High-Throughput Screening Method for Directed Evolution and Characterization of Aldol Activity of D-Threonine Aldolase
A rapid and reliable method for the determination of aldol condensation activity of threonine aldolases (TAs) toward aldehydes and glycine was developed. This 2,4-dinitrophenylhydrazine (DNPH) method has high sensitivity and low background disturbance and can be spectrophotometrically measured for h...
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| Veröffentlicht in: | Applied biochemistry and biotechnology 2021-02, Vol.193 (2), p.417-429 |
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| creator | Gong, Lei Xu, Guochao Cao, Xudong Han, Ruizhi Dong, Jinjun Ni, Ye |
| description | A rapid and reliable method for the determination of aldol condensation activity of threonine aldolases (TAs) toward aldehydes and glycine was developed. This 2,4-dinitrophenylhydrazine (DNPH) method has high sensitivity and low background disturbance and can be spectrophotometrically measured for high-throughput screening and characterization of TAs. For 4-methylsulfonyl benzaldehyde (MSB), the maximum absorbance peak was observed at around 485 nm. Site-directed saturation mutagenesis libraries of
d
-threonine aldolase from
Alcaligenes xylosoxidans
CGMCC 1.4257 (
Ax
DTA) was constructed and screened with this DNPH method for increased aldol activity toward MSB. Two beneficial variants
Ax
DTA
D321C
and
Ax
DTA
N101G
were identified. Substrate specificity of
Ax
DTA and variants toward nineteen aldehydes with different substituents was facilely characterized employing this DNPH method. Furthermore,
Ax
DTA variants displayed enhanced catalytic performance and selectivity in aldol reaction. Consequently, our study provides a rapid screening and characterization method for TAs with potential applications in preparation of chiral β-hydroxy-α-amino acids. |
| doi_str_mv | 10.1007/s12010-020-03447-y |
| format | Article |
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d
-threonine aldolase from
Alcaligenes xylosoxidans
CGMCC 1.4257 (
Ax
DTA) was constructed and screened with this DNPH method for increased aldol activity toward MSB. Two beneficial variants
Ax
DTA
D321C
and
Ax
DTA
N101G
were identified. Substrate specificity of
Ax
DTA and variants toward nineteen aldehydes with different substituents was facilely characterized employing this DNPH method. Furthermore,
Ax
DTA variants displayed enhanced catalytic performance and selectivity in aldol reaction. Consequently, our study provides a rapid screening and characterization method for TAs with potential applications in preparation of chiral β-hydroxy-α-amino acids.</description><identifier>ISSN: 0273-2289</identifier><identifier>EISSN: 1559-0291</identifier><identifier>DOI: 10.1007/s12010-020-03447-y</identifier><identifier>PMID: 33015743</identifier><language>eng</language><publisher>New York: Springer US</publisher><subject>Alcaligenes ; Aldehydes ; Aldolase ; Amino acids ; Benzaldehyde ; Biochemistry ; Biotechnology ; Carbon ; Chemistry ; Chemistry and Materials Science ; Cloning ; Dehydrogenases ; Directed evolution ; Evolution ; Glycine ; High-throughput screening ; Mutagenesis ; Saturation mutagenesis ; Screening ; Selectivity ; Spectrophotometry ; Substrate specificity ; Substrates ; Threonine aldolase</subject><ispartof>Applied biochemistry and biotechnology, 2021-02, Vol.193 (2), p.417-429</ispartof><rights>Springer Science+Business Media, LLC, part of Springer Nature 2020</rights><rights>Springer Science+Business Media, LLC, part of Springer Nature 2020.