Cloning of the full-length isoamylase3 gene from cassava Manihot esculenta Crantz 'KU50' and its heterologous expression in E. coli
Copyright © 2018 Elsevier Masson SAS. All rights reserved.
| Veröffentlicht in: | Plant physiology and biochemistry : PPB. - 1991. - 132(2018) vom: 05. Nov., Seite 281-286 |
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| Format: | Online-Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2018
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| Zugriff auf das übergeordnete Werk: | Plant physiology and biochemistry : PPB |
| Schlagworte: | Journal Article 5′ RACE Cassava Isoamylase 3 Recombinant MeISA3 Starch debranching enzyme Recombinant Proteins Isoamylase EC 3.2.1.68 |
| Zusammenfassung: | Copyright © 2018 Elsevier Masson SAS. All rights reserved. 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 |
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| Beschreibung: | Date Completed 26.11.2018 Date Revised 16.07.2022 published: Print-Electronic Citation Status MEDLINE |
| ISSN: | 1873-2690 |
| DOI: | 10.1016/j.plaphy.2018.09.010 |