کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10143006 | 1646126 | 2018 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Cloning of the full-length isoamylase3 gene from cassava Manihot esculenta Crantz 'KU50' and its heterologous expression in E. coli
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
علوم کشاورزی و بیولوژیک
دانش گیاه شناسی
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چکیده انگلیسی
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 5â² Rapid Amplification of cDNA Ends (5â² 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 75â¯kDa on SDS-PAGE and Western blot, while 80â¯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 37â¯Â°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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Plant Physiology and Biochemistry - Volume 132, November 2018, Pages 281-286
Journal: Plant Physiology and Biochemistry - Volume 132, November 2018, Pages 281-286
نویسندگان
Pawinee Panpetch, Robert A. Field, Tipaporn Limpaseni,