Gene

Ecology

Biology

Oligosaccharide

Biochemistry

Substrate (aquarium)

Polysaccharide

Lyase

Enzyme

Escherichia coli

Molecular mass

Chemistry

Peptide sequence

Uronic acid

Recombinant DNA

Molecular cloning

Protein Expression and Purification

A new alginate lyase gene of algl17 was cloned from an alginate-degrading marine bacterium Microbulbifer sp. ALW1. The gene contained 2220 bp and encoded a 739-amino acid protein classified into the PL-17 family. The recombinant alginate lyase AlgL17 was overexpressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 84.9 kDa. This enzyme showed activities towards sodium alginate, polyM and alginate oligosaccharide, but very low activity towards polyG. These results indicate that AlgL17 is a polyM-specific oligoalginate lyase. When sodium alginate was used as a substrate, the optimum reaction temperature and pH for the enzyme were 35 °C and pH 8.0, respectively. Recombinant AlgL17 was stable at 25 °C, but not stable at 30 °C and 35 °C. It showed good stability over a pH range of 5.0-8.0. The enzyme activity was increased to 1.7 times by adding NaCl to a final concentration of 0.7 M. The ability of the recombinant AlgL17 producing 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) from sodium alginate and polyM block indicates that AlgL17 is an exo-type alginate lyase.

Molecular cloning and characterization of AlgL17, a new exo-oligoalginate lyase from Microbulbifer sp. ALW1