Computation-aided engineering of starch-debranching pullulanase from Bacillus thermoleovorans for enhanced thermostability
Pullulanases are widely used in food, medicine, and other industries because they specifically hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides. In addition, high-temperature thermostable pullulanase has multiple advantages, including decreasing saccharification solution viscosity...
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| Veröffentlicht in: | Applied microbiology and biotechnology 2020-09, Vol.104 (17), p.7551-7562 |
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| creator | Bi, Jiahua Chen, Shuhui Zhao, Xianghan Nie, Yao Xu, Yan |
| description | Pullulanases are widely used in food, medicine, and other industries because they specifically hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides. In addition, high-temperature thermostable pullulanase has multiple advantages, including decreasing saccharification solution viscosity accompanied with enhanced mass transfer and reducing microbial contamination in starch hydrolysis. However, thermophilic pullulanase availability remains limited. Additionally, most do not meet starch-manufacturing requirements due to weak thermostability. Here, we developed a computation-aided strategy to engineer the thermophilic pullulanase from
Bacillus thermoleovorans
. First, three computational design predictors (FoldX, I-Mutant 3.0, and dDFIRE) were combined to predict stability changes introduced by mutations. After excluding conserved and catalytic sites, 17 mutants were identified. After further experimental verification, we confirmed six positive mutants. Among them, the G692M mutant had the highest thermostability improvement, with 3.8 °C increased
T
m
and 2.1-fold longer half-life than the wild type at 70 °C. We then characterized the mechanism underlying increased thermostability, such as rigidity enhancement, closer conformation, and strengthened motion correlation using root mean square fluctuation (RMSF), principal component analysis (PCA), dynamic cross-correlation map (DCCM), and free energy landscape (FEL) analysis.
Key points
•
A computation-aided strategy was developed to engineer pullulanase thermostability.
•
Seventeen mutants were identified by combining three computational design predictors.
•
The G692M mutant was obtained with increased T
m
and half-life at 70 °C. |
| doi_str_mv | 10.1007/s00253-020-10764-z |
| format | Article |
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Bacillus thermoleovorans
. First, three computational design predictors (FoldX, I-Mutant 3.0, and dDFIRE) were combined to predict stability changes introduced by mutations. After excluding conserved and catalytic sites, 17 mutants were identified. After further experimental verification, we confirmed six positive mutants. Among them, the G692M mutant had the highest thermostability improvement, with 3.8 °C increased
T
m
and 2.1-fold longer half-life than the wild type at 70 °C. We then characterized the mechanism underlying increased thermostability, such as rigidity enhancement, closer conformation, and strengthened motion correlation using root mean square fluctuation (RMSF), principal component analysis (PCA), dynamic cross-correlation map (DCCM), and free energy landscape (FEL) analysis.
Key points
•
A computation-aided strategy was developed to engineer pullulanase thermostability.
•
Seventeen mutants were identified by combining three computational design predictors.
•
The G692M mutant was obtained with increased T
m
and half-life at 70 °C.</description><identifier>ISSN: 0175-7598</identifier><identifier>EISSN: 1432-0614</identifier><identifier>DOI: 10.1007/s00253-020-10764-z</identifier><identifier>PMID: 32632476</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer Berlin Heidelberg</publisher><subject>Active sites ; Bacillus thermoleovorans ; Biomedical and Life Sciences ; Biotechnologically Relevant Enzymes and Proteins ; Biotechnology ; Computation ; Computer applications ; Conformation ; Correlation analysis ; Engineers ; Enzyme Stability ; Food contamination ; Food industry ; Free energy ; Geobacillus ; Glycoside Hydrolases - genetics ; Glycoside Hydrolases - metabolism ; Half-life ; High temperature ; Hydrolysis ; Life Sciences ; Mass transfer ; Microbial contamination ; Microbial Genetics and Genomics ; Microbiology ; Microorganisms ; Mutants ; Mutation ; Oligosaccharides ; Principal components analysis ; Pullulanase ; Rigidity ; Saccharification ; Starch ; Temperature ; Thermal stability</subject><ispartof>Applied microbiology and biotechnology, 2020-09, Vol.104 (17), p.7551-7562</ispartof><rights>Springer-Verlag GmbH Germany, part of Springer Nature 2020</rights><rights>COPYRIGHT 2020 Springer</rights><rights>Springer-Verlag GmbH Germany, part of Springer Nature 2020.