Biochemical properties and substrate specificities of a recombinantly produced Azotobacter vinelandii alginate lyase.
Alginate is a polysaccharide composed of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). An Azotobacter vinelandii alginate lyase gene, algL, was cloned, sequenced, and expressed in Escherichia coli. The deduced molecular mass of the corresponding protein is 41.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 39 kDa. Sixty-three percent of the amino acids in this mature protein are identical to those in AlgL from Pseudomonas aeruginosa. AlgL was partially purified, and the activity was found to be optimal at a pH of 8.1 to 8.4 and at 0.35 M NaCl. Divalent cations are not necessary for activity. The pI of the enzyme is 5.1. When an alginate rich in mannuronic acid was used as the substrate, the Km was found to be 4.6 x 10(-4) M (sugar residues). AlgL was found to cleave M-M and M-G bonds but not G-M or G-G bonds. Bonds involving acetylated residues were also cleaved, but this activity may be sensitive to the extent of acetylation.
Ertesvåg
H
H
Unigen Center for Molecular Biology, Norwegian University of Science and Technology, N-7005 Trondheim, Norway. helga.ertesvag@unigen.ntnu.no
Erlien
F
F
Skjåk-Braek
G
G
Rehm
B H
BH
Valla
S
S
D000464:Alginates / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology*; D002240:Carbohydrate Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D009994:Osmolar Concentration; D011133:Polysaccharide-Lyases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity
D000464:Alginates; D002413:Cations, Divalent; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase