{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,26]],"date-time":"2025-12-26T22:31:41Z","timestamp":1766788301114,"version":"build-2065373602"},"reference-count":34,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2015,3,31]],"date-time":"2015-03-31T00:00:00Z","timestamp":1427760000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Thermostability and substrate specificity are important characteristics of enzymes for industrial application, which can be improved by protein engineering. SMG1 lipase from Malassezia globosa is a mono- and diacylglycerol lipase (MDL) that shows activity toward mono- and diacylglycerols, but no activity toward triacylglycerols. SMG1 lipase is considered a potential biocatalyst applied in oil\/fat modification and its crystal structure revealed that an interesting residue-Asn277 may contribute to stabilize loop 273\u2013278 and the 3104 helix which are important to enzyme characterization. In this study, to explore  its role in affecting the stability and catalytic activity, mutagenesis of N277 with Asp (D),  Val (V), Leu (L) and Phe (F) was conducted. Circular dichroism (CD) spectral analysis  and half-life measurement showed that the N277D mutant has better thermostability.  The melting temperature and half-life of the N277D mutant were 56.6 \u00b0C and 187 min, respectively, while that was 54.6 \u00b0C and 121 min for SMG1 wild type (WT). Biochemical characterization of SMG1 mutants were carried out to test whether catalytic properties were affected by mutagenesis. N277D had similar enzymatic properties as SMG1 WT, but N277F showed a different substrate selectivity profile as compared to other SMG1 mutants. Analysis of the SMG1 3D model suggested that N277D formed a salt bridge via its negative charged carboxyl group with a positively charged guanidino group of R227, which might contribute to confer N277D higher temperature stability. These findings not only provide some clues to understand the molecular basis of the lipase structure\/function relationship but also lay the framework for engineering suitable MDL lipases for industrial applications.<\/jats:p>","DOI":"10.3390\/ijms16047273","type":"journal-article","created":{"date-parts":[[2015,4,7]],"date-time":"2015-04-07T03:47:46Z","timestamp":1428378466000},"page":"7273-7288","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["Residue Asn277 Affects the Stability and Substrate Specificity of the SMG1 Lipase from Malassezia globosa"],"prefix":"10.3390","volume":"16","author":[{"given":"Dongming","family":"Lan","sequence":"first","affiliation":[{"name":"School of Bioscience and Bioengineering, South China University of Technology,  Guangzhou 510006, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qian","family":"Wang","sequence":"additional","affiliation":[{"name":"School of Bioscience and Bioengineering, South China University of Technology,  Guangzhou 510006, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jinxin","family":"Xu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pengfei","family":"Zhou","sequence":"additional","affiliation":[{"name":"School of Bioscience and Bioengineering, South China University of Technology,  Guangzhou 510006, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bo","family":"Yang","sequence":"additional","affiliation":[{"name":"School of Bioscience and Bioengineering, South China University of Technology,  Guangzhou 510006, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yonghua","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Light Industry and Food Sciences, South China University of Technology,  Guangzhou 510640, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2015,3,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1385\/ABAB:118:1-3:155","article-title":"Lipases and their industrial applications\u2014An overview","volume":"118","author":"Houde","year":"2004","journal-title":"Appl. Biochem. Biotechnol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"390","DOI":"10.1016\/S0958-1669(02)00341-5","article-title":"Lipases for biotechnology","volume":"13","author":"Jaeger","year":"2002","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1016\/S0167-7799(96)10064-0","article-title":"\u201cInterfacial activation\u201d of lipases: Facts and artifacts","volume":"15","author":"Verger","year":"1997","journal-title":"Trends Biotechnol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2122","DOI":"10.1002\/pro.493","article-title":"Solvent-induced lid opening in lipases: A molecular dynamics study","volume":"19","author":"Rehm","year":"2010","journal-title":"Protein Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1061","DOI":"10.1016\/j.procbio.2008.05.009","article-title":"Interfacially activated lipases against hydrophobic supports: Effect of the support nature on the biocatalytic properties","volume":"43","author":"Cabrera","year":"2008","journal-title":"Process Biochem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.enzmictec.2015.02.001","article-title":"Immobilization of lipases on hydrophobic supports involves the open form of the enzyme","volume":"71","author":"Manoel","year":"2015","journal-title":"Enzym. Microb. Technol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"242","DOI":"10.1002\/bit.21230","article-title":"Improved catalytic properties of immobilized lipases by the presence of very low concentrations of detergents in the reaction medium","volume":"97","author":"Palomo","year":"2007","journal-title":"Biotechnol. Bioeng."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1007\/s11745-004-1258-7","article-title":"Protein engineering and applications of Candida rugosa lipase isoforms","volume":"39","author":"Akoh","year":"2004","journal-title":"Lipids"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"433","DOI":"10.1016\/S0167-7799(02)02046-2","article-title":"Optimizing lipases and related enzymes for efficient application","volume":"20","author":"Bornscheuer","year":"2002","journal-title":"Trends Biotechnol"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1002\/bit.25162","article-title":"Lipase-catalyzed process for biodiesel production: Protein engineering and lipase production","volume":"111","author":"Hwang","year":"2014","journal-title":"Biotechnol. Bioeng."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2383","DOI":"10.1016\/j.febslet.2005.03.037","article-title":"Mutations in the \u201clid\u201d region affect chain length specificity and thermostability of a Pseudomonas fragi lipase","volume":"579","author":"Santarossa","year":"2005","journal-title":"FEBS Lett."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"7994","DOI":"10.1074\/jbc.M113.536045","article-title":"Enhanced enzyme kinetic stability by increasing rigidity within the active site","volume":"289","author":"Xie","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4192","DOI":"10.1002\/anie.200500767","article-title":"Expanding the range of substrate acceptance of enzymes: Combinatorial active-site saturation test","volume":"44","author":"Reetz","year":"2005","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2885","DOI":"10.1002\/adsc.201100163","article-title":"Potential of different enzyme immobilization strategies to improve enzyme performance","volume":"353","author":"Rodrigues","year":"2011","journal-title":"Adv. Synth. Catal."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1451","DOI":"10.1016\/j.enzmictec.2007.01.018","article-title":"Improvement of enzyme activity, stability and selectivity via immobilization techniques","volume":"40","author":"Mateo","year":"2007","journal-title":"Enzym. Microb. Technol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1016\/j.molcatb.2012.01.013","article-title":"Production of lipase SMG1 and its application in synthesizing diacylglyecrol","volume":"77","author":"Wang","year":"2012","journal-title":"J. Mol. Catal. B-Enzym."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1007\/s11746-013-2365-2","article-title":"Production of diacylglycerol-mixture of regioisomers with high purity by two-step enzymatic reactions combined with molecular distillation","volume":"91","author":"Zheng","year":"2014","journal-title":"J. Am. Oil Chem. Soc."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1259","DOI":"10.1007\/s11746-012-2018-x","article-title":"Enzymatic synthesis of diacylglycerols enriched with conjugated linoleic acid by a novel lipase from Malassezia globosa","volume":"89","author":"Xu","year":"2012","journal-title":"J. Am. Oil Chem. Soc."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"792","DOI":"10.1093\/jn\/130.4.792","article-title":"Dietary diacylglycerol suppresses accumulation of body fat compared to triacylglycerol in men in a double-blind controlled trial","volume":"130","author":"Nagao","year":"2000","journal-title":"J. Nutr."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1007\/s11745-003-1042-8","article-title":"Nutritional characteristics of DAG oil","volume":"38","author":"Flickinger","year":"2003","journal-title":"Lipids"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1016\/j.jsb.2012.03.006","article-title":"Crystal structure of a mono- and diacylglycerol lipase from Malassezia globosa reveals a novel lid conformation and insights into the substrate specificity","volume":"178","author":"Xu","year":"2012","journal-title":"J. Struct. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1016\/j.bbrc.2012.06.108","article-title":"Molecular basis for substrate selectivity of a mono- and diacylglycerol lipase from Malassezia globosa","volume":"424","author":"Liu","year":"2012","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/j.biochi.2014.02.004","article-title":"Site-directed mutagenesis studies of the aromatic residues at the active site of a lipase from Malassezia globosa","volume":"102","author":"Gao","year":"2014","journal-title":"Biochimie"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"18682","DOI":"10.1021\/ja4106122","article-title":"Interplay of hydrogen bonds and n\u2192\u03c0* interactions in proteins","volume":"135","author":"Bartlett","year":"2013","journal-title":"J. Am. Chem. Soc."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"15458","DOI":"10.1074\/jbc.273.25.15458","article-title":"\u03c0-Stacking interactions. Alive and well in proteins","volume":"273","author":"McGaughey","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1002\/(SICI)1097-0134(19980701)32:1<111::AID-PROT12>3.0.CO;2-H","article-title":"A set of van der Waals and coulombic radii of protein atoms for molecular and solvent-accessible surface calculation, packing evaluation, and docking","volume":"32","author":"Li","year":"1998","journal-title":"Proteins"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1002\/pro.5560030206","article-title":"Do salt bridges stabilize proteins?A continuum electrostatic analysis","volume":"3","author":"Hendsch","year":"1994","journal-title":"Protein Sci."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"15396","DOI":"10.1073\/pnas.1407768111","article-title":"Loss of conformational entropy in protein folding calculated using realistic ensembles and its implications for NMR-based calculations","volume":"111","author":"Baxa","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1016\/j.jfoodeng.2011.11.020","article-title":"A process for the synthesis of PUFA-enriched triglycerides from high-acid crude fish oil","volume":"109","author":"Wang","year":"2012","journal-title":"J. Food Eng."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1614","DOI":"10.1002\/jsfa.6468","article-title":"Fatty acid specificity of T1 lipase and its potential in acylglycerol synthesis","volume":"94","author":"Qin","year":"2014","journal-title":"J. Sci. Food Agric."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"531","DOI":"10.1007\/s11746-009-1533-x","article-title":"Enzymatic production of monoacylglycerols with camellia oil by the glycerolysis reaction","volume":"87","author":"Zeng","year":"2010","journal-title":"J. Am. Oil Chem. Soc."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/0005-2760(89)90207-5","article-title":"Multi-competitive enzymatic reactions in organic media: A simple test for the determination of lipase fatty acid specificity","volume":"1004","author":"Rangheard","year":"1989","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_33","unstructured":"DeLano, W.L. Available online: http:\/\/www.pymol.org."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1063","DOI":"10.1002\/ejlt.201300442","article-title":"Immobilization of lipase SMG1 and its application in synthesis of partial glycerides","volume":"116","author":"Wang","year":"2014","journal-title":"Eur. J. Lipid Sci. Technol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/16\/4\/7273\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T20:44:12Z","timestamp":1760215452000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/16\/4\/7273"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2015,3,31]]},"references-count":34,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2015,4]]}},"alternative-id":["ijms16047273"],"URL":"https:\/\/doi.org\/10.3390\/ijms16047273","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2015,3,31]]}}}