Novel Detection Method for Evaluating the Activity of an Alkaline Serine Protease from Bacillus clausii
Until now, the detection methods for serine proteases have been quite time-consuming or cannot indicate the “real” protease activity. Here, a rapid and simple method for determining the “real” activity of serine proteases toward AAPX (a kind of mixed polypeptide substrates, with X representing 20 st...
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| Veröffentlicht in: | Journal of agricultural and food chemistry 2022-03, Vol.70 (12), p.3765-3774 |
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| container_issue | 12 |
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| container_title | Journal of agricultural and food chemistry |
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| creator | Li, Xinyue Ren, Shaodong Song, Guangchao Liu, Yihan Li, Yu Lu, Fuping |
| description | Until now, the detection methods for serine proteases have been quite time-consuming or cannot indicate the “real” protease activity. Here, a rapid and simple method for determining the “real” activity of serine proteases toward AAPX (a kind of mixed polypeptide substrates, with X representing 20 standard amino acids) was developed. This AAPX method has high reliability, sensitivity, and repeatability and can be used for detecting the serine protease activity spectrophotometrically. Additionally, the site-directed saturation mutagenesis library of alkaline serine protease PRO (BcPRO) from Bacillus clausii was screened with this AAPX method. Three beneficial mutants S99R, S99H, and S99W were identified, and S99W displayed the highest activity. In comparison to wild-type BcPRO, S99W exhibited enhanced catalytic performance toward eight AAPX monomers, and the molecular dynamics simulation revealed the mechanism responsible for its improved activity toward AAPM. Consequently, this work provides an efficient method for detecting, characterizing, mining, and high-throughput screening of serine proteases. |
| doi_str_mv | 10.1021/acs.jafc.2c00358 |
| format | Article |
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Here, a rapid and simple method for determining the “real” activity of serine proteases toward AAPX (a kind of mixed polypeptide substrates, with X representing 20 standard amino acids) was developed. This AAPX method has high reliability, sensitivity, and repeatability and can be used for detecting the serine protease activity spectrophotometrically. Additionally, the site-directed saturation mutagenesis library of alkaline serine protease PRO (BcPRO) from Bacillus clausii was screened with this AAPX method. Three beneficial mutants S99R, S99H, and S99W were identified, and S99W displayed the highest activity. In comparison to wild-type BcPRO, S99W exhibited enhanced catalytic performance toward eight AAPX monomers, and the molecular dynamics simulation revealed the mechanism responsible for its improved activity toward AAPM. Consequently, this work provides an efficient method for detecting, characterizing, mining, and high-throughput screening of serine proteases.</description><identifier>ISSN: 0021-8561</identifier><identifier>EISSN: 1520-5118</identifier><identifier>DOI: 10.1021/acs.jafc.2c00358</identifier><identifier>PMID: 35311282</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Bacillus - genetics ; Bacillus - metabolism ; Bacillus clausii - metabolism ; Biotechnology and Biological Transformations ; Reproducibility of Results ; Serine - metabolism ; Serine Endopeptidases - genetics ; Serine Endopeptidases - metabolism ; Serine Proteases - genetics ; Serine Proteases - metabolism</subject><ispartof>Journal of agricultural and food chemistry, 2022-03, Vol.70 (12), p.3765-3774</ispartof><rights>2022 American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a336t-b9eebf5fa37dccf3380411b691296b05829959fd2f4316c366ffe3ffc84e21753</citedby><cites>FETCH-LOGICAL-a336t-b9eebf5fa37dccf3380411b691296b05829959fd2f4316c366ffe3ffc84e21753</cites><orcidid>0000-0002-0610-8827 ; 0000-0002-6413-0889</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/acs.jafc.2c00358$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/acs.jafc.2c00358$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>315,781,785,2766,27080,27928,27929,56742,56792</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/35311282$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Li, Xinyue</creatorcontrib><creatorcontrib>Ren, Shaodong</creatorcontrib><creatorcontrib>Song, Guangchao</creatorcontrib><creatorcontrib>Liu, Yihan</creatorcontrib><creatorcontrib>Li, Yu</creatorcontrib><creatorcontrib>Lu, Fuping</creatorcontrib><title>Novel Detection Method for Evaluating the Activity of an Alkaline Serine Protease from Bacillus clausii</title><title>Journal of agricultural and food chemistry</title><addtitle>J. Agric. Food Chem</addtitle><description>Until now, the detection methods for serine proteases have been quite time-consuming or cannot indicate the “real” protease activity. Here, a rapid and simple method for determining the “real” activity of serine proteases toward AAPX (a kind of mixed polypeptide substrates, with X representing 20 standard amino acids) was developed. This AAPX method has high reliability, sensitivity, and repeatability and can be used for detecting the serine protease activity spectrophotometrically. Additionally, the site-directed saturation mutagenesis library of alkaline serine protease PRO (BcPRO) from Bacillus clausii was screened with this AAPX method. Three beneficial mutants S99R, S99H, and S99W were identified, and S99W displayed the highest activity. In comparison to wild-type BcPRO, S99W exhibited enhanced catalytic performance toward eight AAPX monomers, and the molecular dynamics simulation revealed the mechanism responsible for its improved activity toward AAPM. Consequently, this work provides an efficient method for detecting, characterizing, mining, and high-throughput screening of serine proteases.