Heterologous co-expression in E. coli of isoamylase genes from cassava Manihot esculenta Crantz ‘KU50’ achieves enzyme-active heteromeric complex formation
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Cloning of two isoamylase genes, MeISA1 and MeISA2, from cassava (Manihot esculenta Crantz) tubers, accompanied by their co-expression in E. coli demonstrates a requirement for heteromeric complex formation to achieve debranching activity.
Starch debranching enzyme (DBE) or isoamylase (ISA) (EC.184.108.40.206), an important enzyme in starch metabolism, catalyses the hydrolysis of α-1,6 glycosidic linkages of amylopectin. Isoforms of ISAs have been reported in higher plants and algae (Fujita et al. in Planta 208:283–293, 1999; Hussain et al. in Plant Cell 15:133–149, 2003; Ishizaki et al. in Agric Biol Chem 47:771–779, 1983; Mouille et al. in Plant Cell 8:1353–1366, 1996). In the current work, cassava ISA genes were isolated from cDNA generated from total RNA from tubers of Manihot esculanta Crantz cultivar KU50. MeISA1 and MeISA2 were successfully amplified and cloned into a pETDuet1 vector. The putative MeISA1 and MeISA2 proteins comprised 763 and 882 amino acids, with substantial similarity to StISA1 and StISA2 from potato (84.4% and 68.9%, respectively). Recombinant MeISA1 and MeISA2 were co-expressed in Escherichia coli SoluBL21 (DE3). HistrapTM-Purified rMeISA1 and rMeISA2 showed approximate molecular weights of 87 and 99 kDa, respectively, by SDS-PAGE. Debranching activity was only detectable in the column fractions where both recombinant ISA isoforms were present. The heteromeric DBE from crude extracts of 4–5 h induced cultures analysed by gel filtration chromatography and western blot showed combinations of rMeISA1 and rMeISA2 at ratios of 1:1 to 4:1. Pooled fractions with DBE activity were used for enzyme characterisation, which showed that the enzyme was specific for amylopectin, with optimum activity at 37 °C and pH 7.0. Enzyme activity was enhanced by Co2+, Mg2+ and Ca2+, but was strongly inhibited by Cu2+. Debranched amylopectin products showed chain length distributions typical of plant DBE.
KeywordsCassava Starch debranching enzyme Isoamylase Co-expression Recombinant MeISA
We would like to thank Mr. Prasert Thala from National Research Centre of Millet and Corn (Suwan Farm) in Nakhon Ratchasima province for kindly providing us cassava ‘KU50’ tubers. We are very thankful to Prof. Alison M. Smith at John Innes Centre for providing suggestions and support.
TL, PP and RF designed the experiments, which PP conducted; all authors contributed to data analysis; TL and PP led on manuscript preparation, to which all authors contributed.
This work was supported by The Royal Golden Jubilee Ph.D. Program, Thailand Research Fund (TRF) (Grant IRG 5780008) and Chulalongkorn University, the 90TH 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). Studies at the John Innes Centre are supported by the UK BBSRC Institute Strategic Program on Understanding and Exploiting Metabolism (MET) [BB/J004561/1] and the John Innes Foundation.
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Conflict of interest
The authors declare that they have no conflict of interest.
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