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c478t-9859ffcb52ae49939762da8e57d0f132449f119f5b42d0e4c71096cdbf539cd43</citedby><cites>FETCH-LOGICAL-c478t-9859ffcb52ae49939762da8e57d0f132449f119f5b42d0e4c71096cdbf539cd43</cites><orcidid>0000-0003-4887-7517</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/s12010-020-03447-y$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s12010-020-03447-y$$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/33015743$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Gong, Lei</creatorcontrib><creatorcontrib>Xu, Guochao</creatorcontrib><creatorcontrib>Cao, Xudong</creatorcontrib><creatorcontrib>Han, Ruizhi</creatorcontrib><creatorcontrib>Dong, Jinjun</creatorcontrib><creatorcontrib>Ni, Ye</creatorcontrib><title>High-Throughput Screening Method for Directed Evolution and Characterization of Aldol Activity of D-Threonine Aldolase</title><title>Applied biochemistry and biotechnology</title><addtitle>Appl Biochem Biotechnol</addtitle><addtitle>Appl Biochem Biotechnol</addtitle><description>A rapid and reliable method for the determination of aldol condensation activity of threonine aldolases (TAs) toward aldehydes and glycine was developed. This 2,4-dinitrophenylhydrazine (DNPH) method has high sensitivity and low background disturbance and can be spectrophotometrically measured for high-throughput screening and characterization of TAs. For 4-methylsulfonyl benzaldehyde (MSB), the maximum absorbance peak was observed at around 485 nm. Site-directed saturation mutagenesis libraries of
d
-threonine aldolase from
Alcaligenes xylosoxidans
CGMCC 1.4257 (
Ax
DTA) was constructed and screened with this DNPH method for increased aldol activity toward MSB. Two beneficial variants
Ax
DTA
D321C
and
Ax
DTA
N101G
were identified. Substrate specificity of
Ax
DTA and variants toward nineteen aldehydes with different substituents was facilely characterized employing this DNPH method. Furthermore,
Ax
DTA variants displayed enhanced catalytic performance and selectivity in aldol reaction. Consequently, our study provides a rapid screening and characterization method for TAs with potential applications in preparation of chiral β-hydroxy-α-amino acids.</description><subject>Alcaligenes</subject><subject>Aldehydes</subject><subject>Aldolase</subject><subject>Amino acids</subject><subject>Benzaldehyde</subject><subject>Biochemistry</subject><subject>Biotechnology</subject><subject>Carbon</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Cloning</subject><subject>Dehydrogenases</subject><subject>Directed evolution</subject><subject>Evolution</subject><subject>Glycine</subject><subject>High-throughput screening</subject><subject>Mutagenesis</subject><subject>Saturation mutagenesis</subject><subject>Screening</subject><subject>Selectivity</subject><subject>Spectrophotometry</subject><subject>Substrate specificity</subject><subject>Substrates</subject><subject>Threonine aldolase</subject><issn>0273-2289</issn><issn>1559-0291</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>BENPR</sourceid><recordid>eNp9kMtKAzEUhoMotl5ewIUEXI_m2kyWpV6h4sK6DmkunZE6qclMoT69aafqzkUI_Oc_34EPgAuMrjFC4iZhgjAqEMmPMiaKzQEYYs5ljiQ-BENEBC0IKeUAnKT0jhAmJRfHYEApwlwwOgTrx3pRFbMqhm5RrboWvproXFM3C_js2ipY6EOEt3V0pnUW3q3Dsmvr0EDdWDipdNQ5j_WX3oXBw_HShiUcm7Ze1-1mm9xu8S5kpuunOrkzcOT1Mrnz_X8K3u7vZpPHYvry8DQZTwvDRNkWsuTSezPnRDsmJZViRKwuHRcWeUwJY9JjLD2fM2KRY0ZgJEfGzj2n0lhGT8FVz13F8Nm51Kr30MUmn1SElaiUTEicW6RvmRhSis6rVaw_dNwojNRWtepVq6xa7VSrTV663KO7-Yezvys_bnOB9oWUR83Cxb_b_2C_AVoyisM</recordid><startdate>20210201</startdate><enddate>20210201</enddate><creator>Gong, Lei</creator><creator>Xu, Guochao</creator><creator>Cao, Xudong</creator><creator>Han, Ruizhi</creator><creator>Dong, Jinjun</creator><creator>Ni, Ye</creator><general>Springer US</general><general>Springer Nature B.V</general><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7ST</scope><scope>7T7</scope><scope>7TM</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>M7P</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>RC3</scope><scope>SOI</scope><orcidid>https://orcid.org/0000-0003-4887-7517</orcidid></search><sort><creationdate>20210201</creationdate><title>High-Throughput Screening Method for Directed Evolution and Characterization of Aldol Activity of D-Threonine Aldolase</title><author>Gong, Lei ; Xu, Guochao ; Cao, Xudong ; Han, Ruizhi ; Dong, Jinjun ; Ni, Ye</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c478t-9859ffcb52ae49939762da8e57d0f132449f119f5b42d0e4c71096cdbf539cd43</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Alcaligenes</topic><topic>Aldehydes</topic><topic>Aldolase</topic><topic>Amino acids</topic><topic>Benzaldehyde</topic><topic>Biochemistry</topic><topic>Biotechnology</topic><topic>Carbon</topic><topic>Chemistry</topic><topic>Chemistry