Heterologous Expression of Cyclodextrin Glycosyltransferase my20 in Escherichia coli and Its Application in 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid Production
In this study, the cgt gene my20, which encodes cyclodextrin glycosyltransferase (CGTase) and was obtained by the metagenome sequencing of marine microorganisms from the Mariana Trench, was codon optimized and connected to pET-24a for heterologous expression in Escherichia coli BL21(DE3). Through sh...
Ausführliche Beschreibung
Autor*in: |
Kai Song [verfasserIn] Jingjing Sun [verfasserIn] Wei Wang [verfasserIn] Jianhua Hao [verfasserIn] |
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Format: |
E-Artikel |
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Sprache: |
Englisch |
Erschienen: |
2021 |
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Schlagwörter: |
2-O-α-D-glucopyranosyl-L-ascorbic acid |
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Übergeordnetes Werk: |
In: Frontiers in Microbiology - Frontiers Media S.A., 2011, 12(2021) |
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Übergeordnetes Werk: |
volume:12 ; year:2021 |
Links: |
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DOI / URN: |
10.3389/fmicb.2021.664339 |
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Katalog-ID: |
DOAJ055210171 |
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10.3389/fmicb.2021.664339 doi (DE-627)DOAJ055210171 (DE-599)DOAJbde84c426ed54ecc954053143382731d DE-627 ger DE-627 rakwb eng QR1-502 Kai Song verfasserin aut Heterologous Expression of Cyclodextrin Glycosyltransferase my20 in Escherichia coli and Its Application in 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid Production 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier In this study, the cgt gene my20, which encodes cyclodextrin glycosyltransferase (CGTase) and was obtained by the metagenome sequencing of marine microorganisms from the Mariana Trench, was codon optimized and connected to pET-24a for heterologous expression in Escherichia coli BL21(DE3). Through shaking flask fermentation, the optimized condition for recombinant CGTase expression was identified as 20°C for 18 h with 0.4 mM of isopropyl β-D-L-thiogalactopyranoside. The recombinant CGTase was purified by Ni2+-NTA resin, and the optimum pH and temperature were identified as pH 7 and 80°C, respectively. Activity was stable over wide temperature and pH ranges. After purification by Ni2+-NTA resin, the specific activity of the CGTase was 63.3 U/mg after 67.3-fold purification, with a final yield of 43.7%. In addition, the enzyme was used to transform L-ascorbic acid into 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G). The maximal AA-2G production reached 28 g/L, at 40°C, pH 4, 24 h reaction time, 50 g/L donor concentration, and 50 U/g enzyme dosage. The superior properties of recombinant CGTase strongly facilitate the industrial production of AA-2G. 2-O-α-D-glucopyranosyl-L-ascorbic acid cyclodextrin glucosyltransferase E. coli heterologous expression l-ascorbic acid Microbiology Jingjing Sun verfasserin aut Jingjing Sun verfasserin aut Jingjing Sun verfasserin aut Wei Wang verfasserin aut Wei Wang verfasserin aut Wei Wang verfasserin aut Jianhua Hao verfasserin aut Jianhua Hao verfasserin aut Jianhua Hao verfasserin aut In Frontiers in Microbiology Frontiers Media S.A., 2011 12(2021) (DE-627)642889384 (DE-600)2587354-4 1664302X nnns volume:12 year:2021 https://doi.org/10.3389/fmicb.2021.664339 kostenfrei https://doaj.org/article/bde84c426ed54ecc954053143382731d kostenfrei https://www.frontiersin.org/articles/10.3389/fmicb.2021.664339/full kostenfrei https://doaj.org/toc/1664-302X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 12 2021 |
