title: [Effect of changes of amino acids of N-terminal region of the mature protein on secretion of alpha-amylase in B. subtilis].
abstract: The mutant plasmid pAmy413C, in which G takes the place of A at the 271 position of alpha-amylase gene on the pAmy413 from B. licheniformis, was constructed by site-direct mutagenesis. At the N-terminus of the mature alpha-amylase, amino acid +2Asn was substituted by +3Asp in the wild type protein. Then, the alpha-amylase output of the mutant plasmid pAmy413C in B. subtilis was 2.02-2.57 times higher than that of the wild type pAmy413C in the same strain. The amino acid sequencing at the N-terminus of the matural alpha-amylase revealsed that the recognition site of signal peptidase I moved one amino acid upstream, from Ala-(+2)Asn to AlaAla-(+3) Asp. That is, the +2Asn of the wild type was changed to the +3Asp of the mutant. The secondary structural analysis showed that a 14-cycle structure formed in the alpha-amylase mRNA when the free energy was -51.7 kcal. In this case, the mutant is identical with the wild type. The difference between them is that G at 271 position is no longer paired with U at 211 position, hence, a G-overhang is formed. The secondary structural analysis of protein showed that one amino acid diminished in the turn structure of amino acid at 33-37 position, and this very amino acid is involed in an alpha-helix structure. In short, all the changes mentioned above in conformation and charged amino acids contribute to the increase in the protein secretion in B. subtilis.
authors.affiliation: College of Life Sciences, Nankai University.
authors.forename: Q M
authors.initials: QM
authors.lastname: Chen
authors.forename: S H
authors.initials: SH
authors.lastname: Liu
authors.forename: Y G
authors.initials: YG
authors.lastname: Ji
authors.forename: Z H
authors.initials: ZH
authors.lastname: Xue
authors.forename: P
authors.initials: P
authors.lastname: Fu
authors.forename: H R
authors.initials: HR
authors.lastname: Geng
authors.forename: M
authors.initials: M
authors.lastname: Ma
authors.forename: Q
authors.initials: Q
authors.lastname: Sun
authors.forename: D
authors.initials: D
authors.lastname: Liang
authors.forename: Y Q
authors.initials: YQ
authors.lastname: Geng
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D001483:Base Sequence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D000516:alpha-Amylases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000516:alpha-Amylases