Molecular cloning, nucleotide sequence and expression in Escherichia coli of the beta-cyclodextrin glycosyltransferase gene from Bacillus circulans strain no. 8.
The beta-cyclodextrin glycosyltransferase (beta-CGTase) gene was isolated from a lambda-library prepared from Bacillus circulans strain no. 8. It was subcloned into plasmid pTZ and expressed by its endogenous regulatory sequences in Escherichia coli JM 103. The structural gene was sequenced and showed an open reading frame for a polypeptide of 718 amino acid residues. The recombinant beta-CGTase had the same enzymatic properties as the extracellular CGTase (684 amino acid residues, corresponding to a mol. wt. of 74416) produced by B. circulans strain no. 8. The amino acid sequence showed the highest homology (74.6% identical amino acids) with the CGTase of B. circulans strain F-2, which had been erroneously described as an amylase. The homology with the enzyme from the alkalophilic Bacillus sp. strain no. 1011 was 71.4%. The amino acid sequence derived will be used for elucidating the three-dimensional structure of the enzyme.
Institut für Organische Chemie und Biochemie, Freiburg, Federal Republic of Germany.
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L
Nitschke
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K
Heeger
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H
Bender
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GE
Schulz
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D015698:Genomic Library; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D015183:Restriction Mapping
D000596:Amino Acids; D004269:DNA, Bacterial; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase