Molecular cloning of a cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. TS1-1 and characterization of the recombinant enzyme

Kamalesh Rahman, Rosli Md Illias, Osman Hassan, Nik Azmi Nik Mahmood, Noor Aini Abdul Rashid

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

A cyclodextrin glucanotransferase (CGTase) gene from Bacillus sp. TS1-1 was isolated and cloned into Escherichia coli. Starting from TTG codon, there was an open reading frame composed of 2163 bp (721 amino acids). The NH2 terminal position encoded a 46-amino acid of a signal peptide and followed by the mature enzyme (675 amino acids). The deduced amino acid sequence of the mature CGTase from Bacillus sp. TS1-1 exhibited 98.7% homology with 96% identity to the CGTase sequence from alkalophilic Bacillus sp. 1-1. The recombinant CGTase of Bacillus sp. TS1-1 expressed in E. coli was successfully purified to homogeneity using ammonium sulfate precipitation, followed by α-cyclodextrin-bound-epoxy-activated Sepharose 6B affinity chromatography. The purified CGTase enzymes exhibited a single band with molecular weight of 75 kDa on SDS-PAGE. Biochemical characterization of the enzyme shows an optimum temperature of 60 °C and optimum pH of 6.0. The enzyme was stable between pH 7 and 9 and temperature up to 70 °C. The Km and Vmax values calculated were 0.52 mg/ml and 54.35 mg of β-cyclodextrin/ml/min. The yield of the products from soluble starch as the substrate were 86% for β-cyclodextrin and 14% for γ-cyclodextrin after 24 h incubation at 60 °C, without adding any selective agent. The total β-CD produced under the conditions mentioned above was 3.65 g/l.

Original languageEnglish
Pages (from-to)74-84
Number of pages11
JournalEnzyme and Microbial Technology
Volume39
Issue number1
DOIs
Publication statusPublished - 1 Jun 2006

Fingerprint

Cloning
Cyclodextrins
Molecular Cloning
Bacilli
Bacillus
Enzymes
Genes
Amino Acids
Amino acids
Escherichia coli
Affinity chromatography
Temperature
Ammonium Sulfate
Protein Sorting Signals
Affinity Chromatography
Codon
Starch
Sepharose
Open Reading Frames
Polyacrylamide Gel Electrophoresis

Keywords

  • Cyclodextrin glucanotransferase

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology
  • Applied Microbiology and Biotechnology

Cite this

Molecular cloning of a cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. TS1-1 and characterization of the recombinant enzyme. / Rahman, Kamalesh; Illias, Rosli Md; Hassan, Osman; Mahmood, Nik Azmi Nik; Rashid, Noor Aini Abdul.

In: Enzyme and Microbial Technology, Vol. 39, No. 1, 01.06.2006, p. 74-84.

Research output: Contribution to journalArticle

Rahman, Kamalesh ; Illias, Rosli Md ; Hassan, Osman ; Mahmood, Nik Azmi Nik ; Rashid, Noor Aini Abdul. / Molecular cloning of a cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. TS1-1 and characterization of the recombinant enzyme. In: Enzyme and Microbial Technology. 2006 ; Vol. 39, No. 1. pp. 74-84.
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