Efficient production of human alpha-amylase by a Bacillus brevis mutant.
A cDNA for mature human salivary alpha-amylase was directly joined to a sequence encoding the signal peptide of the middle wall protein (MWP) gene of Bacillus brevis 47. This hybrid gene was placed downstream from the multiple promoter region of the MWP gene on a low copy-number plasmid vector, pHW1. B. brevis 47 carrying the plasmid produced 0.9 mg/l of active human alpha-amylase in the medium. A B. brevis 47 mutant obtained on mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine produced an increased amount of the alpha-amylase (6 mg/l). When the fused gene was inserted into a high copy-number expression vector, pNU200, and then introduced into the mutant, a large amount (60 mg/l) of the alpha-amylase was produced in the medium. The alpha-amylase showed approximately the same specific activity and molecular weight as those of the natural enzyme. The mutant showed higher sensitivity to various antibiotics than the original strain, and altered cell wall and cytoplasmic membrane protein compositions. The results of reversion analysis suggested that a single mutation is responsible for the above phenotypes and hyper-productivity of human alpha-amylase.
Konishi
H
H
Department of Food Science and Technology, Faculty of Agriculture, Nagoya University, Japan.
Sato
T
T
Yamagata
H
H
Udaka
S
S
D000595:Amino Acid Sequence; D001407:Bacillus / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D005784:Gene Amplification; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000235:genetics
D004247:DNA; D000516:alpha-Amylases