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Panpetch, Pawinee, Field, Robert A. 
ORCID: https://orcid.org/0000-0001-8574-0275 and Limpaseni, Tipaporn
(2018)
Cloning of the full-length isoamylase3 gene from cassava Manihot esculenta Crantz ‘KU50’ and its heterologous expression in E. coli.
Plant Physiology and Biochemistry, 132.
pp. 281-286.
ISSN 0981-9428
Abstract
Isoamylase (EC.3.2.1.68), an essential enzyme in starch metabolism, catalyses the cleavage of α-1,6 glucosidic linkages of branched α-polyglucans such as beta-limit dextrin and amylopectin, but not pullulan. Three different isoamylase isoforms have been reported in plants and algae. We herein report on the first success in preparation of full-length isoamylase3 gene (MeISA3) of cassava Manihot esculenta Crantz ‘KU50’ from 5′ Rapid Amplification of cDNA Ends (5′ RACE). The MeISA3 was cloned to pET21b and expressed in E. coli. The HistrapTM-purified rMeISA3 appeared as a single band protein with approximate molecular size of 75 kDa on SDS-PAGE and Western blot, while 80 kDa was shown by gel filtration chromatography. This indicated the existence of a monomeric enzyme. Biochemical characterisation of rMeISA3 showed that the enzyme was specific towards beta-limit dextrin, with optimal activity at 37 °C pH 6.0. Activity of rMeISA3 could be significantly promoted by Mg2+ and Co2+. rMeISA3 debranched glucan chains of amylopectin were confirmed by HPAEC-PAD analysis.
| Item Type: | Article |
|---|---|
| Additional Information: | Funding Information: This work was mainly supported by The Royal Golden Jubilee Ph.D. Program, Thailand Research Fund (Grant IRG 5780008 ); Chulalongkorn University , the 90 TH Anniversary of Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University (Grant GCUGR1125601025D ); The IIAC of the CU Centenary Academic Development Project and Thailand Research Fund ( IRG 5780008 ); The UK BBSRC Institute Strategic Program on Understanding and Exploiting Metabolism (MET) [ BB/J004561/1 ] and the John Innes Foundation. Publisher Copyright: © 2018 Elsevier Masson SAS |
| Uncontrolled Keywords: | 5′ race,cassava,isoamylase 3,recombinant meisa3,starch debranching enzyme,physiology,genetics,plant science ,/dk/atira/pure/subjectarea/asjc/1300/1314 |
| Faculty \ School: | Faculty of Science > School of Chemistry, Pharmacy and Pharmacology |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 04 Sep 2024 08:33 |
| Last Modified: | 25 Sep 2024 18:05 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/96500 |
| DOI: | 10.1016/j.plaphy.2018.09.010 |
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