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c428z-320829b455b5037ad6ba991191411c347f8addcb4f6cc0e23372d5c3da4728e53</citedby><cites>FETCH-LOGICAL-c428z-320829b455b5037ad6ba991191411c347f8addcb4f6cc0e23372d5c3da4728e53</cites><orcidid>0000-0001-8065-7640</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/s00253-020-10764-z$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00253-020-10764-z$$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/32632476$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Bi, Jiahua</creatorcontrib><creatorcontrib>Chen, Shuhui</creatorcontrib><creatorcontrib>Zhao, Xianghan</creatorcontrib><creatorcontrib>Nie, Yao</creatorcontrib><creatorcontrib>Xu, Yan</creatorcontrib><title>Computation-aided engineering of starch-debranching pullulanase from Bacillus thermoleovorans for enhanced thermostability</title><title>Applied microbiology and biotechnology</title><addtitle>Appl Microbiol Biotechnol</addtitle><addtitle>Appl Microbiol Biotechnol</addtitle><description>Pullulanases are widely used in food, medicine, and other industries because they specifically hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides. In addition, high-temperature thermostable pullulanase has multiple advantages, including decreasing saccharification solution viscosity accompanied with enhanced mass transfer and reducing microbial contamination in starch hydrolysis. However, thermophilic pullulanase availability remains limited. Additionally, most do not meet starch-manufacturing requirements due to weak thermostability. Here, we developed a computation-aided strategy to engineer the thermophilic pullulanase from
Bacillus thermoleovorans
. First, three computational design predictors (FoldX, I-Mutant 3.0, and dDFIRE) were combined to predict stability changes introduced by mutations. After excluding conserved and catalytic sites, 17 mutants were identified. After further experimental verification, we confirmed six positive mutants. Among them, the G692M mutant had the highest thermostability improvement, with 3.8 °C increased
T
m
and 2.1-fold longer half-life than the wild type at 70 °C. We then characterized the mechanism underlying increased thermostability, such as rigidity enhancement, closer conformation, and strengthened motion correlation using root mean square fluctuation (RMSF), principal component analysis (PCA), dynamic cross-correlation map (DCCM), and free energy landscape (FEL) analysis.
Key points
•
A computation-aided strategy was developed to engineer pullulanase thermostability.
•
Seventeen mutants were identified by combining three computational design predictors.
•
The G692M mutant was obtained with increased T
m
and half-life at 70 °C.</description><subject>Active sites</subject><subject>Bacillus thermoleovorans</subject><subject>Biomedical and Life Sciences</subject><subject>Biotechnologically Relevant Enzymes and Proteins</subject><subject>Biotechnology</subject><subject>Computation</subject><subject>Computer applications</subject><subject>Conformation</subject><subject>Correlation analysis</subject><subject>Engineers</subject><subject>Enzyme Stability</subject><subject>Food contamination</subject><subject>Food industry</subject><subject>Free energy</subject><subject>Geobacillus</subject><subject>Glycoside Hydrolases - genetics</subject><subject>Glycoside Hydrolases - metabolism</subject><subject>Half-life</subject><subject>High temperature</subject><subject>Hydrolysis</subject><subject>Life Sciences</subject><subject>Mass transfer</subject><subject>Microbial contamination</subject><subject>Microbial Genetics and Genomics</subject><subject>Microbiology</subject><subject>Microorganisms</subject><subject>Mutants</subject><subject>Mutation</subject><subject>Oligosaccharides</subject><subject>Principal components analysis</subject><subject>Pullulanase</subject><subject>Rigidity</subject><subject>Saccharification</subject><subject>Starch</subject><subject>Temperature</subject><subject>Thermal stability</subject><issn>0175-7598</issn><issn>1432-0614</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNp9kk1v1DAQhi0EokvhD3BAkbjAwcVfcZJju6JQqRISH2fLsSe7rpJ4sRNE99d3lhSqRQj5YGn8vK9mPC8hLzk744xV7zJjopSUCUY5q7Si-0dkxZUUlGmuHpMV41VJq7KpT8iznG8Y46LW-ik5kUJLoSq9Ivt1HHbzZKcQR2qDB1_AuAkjQArjpohdkSeb3JZ6aJMd3fZQ3c19P_d2tBmKLsWhuLAuYCkX0xbSEHuIPyLSuehiQr8tCtF4eUS_NvRhun1OnnS2z_Di_j4l3y7ff11_pNefPlytz6-pU6LeUylYLZpWlWVbMllZr1vbNJw3XHHupKq62nrvWtVp5xgIKSvhSye9VZWooZSn5M3iu0vx-wx5MkPIDnocAOKcjVCC4_81kiH6-i_0Js5pxO6QkpxjK6V6oDa2BxPGLk7JuoOpOdeSNxJ3oJE6-weFx8MQXByhC1g_Erw9EiAzwc9pY-eczdWXz8esWFiXYs4JOrNLYbDp1nBmDuEwSzgMhsP8CofZo-jV_XRzO4D_I_mdBgTkAuTdYfuQHsb_j-0dfmDExw</recordid><startdate>20200901</startdate><enddate>20200901</enddate><creator>Bi, Jiahua</creator><creator>Chen, Shuhui</creator><creator>Zhao, Xianghan</creator><creator>Nie, Yao</creator><creator>Xu, Yan</creator><general>Springer Berlin Heidelberg</general><general>Springer</general><general>Springer Nature B.V</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>ISR</scope><scope>3V.</scope><scope>7QL</scope><scope>7T7</scope><scope>7WY</scope><scope>7WZ</scope><scope>7X7</scope><scope>7XB</scope><scope>87Z</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>8FL</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BEZIV</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FRNLG</scope><scope>FYUFA</scope><scope>F~G</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K60</scope><scope>K6~</scope><scope>K9.