BIOCHEMICAL-PROPERTIES AND SUBSTRATE SPECIFICITIES OF A RECOMBINANTLYPRODUCED AZOTOBACTER-VINELANDII ALGINATE LYASE

Alginate is a polysaccharide composed of beta-D-mannuronic acid (M) an d 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 kD a. Sixty-three percent of the amino acids in this mature protein are i dentical to those in AlgL from Pseudomonas aeruginosa. AlgL was partia lly 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 acti vity. The pI of the enzyme is 5.1. When an alginate rich in mannuronic acid was used as the substrate, the K-m 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 cleav ed, but this activity may be sensitive to the extent of acetylation.