{"title":"动力学指导下的环糊精糖基转移酶工程,具有更强的分子间转糖基化活性","authors":"Hanchi Chen, Lingjun Ju, Yangyang Dong, Shijie Lu, Yingling Bao, Linjiang Zhu, Xiaolong Chen","doi":"10.1002/aic.18512","DOIUrl":null,"url":null,"abstract":"<p>Cyclodextrin glycosyltransferase (CGTase) catalyzes intermolecular transglycosylation through either disproportionation or cyclization-coupling pathway. Kinetics analysis reveals that the hesperidin glycosylation process catalyzed by a CGTase variant (M1) is primarily accomplished through the disproportionation pathway. The cyclization-coupling pathway exhibits a lower reaction rate and competitively consumes glycosyl donor and yield byproducts that impair disproportionation. Under the guidance of reaction kinetics, mutagenesis was targeted at residues in the −3, +1, and +2 subsites, known to control the selectivity between disproportionation and cyclization. A quadruple variant was identified with 2.9 times hesperidin glycosylation activity compared to M1, and 20.3 times compared to the wild-type. Kinetic analysis reveals a fourfold improvement of <i>k</i><sub>cat</sub>/<i>K</i><sub>mA</sub> for disproportionation and an 85.5% reduction in <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> for cyclization after mutagenesis. Binding free energy analysis further confirms that the mutagenesis favors the binding of hesperidin, and destabilizes the binding of cyclodextrin.</p>","PeriodicalId":120,"journal":{"name":"AIChE Journal","volume":null,"pages":null},"PeriodicalIF":3.5000,"publicationDate":"2024-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Kinetics guided engineering of cyclodextrin glycosyltransferase with enhanced intermolecular transglycosylation activity\",\"authors\":\"Hanchi Chen, Lingjun Ju, Yangyang Dong, Shijie Lu, Yingling Bao, Linjiang Zhu, Xiaolong Chen\",\"doi\":\"10.1002/aic.18512\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Cyclodextrin glycosyltransferase (CGTase) catalyzes intermolecular transglycosylation through either disproportionation or cyclization-coupling pathway. Kinetics analysis reveals that the hesperidin glycosylation process catalyzed by a CGTase variant (M1) is primarily accomplished through the disproportionation pathway. The cyclization-coupling pathway exhibits a lower reaction rate and competitively consumes glycosyl donor and yield byproducts that impair disproportionation. Under the guidance of reaction kinetics, mutagenesis was targeted at residues in the −3, +1, and +2 subsites, known to control the selectivity between disproportionation and cyclization. A quadruple variant was identified with 2.9 times hesperidin glycosylation activity compared to M1, and 20.3 times compared to the wild-type. Kinetic analysis reveals a fourfold improvement of <i>k</i><sub>cat</sub>/<i>K</i><sub>mA</sub> for disproportionation and an 85.5% reduction in <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> for cyclization after mutagenesis. Binding free energy analysis further confirms that the mutagenesis favors the binding of hesperidin, and destabilizes the binding of cyclodextrin.</p>\",\"PeriodicalId\":120,\"journal\":{\"name\":\"AIChE Journal\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.5000,\"publicationDate\":\"2024-06-14\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"AIChE Journal\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/aic.18512\",\"RegionNum\":3,\"RegionCategory\":\"工程技术\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"ENGINEERING, CHEMICAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"AIChE Journal","FirstCategoryId":"5","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/aic.18512","RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"ENGINEERING, CHEMICAL","Score":null,"Total":0}
Kinetics guided engineering of cyclodextrin glycosyltransferase with enhanced intermolecular transglycosylation activity
Cyclodextrin glycosyltransferase (CGTase) catalyzes intermolecular transglycosylation through either disproportionation or cyclization-coupling pathway. Kinetics analysis reveals that the hesperidin glycosylation process catalyzed by a CGTase variant (M1) is primarily accomplished through the disproportionation pathway. The cyclization-coupling pathway exhibits a lower reaction rate and competitively consumes glycosyl donor and yield byproducts that impair disproportionation. Under the guidance of reaction kinetics, mutagenesis was targeted at residues in the −3, +1, and +2 subsites, known to control the selectivity between disproportionation and cyclization. A quadruple variant was identified with 2.9 times hesperidin glycosylation activity compared to M1, and 20.3 times compared to the wild-type. Kinetic analysis reveals a fourfold improvement of kcat/KmA for disproportionation and an 85.5% reduction in kcat/Km for cyclization after mutagenesis. Binding free energy analysis further confirms that the mutagenesis favors the binding of hesperidin, and destabilizes the binding of cyclodextrin.
期刊介绍:
The AIChE Journal is the premier research monthly in chemical engineering and related fields. This peer-reviewed and broad-based journal reports on the most important and latest technological advances in core areas of chemical engineering as well as in other relevant engineering disciplines. To keep abreast with the progressive outlook of the profession, the Journal has been expanding the scope of its editorial contents to include such fast developing areas as biotechnology, electrochemical engineering, and environmental engineering.
The AIChE Journal is indeed the global communications vehicle for the world-renowned researchers to exchange top-notch research findings with one another. Subscribing to the AIChE Journal is like having immediate access to nine topical journals in the field.
Articles are categorized according to the following topical areas:
Biomolecular Engineering, Bioengineering, Biochemicals, Biofuels, and Food
Inorganic Materials: Synthesis and Processing
Particle Technology and Fluidization
Process Systems Engineering
Reaction Engineering, Kinetics and Catalysis
Separations: Materials, Devices and Processes
Soft Materials: Synthesis, Processing and Products
Thermodynamics and Molecular-Scale Phenomena
Transport Phenomena and Fluid Mechanics.