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10.3389/fmicb.2021.664339 doi (DE-627)DOAJ055210171 (DE-599)DOAJbde84c426ed54ecc954053143382731d DE-627 ger DE-627 rakwb eng QR1-502 Kai Song verfasserin aut Heterologous Expression of Cyclodextrin Glycosyltransferase my20 in Escherichia coli and Its Application in 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid Production 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier In this study, the cgt gene my20, which encodes cyclodextrin glycosyltransferase (CGTase) and was obtained by the metagenome sequencing of marine microorganisms from the Mariana Trench, was codon optimized and connected to pET-24a for heterologous expression in Escherichia coli BL21(DE3). Through shaking flask fermentation, the optimized condition for recombinant CGTase expression was identified as 20°C for 18 h with 0.4 mM of isopropyl β-D-L-thiogalactopyranoside. The recombinant CGTase was purified by Ni2+-NTA resin, and the optimum pH and temperature were identified as pH 7 and 80°C, respectively. Activity was stable over wide temperature and pH ranges. After purification by Ni2+-NTA resin, the specific activity of the CGTase was 63.3 U/mg after 67.3-fold purification, with a final yield of 43.7%. In addition, the enzyme was used to transform L-ascorbic acid into 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G). The maximal AA-2G production reached 28 g/L, at 40°C, pH 4, 24 h reaction time, 50 g/L donor concentration, and 50 U/g enzyme dosage. The superior properties of recombinant CGTase strongly facilitate the industrial production of AA-2G. 2-O-α-D-glucopyranosyl-L-ascorbic acid cyclodextrin glucosyltransferase E. coli heterologous expression l-ascorbic acid Microbiology Jingjing Sun verfasserin aut Jingjing Sun verfasserin aut Jingjing Sun verfasserin aut Wei Wang verfasserin aut Wei Wang verfasserin aut Wei Wang verfasserin aut Jianhua Hao verfasserin aut Jianhua Hao verfasserin aut Jianhua Hao verfasserin aut In Frontiers in Microbiology Frontiers Media S.A., 2011 12(2021) (DE-627)642889384 (DE-600)2587354-4 1664302X nnns volume:12 year:2021 https://doi.org/10.3389/fmicb.2021.664339 kostenfrei https://doaj.org/article/bde84c426ed54ecc954053143382731d kostenfrei https://www.frontiersin.org/articles/10.3389/fmicb.2021.664339/full kostenfrei https://doaj.org/toc/1664-302X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 12 2021 |
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10.3389/fmicb.2021.664339 doi (DE-627)DOAJ055210171 (DE-599)DOAJbde84c426ed54ecc954053143382731d DE-627 ger DE-627 rakwb eng QR1-502 Kai Song verfasserin aut Heterologous Expression of Cyclodextrin Glycosyltransferase my20 in Escherichia coli and Its Application in 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid Production 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier In this study, the cgt gene my20, which encodes cyclodextrin glycosyltransferase (CGTase) and was obtained by the metagenome sequencing of marine microorganisms from the Mariana Trench, was codon optimized and connected to pET-24a for heterologous expression in Escherichia coli BL21(DE3). Through shaking flask fermentation, the optimized condition for recombinant CGTase expression was identified as 20°C for 18 h with 0.4 mM of isopropyl β-D-L-thiogalactopyranoside. The recombinant CGTase was purified by Ni2+-NTA resin, and the optimum pH and temperature were identified as pH 7 and 80°C, respectively. Activity was stable over wide temperature and pH ranges. After purification by Ni2+-NTA resin, the specific activity of the CGTase was 63.3 U/mg after 67.3-fold purification, with a final yield of 43.7%. In addition, the enzyme was used to transform L-ascorbic acid into 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G). The maximal AA-2G production reached 28 g/L, at 40°C, pH 4, 24 h reaction time, 50 g/L donor concentration, and 50 U/g enzyme dosage. The superior properties of recombinant CGTase strongly facilitate the industrial production of AA-2G. 2-O-α-D-glucopyranosyl-L-ascorbic acid cyclodextrin glucosyltransferase E. coli heterologous expression l-ascorbic acid Microbiology Jingjing Sun verfasserin aut Jingjing Sun verfasserin aut Jingjing Sun verfasserin aut Wei Wang verfasserin aut Wei Wang verfasserin aut Wei Wang verfasserin aut Jianhua Hao verfasserin aut Jianhua Hao verfasserin aut Jianhua Hao verfasserin aut In Frontiers in Microbiology Frontiers Media S.A., 2011 12(2021) (DE-627)642889384 (DE-600)2587354-4 1664302X nnns volume:12 year:2021 https://doi.org/10.3389/fmicb.2021.664339 kostenfrei https://doaj.org/article/bde84c426ed54ecc954053143382731d kostenfrei https://www.frontiersin.org/articles/10.3389/fmicb.2021.664339/full kostenfrei https://doaj.org/toc/1664-302X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 12 2021 |