</scope><scope>L.-</scope><scope>LK8</scope><scope>M0C</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQBIZ</scope><scope>PQBZA</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0001-8065-7640</orcidid></search><sort><creationdate>20200901</creationdate><title>Computation-aided engineering of starch-debranching pullulanase from Bacillus thermoleovorans for enhanced thermostability</title><author>Bi, Jiahua ; Chen, Shuhui ; Zhao, Xianghan ; Nie, Yao ; Xu, Yan</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c428z-320829b455b5037ad6ba991191411c347f8addcb4f6cc0e23372d5c3da4728e53</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Active sites</topic><topic>Bacillus thermoleovorans</topic><topic>Biomedical and Life Sciences</topic><topic>Biotechnologically Relevant Enzymes and Proteins</topic><topic>Biotechnology</topic><topic>Computation</topic><topic>Computer applications</topic><topic>Conformation</topic><topic>Correlation analysis</topic><topic>Engineers</topic><topic>Enzyme Stability</topic><topic>Food contamination</topic><topic>Food industry</topic><topic>Free energy</topic><topic>Geobacillus</topic><topic>Glycoside Hydrolases - 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Academic</collection><jtitle>Applied microbiology and biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bi, Jiahua</au><au>Chen, Shuhui</au><au>Zhao, Xianghan</au><au>Nie, Yao</au><au>Xu, Yan</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Computation-aided engineering of starch-debranching pullulanase from Bacillus thermoleovorans for enhanced thermostability</atitle><jtitle>Applied microbiology and biotechnology</jtitle><stitle>Appl Microbiol Biotechnol</stitle><addtitle>Appl Microbiol Biotechnol</addtitle><date>2020-09-01</date><risdate>2020</risdate><volume>104</volume><issue>17</issue><spage>7551</spage><epage>7562</epage><pages>7551-7562</pages><issn>0175-7598</issn><eissn>1432-0614</eissn><abstract>Pullulanases are widely used in food, medicine, and other industries because they specifically hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides. In addition, high-temperature thermostable pullulanase has multiple advantages, including decreasing saccharification solution viscosity accompanied with enhanced mass transfer and reducing microbial contamination in starch hydrolysis. However, thermophilic pullulanase availability remains limited. Additionally, most do not meet starch-manufacturing requirements due to weak thermostability. Here, we developed a computation-aided strategy to engineer the thermophilic pullulanase from
Bacillus thermoleovorans
. First, three computational design predictors (FoldX, I-Mutant 3.0, and dDFIRE) were combined to predict stability changes introduced by mutations. After excluding conserved and catalytic sites, 17 mutants were identified. After further experimental verification, we confirmed six positive mutants. Among them, the G692M mutant had the highest thermostability improvement, with 3.8 °C increased
T
m
and 2.1-fold longer half-life than the wild type at 70 °C. We then characterized the mechanism underlying increased thermostability, such as rigidity enhancement, closer conformation, and strengthened motion correlation using root mean square fluctuation (RMSF), principal component analysis (PCA), dynamic cross-correlation map (DCCM), and free energy landscape (FEL) analysis.
Key points
•
A computation-aided strategy was developed to engineer pullulanase thermostability.
•
Seventeen mutants were identified by combining three computational design predictors.
•
The G692M mutant was obtained with increased T
m
and half-life at 70 °C.</abstract><cop>Berlin/Heidelberg</cop><pub>Springer Berlin Heidelberg</pub><pmid>32632476</pmid><doi>10.1007/s00253-020-10764-z</doi><tpages>12</tpages><orcidid>https://orcid.org/0000-0001-8065-7640</orcidid></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0175-7598 |
| ispartof | Applied microbiology and biotechnology, 2020-09, Vol.104 (17), p.7551-7562 |
| issn | 0175-7598 1432-0614 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_2421107930 |
| source | MEDLINE; Springer Nature - Complete Springer Journals |
| subjects | Active sites Bacillus thermoleovorans Biomedical and Life Sciences Biotechnologically Relevant Enzymes and Proteins Biotechnology Computation Computer applications Conformation Correlation analysis Engineers Enzyme Stability Food contamination Food industry Free energy Geobacillus Glycoside Hydrolases - genetics Glycoside Hydrolases - metabolism Half-life High temperature Hydrolysis Life Sciences Mass transfer Microbial contamination Microbial Genetics and Genomics Microbiology Microorganisms Mutants Mutation Oligosaccharides Principal components analysis Pullulanase Rigidity Saccharification Starch Temperature Thermal stability |
| title | Computation-aided engineering of starch-debranching pullulanase from Bacillus thermoleovorans for enhanced thermostability |
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