Alginate is a copolymer of beta-D-mannuronic acid and alpha-L-guluronic acid(GulA), linked together by 1-4 linkages. The polymer is a well-established industrial product obtained commercially by harvesting brown seaweeds. Some bacteria, mostly derived from Azotobacter vinelandii, A. chroococcum and several species of Pseudomonas, are also capable of producing copious amounts of this polymer. This uniform polymer is then further modified by acetylation at positions O-2 and/or O-3 but not in seaweed alginates.
A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and DNA. Bacteria biofilms cause chronic infections because they show increased tolerance to antibiotics system. Cystic fibrosis patients with chronic lung infection is caused by biofilm-growing mucoid Pseudomonas aeruginosa strains. Therefore, alginate lyase mutants with high enzyme activity are needed to decompose the biofilm efficiently.
Alginate lyase(AlgL) were cloned from the Azotobacter vinelandii using plasmid pET28a(+) for mutagenesis and were prepared 79 mutants. Mutants were expressed as His tag fusion enzymes in E. coli, and purified using Ni-NTA agarose bead. The alginate lyase activity was quantitatively measured by thiobarbituric acid assay using sodium alginate(seaweed alginate) and acetylated algiante(bacterial alginate).
K194E/K245D/K319A was the mutants the highest activity in acetylated alginate and K194E/K245A/R247A/K319A was the mutants the highest activity in sodium alginate. The activity was increased more than 6-fold. However, of various mutants, K63A, K63E, N198A, N198D, H199A, Y253A and Y253F were found to be inactive. Asn-197, His-199 and Tyr-253 of wild type AlgL contributed to its high catalytic activity by interacting alginate.
These improved mutants of AlgL would be valuable for the eradication of biofilm-forming bacteria when combined with antibiotics.