and Materials Science</topic><topic>Cloning</topic><topic>Dehydrogenases</topic><topic>Directed evolution</topic><topic>Evolution</topic><topic>Glycine</topic><topic>High-throughput screening</topic><topic>Mutagenesis</topic><topic>Saturation mutagenesis</topic><topic>Screening</topic><topic>Selectivity</topic><topic>Spectrophotometry</topic><topic>Substrate specificity</topic><topic>Substrates</topic><topic>Threonine aldolase</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Gong, Lei</creatorcontrib><creatorcontrib>Xu, Guochao</creatorcontrib><creatorcontrib>Cao, Xudong</creatorcontrib><creatorcontrib>Han, Ruizhi</creatorcontrib><creatorcontrib>Dong, Jinjun</creatorcontrib><creatorcontrib>Ni, Ye</creatorcontrib><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Environment Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Health & Medical Collection</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 Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</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</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Biological Science Database</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>Genetics Abstracts</collection><collection>Environment Abstracts</collection><jtitle>Applied biochemistry and biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Gong, Lei</au><au>Xu, Guochao</au><au>Cao, Xudong</au><au>Han, Ruizhi</au><au>Dong, Jinjun</au><au>Ni, Ye</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>High-Throughput Screening Method for Directed Evolution and Characterization of Aldol Activity of D-Threonine Aldolase</atitle><jtitle>Applied biochemistry and biotechnology</jtitle><stitle>Appl Biochem Biotechnol</stitle><addtitle>Appl Biochem Biotechnol</addtitle><date>2021-02-01</date><risdate>2021</risdate><volume>193</volume><issue>2</issue><spage>417</spage><epage>429</epage><pages>417-429</pages><issn>0273-2289</issn><eissn>1559-0291</eissn><abstract>A rapid and reliable method for the determination of aldol condensation activity of threonine aldolases (TAs) toward aldehydes and glycine was developed. This 2,4-dinitrophenylhydrazine (DNPH) method has high sensitivity and low background disturbance and can be spectrophotometrically measured for high-throughput screening and characterization of TAs. For 4-methylsulfonyl benzaldehyde (MSB), the maximum absorbance peak was observed at around 485 nm. Site-directed saturation mutagenesis libraries of
d
-threonine aldolase from
Alcaligenes xylosoxidans
CGMCC 1.4257 (
Ax
DTA) was constructed and screened with this DNPH method for increased aldol activity toward MSB. Two beneficial variants
Ax
DTA
D321C
and
Ax
DTA
N101G
were identified. Substrate specificity of
Ax
DTA and variants toward nineteen aldehydes with different substituents was facilely characterized employing this DNPH method. Furthermore,
Ax
DTA variants displayed enhanced catalytic performance and selectivity in aldol reaction. Consequently, our study provides a rapid screening and characterization method for TAs with potential applications in preparation of chiral β-hydroxy-α-amino acids.</abstract><cop>New York</cop><pub>Springer US</pub><pmid>33015743</pmid><doi>10.1007/s12010-020-03447-y</doi><tpages>13</tpages><orcidid>https://orcid.org/0000-0003-4887-7517</orcidid></addata></record> |
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| ispartof | Applied biochemistry and biotechnology, 2021-02, Vol.193 (2), p.417-429 |
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| source | Springer Nature - Complete Springer Journals |
| subjects | Alcaligenes Aldehydes Aldolase Amino acids Benzaldehyde Biochemistry Biotechnology Carbon Chemistry Chemistry and Materials Science Cloning Dehydrogenases Directed evolution Evolution Glycine High-throughput screening Mutagenesis Saturation mutagenesis Screening Selectivity Spectrophotometry Substrate specificity Substrates Threonine aldolase |
| title | High-Throughput Screening Method for Directed Evolution and Characterization of Aldol Activity of D-Threonine Aldolase |
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