Molecular cloning of a glucoamylase gene from a thermophilic Clostridium and kinetics of the cloned enzyme.
Clostridium sp. G0005 produces a cell-bound glucoamylase (CGA). The gene encoding CGA has been sequenced. The deduced amino acid sequence begins with a putative 21-residue signal sequence for secretion of bacterial lipoproteins, which suggests that a putative CGA precursor is modified and secreted like other bacterial lipoproteins in Clostridium sp. G0005, and that the modified residue is important in the cell-bound form of mature CGA. Comparison of the amino acid sequence of the CGA precursor with known eukaryotic enzymes showed several regions of high similarity in spite of low similarity throughout the overall primary structure. CGA is the first bacterial glucoamylase to be cloned. The CGA gene was expressed in Escherichia coli cells with an inducible expression plasmid, in which the 5' non-coding region and the N-terminal coding region of the gene were replaced with the lac promoter. Kinetic studies of the cloned enzyme purified from E. coli were performed with a set of linear malto-oligosaccharides as substrates, and the subsite affinity was calculated from the kinetic parameters. CGA had typical kinetic properties for a glucoamylase, but this bacterial enzyme had higher isomaltose-hydrolyzing activity than other eukaryotic glucoamylases.
Ohnishi
H
H
Department of Agricultural Chemistry, University of Tokyo, Japan.
Kitamura
H
H
Minowa
T
T
Sakai
H
H
Ohta
T
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D015183:Restriction Mapping
D001426:Bacterial Proteins; D005087:Glucan 1,4-alpha-Glucosidase