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Updated: Jul 2, 2026
Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles
Published on: December 31, 2019
Cloning, extracellular expression and characterization of a predominant beta-CGTase from Bacillus sp. G1 in E. coli
Rui Min Ong1, Kian Mau Goh, Nor Muhammad Mahadi
1Department of Bioprocess Engineering, Universiti Teknologi Malaysia, 81310, UTM Skudai, Johor, Malaysia.
Abstract:
The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60 degrees C. Heterologous recombinant protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% beta-cyclodextrin (CD) and 10% gamma-CD. This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could potentially serve as an industrial enzyme for the production of beta-CD.
