Purification and characterization of the recombinant alginate lyase from Pseudomonas sp. leaked by Escherichia coli upon addition of glycine.
The plasmid pAL205 encodes an alginate lyase gene of Pseudomonas sp. OS-ALG-9, fused in frame to the beta-galactosidase alpha-peptide gene. The alginate lyase (Aly) expressed in Escherichia coli (pAL205) was significantly secreted into the medium by the addition of glycine. The extracellular enzyme isolated from the culture of E. coli JM109 (pAL205) was purified over 15,000-fold by successive chromatography and subjected to amino acid sequence analysis. The sequence determined was identical to that of the intracellular protein. Since the activity and molecular size of the extracellular Aly is identical to the intracellular protein and to the Aly isolated from Pseudomonas, the glycine does not affect or modify the Aly during its leakage into the medium.
Fujiyama
K
K
International Center of Cooperative Research in Biotechnology, Faculty of Engineering, Osaka University, Japan.
Maki
H
H
Kinoshita
S
S
Yoshida
T
T
D000464:Alginates / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005998:Glycine / Q000494:pharmacology; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis
D000464:Alginates; D011993:Recombinant Fusion Proteins; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase; D005998:Glycine