Crystal structure and genetic modifications of FI-CMCase from Aspergillus aculeatus F-50
Cellulose is the major component of the plant cell wall and the most abundant renewable biomass on earth, and its decomposition has proven to be very useful in many commercial applications. Endo-1,4-β-d-glucanase (EC 3.2.1.4; endoglucanase), which catalyzes the random hydrolysis of 1,4-β-glycosidic...
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| Veröffentlicht in: | Biochemical and biophysical research communications 2016-09, Vol.478 (2), p.565-572 |
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| creator | Huang, Jian-Wen Liu, Weidong Lai, Hui-Lin Cheng, Ya-Shan Zheng, Yingying Li, Qian Sun, Hong Kuo, Chih-Jung Guo, Rey-Ting Chen, Chun-Chi |
| description | Cellulose is the major component of the plant cell wall and the most abundant renewable biomass on earth, and its decomposition has proven to be very useful in many commercial applications. Endo-1,4-β-d-glucanase (EC 3.2.1.4; endoglucanase), which catalyzes the random hydrolysis of 1,4-β-glycosidic bonds of the cellulose main chain to cleave cellulose into smaller fragments, is the key cellulolytic enzyme. An endoglucanase isolated from Aspergillus aculeatus F-50 (FI-CMCase), which is classified into the glycoside hydrolase (GH) family 12, was demonstrated to be effectively expressed in the industrial strain Pichia pastoris. Here, the crystal structure and complex structures of P. pastoris-expressed FI-CMCase were solved to high resolution. The overall structure is analyzed and compared to other GH12 members. In addition, the substrate-surrounding residues were engineered to search for variants with improved enzymatic activity. Among 14 mutants constructed, one with two-fold increase in protein expression was identified, which possesses a potential to be further developed as a commercial enzyme product.
•An GH12 endoglucanase FI-CMCase from Aspergillus aculeatus was expressed in Pichia.•Crystal structures of FI-CMCase in apo and substrate-bound form were solved.•Substrate-surrounding residues were engineered to enhance enzyme performance. |
| doi_str_mv | 10.1016/j.bbrc.2016.07.101 |
| format | Article |
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•An GH12 endoglucanase FI-CMCase from Aspergillus aculeatus was expressed in Pichia.•Crystal structures of FI-CMCase in apo and substrate-bound form were solved.•Substrate-surrounding residues were engineered to enhance enzyme performance.</description><identifier>ISSN: 0006-291X</identifier><identifier>EISSN: 1090-2104</identifier><identifier>DOI: 10.1016/j.bbrc.2016.07.101</identifier><identifier>PMID: 27470581</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Amino Acid Sequence ; Aspergillosis - microbiology ; Aspergillus - chemistry ; Aspergillus - enzymology ; Aspergillus - genetics ; Aspergillus - metabolism ; Aspergillus aculeatus ; Cellulase - chemistry ; Cellulase - genetics ; Cellulase - metabolism ; Crystallography, X-Ray ; Endoglucanase ; Gene Expression ; Glycoside Hydrolases - chemistry ; Glycoside Hydrolases - genetics ; Glycoside Hydrolases - metabolism ; Humans ; Models, Molecular ; Pichia - genetics ; Pichia pastoris ; Protein Conformation ; Protein structure ; Sequence Alignment ; Substrate Specificity ; X-ray crystallography</subject><ispartof>Biochemical and biophysical research communications, 2016-09, Vol.478 (2), p.565-572</ispartof><rights>2016 Elsevier Inc.</rights><rights>Copyright © 2016 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c389t-e8e4489f07c500651766bcf31f0a263d1d11496f91694b73b40e51950f24fa243</citedby><cites>FETCH-LOGICAL-c389t-e8e4489f07c500651766bcf31f0a263d1d11496f91694b73b40e51950f24fa243</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.bbrc.2016.07.101$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>315,781,785,3551,27929,27930,46000</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27470581$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Huang, Jian-Wen</creatorcontrib><creatorcontrib>Liu, Weidong</creatorcontrib><creatorcontrib>Lai, Hui-Lin</creatorcontrib><creatorcontrib>Cheng, Ya-Shan</creatorcontrib><creatorcontrib>Zheng, Yingying</creatorcontrib><creatorcontrib>Li, Qian</creatorcontrib><creatorcontrib>Sun, Hong</creatorcontrib><creatorcontrib>Kuo, Chih-Jung</creatorcontrib><creatorcontrib>Guo, Rey-Ting</creatorcontrib><creatorcontrib>Chen, Chun-Chi</creatorcontrib><title>Crystal structure and genetic modifications of FI-CMCase from Aspergillus aculeatus F-50</title><title>Biochemical and biophysical research communications</title><addtitle>Biochem Biophys Res Commun</addtitle><description>Cellulose is the major component of the plant cell wall and the most abundant renewable biomass on earth, and its decomposition has proven to be very useful in many commercial applications. Endo-1,4-β-d-glucanase (EC 3.2.1.4; endoglucanase), which catalyzes the random hydrolysis of 1,4-β-glycosidic bonds of the cellulose main chain to cleave cellulose into smaller fragments, is the key cellulolytic enzyme. An endoglucanase isolated from Aspergillus aculeatus F-50 (FI-CMCase), which is classified into the glycoside hydrolase (GH) family 12, was demonstrated to be effectively expressed in the industrial strain Pichia pastoris. Here, the crystal structure and complex structures of P. pastoris-expressed FI-CMCase were solved to high resolution. The overall structure is analyzed and compared to other GH12 members. In addition, the substrate-surrounding residues were engineered to search for variants with improved enzymatic activity. Among 14 mutants constructed, one with two-fold increase in protein expression was identified, which possesses a potential to be further developed as a commercial enzyme product.
•An GH12 endoglucanase FI-CMCase from Aspergillus aculeatus was expressed in Pichia.•Crystal structures of FI-CMCase in apo and substrate-bound form were solved.•Substrate-surrounding residues were engineered to enhance enzyme performance.</description><subject>Amino Acid Sequence</subject><subject>Aspergillosis - microbiology</subject><subject>Aspergillus - chemistry</subject><subject>Aspergillus - enzymology</subject><subject>Aspergillus - genetics</subject><subject>Aspergillus - metabolism</subject><subject>Aspergillus aculeatus</subject><subject>Cellulase - chemistry</subject><subject>Cellulase - genetics</subject><subject>Cellulase - metabolism</subject><subject>Crystallography, X-Ray</subject><subject>Endoglucanase</subject><subject>Gene Expression</subject><subject>Glycoside Hydrolases - chemistry</subject><subject>Glycoside Hydrolases - genetics</subject><subject>Glycoside Hydrolases - metabolism</subject><subject>Humans</subject><subject>Models, Molecular</subject><subject>Pichia - genetics</subject><subject>Pichia pastoris</subject><subject>Protein Conformation</subject><subject>Protein structure</subject><subject>Sequence Alignment</subject><subject>Substrate Specificity</subject><subject>X-ray crystallography</subject><issn>0006-291X</issn><issn>1090-2104</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkE1r3DAQhkVpabZJ_0APRcdevJ2RZdmCXoLppoGUXhrITcjyKGjxx1ayC_n3ldk0x9LTDMPzvgwPYx8Q9gioPh_3XRfdXuR9D_V2e8V2CBoKgSBfsx0AqEJofLhg71I6AiBKpd-yC1HLGqoGd-yhjU9psQNPS1zdskbidur5I020BMfHuQ8-OLuEeUp89vxwW7TfW5uI-ziP_DqdKD6GYVgTt24dyC55OxQVXLE33g6J3j_PS3Z_-Pqz_Vbc_bi5ba_vClc2eimoISkb7aF2Vf62wlqpzvkSPVihyh77_LNWXqPSsqvLTgJVqCvwQnorZHnJPp17T3H-tVJazBiSo2GwE81rMtiIWkOFCv8DzXqqRuomo-KMujinFMmbUwyjjU8GwWzyzdFs8s0m30C93XLo43P_2o3Uv0T-2s7AlzNAWcjvQNEkF2hy1IdIbjH9HP7V_wc4kZNJ</recordid><startdate>20160916</startdate><enddate>20160916</enddate><creator>Huang, Jian-Wen</creator><creator>Liu, Weidong</creator><creator>Lai, Hui-Lin</creator><creator>Cheng, Ya-Shan</creator><creator>Zheng, Yingying</creator><creator>Li, Qian</creator><creator>Sun, Hong</creator><creator>Kuo, Chih-Jung</creator><creator>Guo, Rey-Ting</creator><creator>Chen, Chun-Chi</creator><general>Elsevier Inc</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><scope>8FD</scope><scope>FR3</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope></search><sort><creationdate>20160916</creationdate><title>Crystal structure and genetic modifications of FI-CMCase from Aspergillus aculeatus F-50</title><author>Huang, Jian-Wen ; Liu, Weidong ; Lai, Hui-Lin ; Cheng, Ya-Shan ; Zheng, Yingying ; Li, Qian ; Sun, Hong ; Kuo, Chih-Jung ; Guo, Rey-Ting ; Chen, Chun-Chi</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c389t-e8e4489f07c500651766bcf31f0a263d1d11496f91694b73b40e51950f24fa243</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Amino Acid Sequence</topic><topic>Aspergillosis - microbiology</topic><topic>Aspergillus - chemistry</topic><topic>Aspergillus - enzymology</topic><topic>Aspergillus - genetics</topic><topic>Aspergillus - metabolism</topic><topic>Aspergillus aculeatus</topic><topic>Cellulase - chemistry</topic><topic>Cellulase - genetics</topic><topic>Cellulase - metabolism</topic><topic>Crystallography, X-Ray</topic><topic>Endoglucanase</topic><topic>Gene Expression</topic><topic>Glycoside Hydrolases - chemistry</topic><topic>Glycoside Hydrolases - genetics</topic><topic>Glycoside Hydrolases - metabolism</topic><topic>Humans</topic><topic>Models, Molecular</topic><topic>Pichia - genetics</topic><topic>Pichia pastoris</topic><topic>Protein Conformation</topic><topic>Protein structure</topic><topic>Sequence Alignment</topic><topic>Substrate Specificity</topic><topic>X-ray crystallography</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Huang, Jian-Wen</creatorcontrib><creatorcontrib>Liu, Weidong</creatorcontrib><creatorcontrib>Lai, Hui-Lin</creatorcontrib><creatorcontrib>Cheng, Ya-Shan</creatorcontrib><creatorcontrib>Zheng, Yingying</creatorcontrib><creatorcontrib>Li, Qian</creatorcontrib><creatorcontrib>Sun, Hong</creatorcontrib><creatorcontrib>Kuo, Chih-Jung</creatorcontrib><creatorcontrib>Guo, Rey-Ting</creatorcontrib><creatorcontrib>Chen, Chun-Chi</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><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><jtitle>Biochemical and biophysical research communications</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Huang, Jian-Wen</au><au>Liu, Weidong</au><au>Lai, Hui-Lin</au><au>Cheng, Ya-Shan</au><au>Zheng, Yingying</au><au>Li, Qian</au><au>Sun, Hong</au><au>Kuo, Chih-Jung</au><au>Guo, Rey-Ting</au><au>Chen, Chun-Chi</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Crystal structure and genetic modifications of FI-CMCase from Aspergillus aculeatus F-50</atitle><jtitle>Biochemical and biophysical research communications</jtitle><addtitle>Biochem Biophys Res Commun</addtitle><date>2016-09-16</date><risdate>2016</risdate><volume>478</volume><issue>2</issue><spage>565</spage><epage>572</epage><pages>565-572</pages><issn>0006-291X</issn><eissn>1090-2104</eissn><abstract>Cellulose is the major component of the plant cell wall and the most abundant renewable biomass on earth, and its decomposition has proven to be very useful in many commercial applications. Endo-1,4-β-d-glucanase (EC 3.2.1.4; endoglucanase), which catalyzes the random hydrolysis of 1,4-β-glycosidic bonds of the cellulose main chain to cleave cellulose into smaller fragments, is the key cellulolytic enzyme. An endoglucanase isolated from Aspergillus aculeatus F-50 (FI-CMCase), which is classified into the glycoside hydrolase (GH) family 12, was demonstrated to be effectively expressed in the industrial strain Pichia pastoris. Here, the crystal structure and complex structures of P. pastoris-expressed FI-CMCase were solved to high resolution. The overall structure is analyzed and compared to other GH12 members. In addition, the substrate-surrounding residues were engineered to search for variants with improved enzymatic activity. Among 14 mutants constructed, one with two-fold increase in protein expression was identified, which possesses a potential to be further developed as a commercial enzyme product.
•An GH12 endoglucanase FI-CMCase from Aspergillus aculeatus was expressed in Pichia.•Crystal structures of FI-CMCase in apo and substrate-bound form were solved.•Substrate-surrounding residues were engineered to enhance enzyme performance.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>27470581</pmid><doi>10.1016/j.bbrc.2016.07.101</doi><tpages>8</tpages></addata></record> |
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| ispartof | Biochemical and biophysical research communications, 2016-09, Vol.478 (2), p.565-572 |
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| subjects | Amino Acid Sequence Aspergillosis - microbiology Aspergillus - chemistry Aspergillus - enzymology Aspergillus - genetics Aspergillus - metabolism Aspergillus aculeatus Cellulase - chemistry Cellulase - genetics Cellulase - metabolism Crystallography, X-Ray Endoglucanase Gene Expression Glycoside Hydrolases - chemistry Glycoside Hydrolases - genetics Glycoside Hydrolases - metabolism Humans Models, Molecular Pichia - genetics Pichia pastoris Protein Conformation Protein structure Sequence Alignment Substrate Specificity X-ray crystallography |
| title | Crystal structure and genetic modifications of FI-CMCase from Aspergillus aculeatus F-50 |
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