• BMB Reports
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• v.40 no.3
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• pp.333-340
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• 2007

Gene encoding cyclodextrin glycosyltransferase (CGTase), from thermotolerant Paenibacillus sp. T16 isolated from hot spring area in northern Thailand, was cloned and expressed in E. coli (JM109). The nucleotide sequences of both wild type and transformed CGTases consisted of 2139 bp open reading frame, 713 deduced amino acids residues with difference of 4 amino acid residues. The recombinant cells required 24 h culture time and a neutral pH for culture medium to produce compatible amount of CGTase compared to 72 h culture time and pH 10 for wild type. The recombinant and wild-type CGTases were purified by starch adsorption and phenyl sepharose column chromatography and characterized in parallel. Both enzymes showed molecular weight of 77 kDa and similar optimum pHs and temperatures with recombinant enzyme showing broader range. There were some significant difference in pH, temperature stability and kinetic parameters. The presence of high starch concentration resulted in higher thermostability in recombinant enzyme than the wild type. The recombinant enzyme was more stable at higher temperature and lower pH, with lower $K_m$ for coupling reaction using cellobiose and cyclodextrins as substrates.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.92-96
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• 2001

Effect of environmental factors on the expression of soluble forms of Bacillus macerans cyclodextrin glycosyltransferase in recombinant Escherichia coli BL21(DE3)pLysE:pTCGT1 were investigated. The amount of soluble CGTase produced in the cell was measured by determining its enzymatic activity. The soluble fractionof the enzyme was increased by lowering the culture temperature to $30{\circ}C$ and medium pH to 5.8 compared to the enzyme production in LB medium at $37^{\circ}C$ and pH7.0. Addition of 0.2 M NaCl enhanced enzyme expression levels at the expense of cell growth. Glycine betaine that was added after 3 h of induction protected not only the cell growth from hig osmotic pressue but also hepld in vivo folding of CGTase in recombinant E. coli. Addition of 1 mM $CaCl_2$ was also effective in the expression of soluble CGTase, resulting in 15 U/ml of the enzyme activity.