</description><subject>Bacillus - genetics</subject><subject>Bacillus - metabolism</subject><subject>Bacillus clausii - metabolism</subject><subject>Biotechnology and Biological Transformations</subject><subject>Reproducibility of Results</subject><subject>Serine - metabolism</subject><subject>Serine Endopeptidases - genetics</subject><subject>Serine Endopeptidases - metabolism</subject><subject>Serine Proteases - genetics</subject><subject>Serine Proteases - metabolism</subject><issn>0021-8561</issn><issn>1520-5118</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kDtPwzAUhS0EgvLYmZBHBlJ87Tp1xvJG4iUBc-S412BwY7CdSvx7UlrYmM7ynSOdj5B9YENgHI61ScM3bc2QG8aEVGtkAJKzQgKodTJgPVMoWcIW2U7pjTGm5Jhtki0hBQBXfEBe7sIcPT3DjCa70NJbzK9hSm2I9Hyufaeza19ofkU66YG5y180WKpbOvHv2rsW6SPGRTzEkFEnpDaGGT3RxnnfJWq87pJzu2TDap9wb5U75Pni_On0qri5v7w-ndwUWogyF02F2FhptRhPjbFCKDYCaMoKeFU2TCpeVbKyU25HAkojytJaFNYaNUIOYyl2yOFy9yOGzw5TrmcuGfRetxi6VPNyBBKkVAuULVETQ0oRbf0R3UzHrxpYvdBb93rrhd56pbevHKzWu2aG07_Cr88eOFoCP9XQxbY_-__eN4YWhp0</recordid><startdate>20220330</startdate><enddate>20220330</enddate><creator>Li, Xinyue</creator><creator>Ren, Shaodong</creator><creator>Song, Guangchao</creator><creator>Liu, Yihan</creator><creator>Li, Yu</creator><creator>Lu, Fuping</creator><general>American Chemical Society</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>7X8</scope><orcidid>https://orcid.org/0000-0002-0610-8827</orcidid><orcidid>https://orcid.org/0000-0002-6413-0889</orcidid></search><sort><creationdate>20220330</creationdate><title>Novel Detection Method for Evaluating the Activity of an Alkaline Serine Protease from Bacillus clausii</title><author>Li, Xinyue ; Ren, Shaodong ; Song, Guangchao ; Liu, Yihan ; Li, Yu ; Lu, Fuping</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a336t-b9eebf5fa37dccf3380411b691296b05829959fd2f4316c366ffe3ffc84e21753</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2022</creationdate><topic>Bacillus - genetics</topic><topic>Bacillus - metabolism</topic><topic>Bacillus clausii - metabolism</topic><topic>Biotechnology and Biological Transformations</topic><topic>Reproducibility of Results</topic><topic>Serine - metabolism</topic><topic>Serine Endopeptidases - genetics</topic><topic>Serine Endopeptidases - metabolism</topic><topic>Serine Proteases - genetics</topic><topic>Serine Proteases - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Li, Xinyue</creatorcontrib><creatorcontrib>Ren, Shaodong</creatorcontrib><creatorcontrib>Song, Guangchao</creatorcontrib><creatorcontrib>Liu, Yihan</creatorcontrib><creatorcontrib>Li, Yu</creatorcontrib><creatorcontrib>Lu, Fuping</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of agricultural and food chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Li, Xinyue</au><au>Ren, Shaodong</au><au>Song, Guangchao</au><au>Liu, Yihan</au><au>Li, Yu</au><au>Lu, Fuping</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Novel Detection Method for Evaluating the Activity of an Alkaline Serine Protease from Bacillus clausii</atitle><jtitle>Journal of agricultural and food chemistry</jtitle><addtitle>J. Agric. Food Chem</addtitle><date>2022-03-30</date><risdate>2022</risdate><volume>70</volume><issue>12</issue><spage>3765</spage><epage>3774</epage><pages>3765-3774</pages><issn>0021-8561</issn><eissn>1520-5118</eissn><abstract>Until now, the detection methods for serine proteases have been quite time-consuming or cannot indicate the “real” protease activity. Here, a rapid and simple method for determining the “real” activity of serine proteases toward AAPX (a kind of mixed polypeptide substrates, with X representing 20 standard amino acids) was developed. This AAPX method has high reliability, sensitivity, and repeatability and can be used for detecting the serine protease activity spectrophotometrically. Additionally, the site-directed saturation mutagenesis library of alkaline serine protease PRO (BcPRO) from Bacillus clausii was screened with this AAPX method. Three beneficial mutants S99R, S99H, and S99W were identified, and S99W displayed the highest activity. 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| ispartof | Journal of agricultural and food chemistry, 2022-03, Vol.70 (12), p.3765-3774 |
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| source | MEDLINE; American Chemical Society Publications |
| subjects | Bacillus - genetics Bacillus - metabolism Bacillus clausii - metabolism Biotechnology and Biological Transformations Reproducibility of Results Serine - metabolism Serine Endopeptidases - genetics Serine Endopeptidases - metabolism Serine Proteases - genetics Serine Proteases - metabolism |
| title | Novel Detection Method for Evaluating the Activity of an Alkaline Serine Protease from Bacillus clausii |
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