access to the binding site. Based on its biochemical properties, high specific activity, and the possibility of its production in
P. pastoris
, the tannase described could be used in food and beverage processing at low and medium temperatures.</description><subject>Aspergillus niger</subject><subject>Binding sites</subject><subject>Biochemistry</subject><subject>Biological Techniques</subject><subject>Biotechnology</subject><subject>Cell Biology</subject><subject>Chains</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Deglycosylation</subject><subject>Enzymes</subject><subject>Food processing</subject><subject>Gallic acid</subject><subject>Human Genetics</subject><subject>Original Paper</subject><subject>pH effects</subject><subject>Pichia pastoris</subject><subject>Protein Science</subject><subject>Structural 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Engineering of an Aspergillus niger Tannase to Produce in Pichia pastoris a Single-Chain Enzyme with High Specific Activity</title><author>Ordaz-Pérez, Daniela ; Fuentes-Garibay, José Antonio ; Guerrero-Olazarán, Martha ; Viader-Salvadó, José María</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c375t-72c1dc3d31ef43461e2353877eecc437c5ed68bf24880d63a2c0e48fd3272f363</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2022</creationdate><topic>Aspergillus niger</topic><topic>Binding sites</topic><topic>Biochemistry</topic><topic>Biological Techniques</topic><topic>Biotechnology</topic><topic>Cell Biology</topic><topic>Chains</topic><topic>Chemistry</topic><topic>Chemistry and Materials Science</topic><topic>Deglycosylation</topic><topic>Enzymes</topic><topic>Food processing</topic><topic>Gallic acid</topic><topic>Human Genetics</topic><topic>Original Paper</topic><topic>pH effects</topic><topic>Pichia 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Single-Chain Enzyme with High Specific Activity</atitle><jtitle>Molecular biotechnology</jtitle><stitle>Mol Biotechnol</stitle><addtitle>Mol Biotechnol</addtitle><date>2022-04-01</date><risdate>2022</risdate><volume>64</volume><issue>4</issue><spage>388</spage><epage>400</epage><pages>388-400</pages><issn>1073-6085</issn><eissn>1559-0305</eissn><abstract>Tannin acyl hydrolases or tannases (E.C.3.1.1.20) are enzymes that hydrolyze the ester bond of tannins to produce gallic acid and glucose. We engineered the
Aspergillus niger
GH1 tannase sequence and
Pichia pastoris
strains to produce and secrete the enzyme as a single-chain protein. The recombinant tannase was N-glycosylated, had a molecular mass after N-deglycosylation of 65.4 kDa, and showed activity over broad pH and temperature ranges, with optimum pH and temperature of 5.0 and 20 °C. Furthermore, the single-chain tannase had an 11-fold increased specific activity in comparison to the double-chain
A. niger
GH1 tannase, which was also produced in
P. pastoris
. Structural analysis suggested that the high specific activity may be due to the presence of a flexible loop in the lid domain, which can control and drive the substrate to the active site. In contrast, the low specific activity of the double-chain tannase may be due to the presence of a disordered and flexible loop that could hinder the substrate’s access to the binding site. Based on its biochemical properties, high specific activity, and the possibility of its production in
P. pastoris
, the tannase described could be used in food and beverage processing at low and medium temperatures.</abstract><cop>New York</cop><pub>Springer US</pub><pmid>34655039</pmid><doi>10.1007/s12033-021-00416-6</doi><tpages>13</tpages><orcidid>https://orcid.org/0000-0003-4801-9192</orcidid></addata></record> |
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| ispartof | Molecular biotechnology, 2022-04, Vol.64 (4), p.388-400 |
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| subjects | Aspergillus niger Binding sites Biochemistry Biological Techniques Biotechnology Cell Biology Chains Chemistry Chemistry and Materials Science Deglycosylation Enzymes Food processing Gallic acid Human Genetics Original Paper pH effects Pichia pastoris Protein Science Structural analysis Substrates Tannase Tannins |
| title | Sequence Engineering of an Aspergillus niger Tannase to Produce in Pichia pastoris a Single-Chain Enzyme with High Specific Activity |
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