Cloning of the full-length isoamylase3 gene from cassava Manihot esculenta Crantz 'KU50' and its heterologous expression in E. coli.
Cloning of the full-length isoamylase3 gene from cassava Manihot esculenta Crantz 'KU50' and its heterologous expression in E. coli.
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木薯 Manihot esculenta Crantz KU50 全长异淀粉酶 3 基因的克隆及其在大肠杆菌中的异源表达。
DOI：
10.1016/j.plaphy.2018.09.010
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Panpetch P
中科院分区：
文献类型：
--
作者：
Panpetch P
异淀粉酶（EC. 3.2.1.68）是淀粉代谢中的一种必需酶，催化支链α-聚葡聚糖（如β-极限糊精和支链淀粉）的α-1，6糖苷键的裂解，但不催化支链淀粉的裂解。在植物和藻类中已经报道了三种不同的异淀粉酶同工型。本文报道了利用5 ′ RACE技术首次成功地获得木薯‘KU 50’异淀粉酶3基因全长（MeISA 3）。将MeISA 3基因克隆到原核表达载体pET 21 b上，在E.杆菌经SDS-PAGE和Western blot检测，rMeISA 3为单一条带，分子量约为75 kDa，而凝胶过滤层析检测其分子量约为80 kDa。这表明存在单体酶。rMeISA 3的生物化学表征表明，该酶对β-极限糊精具有特异性，在37 °C pH 6.0下具有最佳活性。Mg ~（2+）和Co ~（2+）对rMeISA 3的活性有显著的促进作用。通过HPAEC-PAD分析证实支链淀粉的rMeISA 3去支化葡聚糖链。
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 cassavaManihot esculentaCrantz ‘KU50’ from 5′ Rapid Amplification of cDNA Ends (5′ RACE). TheMeISA3was cloned to pET21b and expressed inE. 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.
来自发育中的玉米粒的两类淀粉脱支酶
DOI：
--
发表时间：
1991
期刊：
影响因子：
--
作者：
D. C. Doehlert;C. A. Knutson
通讯作者：
C. A. Knutson
马铃薯块茎（Solanum tuberosum L.）的脱支酶。
DOI：
--
发表时间：
1983
期刊：
影响因子：
--
作者：
Y. Ishizaki;H. Taniguchi;Y. Maruyama;Michinori Nakamura
通讯作者：
Michinori Nakamura
影响因子：
3.3
作者：
Deschamps, Philippe;Moreau, Herve;Ball, Steven G.
通讯作者：
Ball, Steven G.