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10.3389/fmicb.2021.664339 doi (DE-627)DOAJ055210171 (DE-599)DOAJbde84c426ed54ecc954053143382731d DE-627 ger DE-627 rakwb eng QR1-502 Kai Song verfasserin aut Heterologous Expression of Cyclodextrin Glycosyltransferase my20 in Escherichia coli and Its Application in 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid Production 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier In this study, the cgt gene my20, which encodes cyclodextrin glycosyltransferase (CGTase) and was obtained by the metagenome sequencing of marine microorganisms from the Mariana Trench, was codon optimized and connected to pET-24a for heterologous expression in Escherichia coli BL21(DE3). Through shaking flask fermentation, the optimized condition for recombinant CGTase expression was identified as 20°C for 18 h with 0.4 mM of isopropyl β-D-L-thiogalactopyranoside. The recombinant CGTase was purified by Ni2+-NTA resin, and the optimum pH and temperature were identified as pH 7 and 80°C, respectively. Activity was stable over wide temperature and pH ranges. After purification by Ni2+-NTA resin, the specific activity of the CGTase was 63.3 U/mg after 67.3-fold purification, with a final yield of 43.7%. In addition, the enzyme was used to transform L-ascorbic acid into 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G). The maximal AA-2G production reached 28 g/L, at 40°C, pH 4, 24 h reaction time, 50 g/L donor concentration, and 50 U/g enzyme dosage. The superior properties of recombinant CGTase strongly facilitate the industrial production of AA-2G. 2-O-α-D-glucopyranosyl-L-ascorbic acid cyclodextrin glucosyltransferase E. coli heterologous expression l-ascorbic acid Microbiology Jingjing Sun verfasserin aut Jingjing Sun verfasserin aut Jingjing Sun verfasserin aut Wei Wang verfasserin aut Wei Wang verfasserin aut Wei Wang verfasserin aut Jianhua Hao verfasserin aut Jianhua Hao verfasserin aut Jianhua Hao verfasserin aut In Frontiers in Microbiology Frontiers Media S.A., 2011 12(2021) (DE-627)642889384 (DE-600)2587354-4 1664302X nnns volume:12 year:2021 https://doi.org/10.3389/fmicb.2021.664339 kostenfrei https://doaj.org/article/bde84c426ed54ecc954053143382731d kostenfrei https://www.frontiersin.org/articles/10.3389/fmicb.2021.664339/full kostenfrei https://doaj.org/toc/1664-302X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 12 2021 |
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QR1-502 Heterologous Expression of Cyclodextrin Glycosyltransferase my20 in Escherichia coli and Its Application in 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid Production 2-O-α-D-glucopyranosyl-L-ascorbic acid cyclodextrin glucosyltransferase E. coli heterologous expression l-ascorbic acid |
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Heterologous Expression of Cyclodextrin Glycosyltransferase my20 in Escherichia coli and Its Application in 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid Production |
abstract |
In this study, the cgt gene my20, which encodes cyclodextrin glycosyltransferase (CGTase) and was obtained by the metagenome sequencing of marine microorganisms from the Mariana Trench, was codon optimized and connected to pET-24a for heterologous expression in Escherichia coli BL21(DE3). Through shaking flask fermentation, the optimized condition for recombinant CGTase expression was identified as 20°C for 18 h with 0.4 mM of isopropyl β-D-L-thiogalactopyranoside. The recombinant CGTase was purified by Ni2+-NTA resin, and the optimum pH and temperature were identified as pH 7 and 80°C, respectively. Activity was stable over wide temperature and pH ranges. After purification by Ni2+-NTA resin, the specific activity of the CGTase was 63.3 U/mg after 67.3-fold purification, with a final yield of 43.7%. In addition, the enzyme was used to transform L-ascorbic acid into 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G). The maximal AA-2G production reached 28 g/L, at 40°C, pH 4, 24 h reaction time, 50 g/L donor concentration, and 50 U/g enzyme dosage. The superior properties of recombinant CGTase strongly facilitate the industrial production of AA-2G. |
abstractGer |
