Production of autoproteolytically subunit-assembled 7-beta-(4-carboxybutanamido)cephalosporanic acid (GL-7ACA) acylase from Pseudomonas sp. C427 using a chitin-binding domain.
7-Beta-(4-Carboxybutanamido)cephalosporanic acid (GL-7ACA) acylase from Pseudomonas sp. C427 is known as a proteolytically processed bacterial enzyme. GL-7ACA acylase from Pseudomonas sp. C427 (C427) consists of alpha- and beta-subunits that are processed from a precursor peptide by removing the spacer peptide. A chitin-binding domain (CBD) of chitinase A1 derived from Bacillus circulans was genetically fused into four different positions of the C427-encoding gene. In the four enzymes thereby produced, Nalpha427, SP427, Calpha427, and Cbeta427, it was fused, respectively, to the N-terminal region of the alpha-subunit; the C-terminal region of the alpha-subunit; the three-amino-acid upper region of the C-terminal of the alpha-subunit; and to the C-terminal region of the beta-subunit. All of the fusion enzymes, expressed in Eschericha coli, were successfully processed into active forms and had GL-7ACA acylase activity. The affinity-binding activity to crystalline chitin was affected by the fusing position of CBD. Nalpha427, Calpha427, and Cbeta427 remained fused to the CBD after their processing steps and could bind to chitin, but in the case of SP427 the fused CBD was cleaved away during the processing steps and binding activity was no longer observed. These results indicate that CBD is functional in such autoproteolytically subunit-assembled acylases.
Nagao
Koji
K
Fermentation Development Laboratories, Fujisawa Pharmaceutical Co. Ltd, 156, Nakagawara, Shinkawa-cho, 452-0915, Nishikasugai-gun, Aichi, Japan. koji_nagao@po.fujisawa.co.jp.
Yamashita
Michio
M
Ueda
Mitsuyoshi
M
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010399:Penicillin Amidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D021382:Protein Sorting Signals; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D002686:Chitin; D002688:Chitinases; D010447:Peptide Hydrolases; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase