Cloning, extracellular expression and characterization of a predominant β-CGTase from Bacillus sp. G1 in E. coli

Rui Min Ong, Kian Mau Goh, Nor Muhammad Mahadi, Osman Hassan, Raja Noor Zaliha Raja Abdul Rahman, Rosli Md Illias

Research output: Contribution to journalArticle

43 Citations (Scopus)

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°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% β-cyclodextrin (CD) and 10% γ-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 β-CD.

Original languageEnglish
Pages (from-to)1705-1714
Number of pages10
JournalJournal of Industrial Microbiology and Biotechnology
Volume35
Issue number12
DOIs
Publication statusPublished - Dec 2008

Fingerprint

Cloning
Cyclodextrins
Bacilli
Escherichia coli
Bacillus
Organism Cloning
Starch
Manihot
Enzymes
Inclusion Bodies
Protein Sorting Signals
Recombinant Proteins
Recombinant proteins
Open Reading Frames
Purification
Nucleotides
Genes
Molecular Weight
Molecular weight
Amino acids

Keywords

  • Bacillus sp. G1
  • Cyclodextrin
  • Cyclodextrin glucanotransferase
  • Extracellular expression
  • Predominant β-CGTase
  • Signal peptide

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

Cite this

Cloning, extracellular expression and characterization of a predominant β-CGTase from Bacillus sp. G1 in E. coli. / Ong, Rui Min; Goh, Kian Mau; Mahadi, Nor Muhammad; Hassan, Osman; Rahman, Raja Noor Zaliha Raja Abdul; Illias, Rosli Md.

In: Journal of Industrial Microbiology and Biotechnology, Vol. 35, No. 12, 12.2008, p. 1705-1714.

Research output: Contribution to journalArticle

Ong, Rui Min ; Goh, Kian Mau ; Mahadi, Nor Muhammad ; Hassan, Osman ; Rahman, Raja Noor Zaliha Raja Abdul ; Illias, Rosli Md. / Cloning, extracellular expression and characterization of a predominant β-CGTase from Bacillus sp. G1 in E. coli. In: Journal of Industrial Microbiology and Biotechnology. 2008 ; Vol. 35, No. 12. pp. 1705-1714.
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