Cloning and sequence analysis of the gene for glucodextranase from Arthrobacter globiformis T-3044 and expression in Escherichia coli cells.
The gld gene for glucodextranase from Arthrobacter globiformis T-3044 was cloned by using a combination of gene walking and probe methods and expressed on the recombinant plasmid pGD8, which was constructed with pUC118, in Escherichia coli cells. The enzyme gene consisted of a unique open reading frame of 3,153 bp. The comparison of the DNA sequence data with the N-terminal and 6 internal amino acid sequences of the purified enzyme secreted from A. globiformis T-3044 suggested the enzyme was translated from mRNA as a secretory precursor with a signal peptide of 28 amino acids residues. The deduced amino acids sequence of the mature enzyme contained 1,023 residues, resulting in a polypeptide with a molecular mass of 107,475 daltons. The deduced sequence showed about 38% identity to that of the glucoamylase from Clostridium sp. G0005. The glucodextranase activity of transformant harboring pGD8 was about 40 mU/ml at 30 degrees C for a 16-h culture. Although the GDase that was produced from the transformant was shorter than authentic GDase by 2 amino acid residues at the N-terminal end side, its enzymatic properties were almost same as the authentic one. Two kinds of genes, dex1 and dex2, for endo-dextranases from A. globiformis T-3044 were also cloned into Escherichia coli cells. The N-terminal of the purified endo-dextranase from A. globiformis T-3044 agreed with the deduced amino acid sequence, after the 33rd alanine residue, of only the dex1 gene for edo-dextranase. This result suggests that the endo-dextranase is translated from mRNA as a secretory precursor with a signal peptide of 32 amino acids residues. The deduced sequence of endo-dextranase 1 and endo-dextranase 2 showed about 93% and 65% identity with that of known endo-dextranase from Arthrobacter sp. CB-8, respectively.
Oguma
T
T
Noda Institute for Scientific Research, Chiba, Japan. toguma@mail.kikkoman.co.jp
Kurokawa
T
T
Tobe
K
K
Kitao
S
S
Kobayashi
M
M
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D005959:Glucosidases / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping
D005959:Glucosidases; C023149:exo-1,6-alpha-glucosidase