[Effect of the changes of amino acids on both signal peptide C-terminal and mature protein N-terminal region to the secretion of alpha-amylase in B. subtilis].
By site-directed mutagenesis, G and C have taken the place of T and G at nucleotide sequence 287 and 291 of B. licheniformis alpha-amylase gene to generate pAm-y413B and the N-terminal sequence of mature protein have been changed from 7Leu 8Met to 7Arg8Ile. By the insertion of polylinker into the C-terminal of the signal sequence of alpha-amylase gene of pAmy413, the signal peptide of alpha-amylase produced by pAmy413L is 13 amino acids more than the pAmy413 (which is 29 amino acids long) and also, a new recognition cleavage sequence for signal peptidase I (Ala-Gln-Ala decreases Ser) is created; The secondary structure of the signal peptide has been analyzed by computer programs. The alpha-amylase relative activity of the two mutant strains is 3% and 36% of pAmy413, respectively. The molecular weight of extracellular alpha-amylase is the same as pAmy413. Terminal analysis shows that the N-terminal amino acid of mature protein is Ala, not Ser, and suggests that SPase I prefers to cleavage at the wild type recognition site (Ala-Ala-Ala decreases Ala). Therefore, all of the above results show that the secretion of alpha-amylase in B. subtilis is in accordance with the co-translational transportation model.
Ji
Y
Y
Lab. for Biotechnology, Nankai Univ., Tianjin.
Yang
L
L
Chen
Q
Q
Ma
M
M
Geng
Y
Y
Jiang
R
R
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021382:Protein Sorting Signals / Q000235:genetics*; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D021382:Protein Sorting Signals; D000516:alpha-Amylases