In this study, the cgt gene my20, which encodes cyclodextrin glycosyltransferase (CGTase) and was obtained by the metagenome sequencing of marine microorganisms from the Mariana Trench, was codon optimized and connected to pET-24a for heterologous expression in Escherichia coli BL21(DE3). Through shaking flask fermentation, the optimized condition for recombinant CGTase expression was identified as 20°C for 18 h with 0.4 mM of isopropyl β-D-L-thiogalactopyranoside. The recombinant CGTase was purified by Ni2+-NTA resin, and the optimum pH and temperature were identified as pH 7 and 80°C, respectively. Activity was stable over wide temperature and pH ranges. After purification by Ni2+-NTA resin, the specific activity of the CGTase was 63.3 U/mg after 67.3-fold purification, with a final yield of 43.7%. In addition, the enzyme was used to transform L-ascorbic acid into 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G). The maximal AA-2G production reached 28 g/L, at 40°C, pH 4, 24 h reaction time, 50 g/L donor concentration, and 50 U/g enzyme dosage. The superior properties of recombinant CGTase strongly facilitate the industrial production of AA-2G. |
abstract_unstemmed |
In this study, the cgt gene my20, which encodes cyclodextrin glycosyltransferase (CGTase) and was obtained by the metagenome sequencing of marine microorganisms from the Mariana Trench, was codon optimized and connected to pET-24a for heterologous expression in Escherichia coli BL21(DE3). Through shaking flask fermentation, the optimized condition for recombinant CGTase expression was identified as 20°C for 18 h with 0.4 mM of isopropyl β-D-L-thiogalactopyranoside. The recombinant CGTase was purified by Ni2+-NTA resin, and the optimum pH and temperature were identified as pH 7 and 80°C, respectively. Activity was stable over wide temperature and pH ranges. After purification by Ni2+-NTA resin, the specific activity of the CGTase was 63.3 U/mg after 67.3-fold purification, with a final yield of 43.7%. In addition, the enzyme was used to transform L-ascorbic acid into 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G). The maximal AA-2G production reached 28 g/L, at 40°C, pH 4, 24 h reaction time, 50 g/L donor concentration, and 50 U/g enzyme dosage. The superior properties of recombinant CGTase strongly facilitate the industrial production of AA-2G. |
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<?xml version="1.0" encoding="UTF-8"?><collection xmlns="http://www.loc.gov/MARC21/slim"><record><leader>01000caa a22002652 4500</leader><controlfield tag="001">DOAJ055210171</controlfield><controlfield tag="003">DE-627</controlfield><controlfield tag="005">20230308190056.0</controlfield><controlfield tag="007">cr uuu---uuuuu</controlfield><controlfield tag="008">230227s2021 xx |||||o 00| ||eng c</controlfield><datafield tag="024" ind1="7" ind2=" "><subfield code="a">10.3389/fmicb.2021.664339</subfield><subfield code="2">doi</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-627)DOAJ055210171</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-599)DOAJbde84c426ed54ecc954053143382731d</subfield></datafield><datafield tag="040" ind1=" " ind2=" "><subfield code="a">DE-627</subfield><subfield code="b">ger</subfield><subfield code="c">DE-627</subfield><subfield code="e">rakwb</subfield></datafield><datafield tag="041" ind1=" " ind2=" "><subfield code="a">eng</subfield></datafield><datafield tag="050" ind1=" " ind2="0"><subfield code="a">QR1-502</subfield></datafield><datafield tag="100" ind1="0" ind2=" "><subfield code="a">Kai Song</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">Heterologous Expression of Cyclodextrin Glycosyltransferase my20 in Escherichia coli and Its Application in 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid Production</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="c">2021</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="a">Text</subfield><subfield code="b">txt</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="a">Computermedien</subfield><subfield code="b">c</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="a">Online-Ressource</subfield><subfield code="b">cr</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">In this study, the cgt gene my20, which encodes cyclodextrin glycosyltransferase (CGTase) and was obtained by the metagenome sequencing of marine microorganisms from the Mariana Trench, was codon optimized and connected to pET-24a for heterologous expression in Escherichia coli BL21(DE3). 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