Molecular identification of oligoalginate lyase of Sphingomonas sp. strain A1 as one of the enzymes required for complete depolymerization of alginate

Abstract

A bacterium, Sphingomonas sp. strain A1, can incorporate alginate into cells through a novel ABC (ATP-binding cassette) transporter system specific to the macromolecule. The transported alginate is depolymerized to di- and trisaccharides by three kinds of cytoplasmic alginate lyases (A1-I [66 kDa], A1-II [25 kDa], and A1-III [40 kDa]) generated from a single precursor through posttranslational autoprocessing. The resultant alginate oligosaccharides were degraded to monosaccharides by cytoplasmic oligoalginate lyase. The enzyme and its gene were isolated from the bacterial cells grown in the presence of alginate. The purified enzyme was a monomer with a molecular mass of 85 kDa and cleaved glycosidic bonds not only in oligosaccharides produced from alginate by alginate lyases but also in polysaccharides (alginate, polymannuronate, and polyguluronate) most efficiently at pH 8.0 and 37 degrees C. The reaction catalyzed by the oligoalginate lyase was exolytic and thought to play an important role in the complete depolymerization of alginate in Sphingomonas sp. strain A1. The gene for this novel enzyme consisted of an open reading frame of 2,286 bp encoding a polypeptide with a molecular weight of 86,543 and was located downstream of the genes coding for the precursor of alginate lyases (aly) and the ABC transporter (algS, algM1, and algM2). This result indicates that the genes for proteins required for the transport and complete depolymerization of alginate are assembled to form a cluster.

FIG. 1

Overall pathway for alginate metabolism in Sphingomonas sp. strain A1. Alginate is first concentrated in a mouth-like pit on the cell surface and then incorporated into cells through the ABC transporter system consisting of AlgS, AlgM1, and AlgM2. The incorporated alginate is depolymerized by three kinds of alginate lyase (A1-I, A1-II, and A1-III) to give rise to di- and trisaccharides with an unsaturated uronyl residue at the nonreducing terminus. Alginate lyases are first synthesized as a precursor protein (Po), followed by the transformation to A1-I by excising the N-terminal peptide. A1-I is autocatalytically processed to A1-II and A1-III. The resultant oligosaccharides by the actions of A1-I, A1-II, and A1-III are degraded by oligoalginate lyase to an unsaturated monosaccharide, which is nonenzymatically converted into α-keto acid. Genes encoding proteins responsible for alginate transport and assimilation form a cluster. aly, alginate lyase (Po) gene; algS, algM1, and algM2, alginate ABC transporter genes; algQ1 and algQ2, genes with unknown functions; oal, oligoalginate lyase gene.

FIG. 2

Degradation of alginate trisaccharide by cytoplasmic enzyme fraction. Alginate trisaccharide (10 μg) was incubated with the enzyme fraction (5.6 μg of protein) for 15 min (lane 3), 1 h (lane 4), and 24 h (lane 5), and the products were analyzed by TLC. Tri, Di, and Mono indicate the tri-, di-, and monosaccharides generated from alginate, respectively. Lane 1, alginate disaccharide (10 μg); lane 2, alginate trisaccharide (10 μg).

FIG. 3

Electrophoretic profiles of oligoalginate lyase. (A) SDS-PAGE. Lane 1, molecular mass standards (synthetic polypeptides with molecular masses of 225, 150, 100, 75, 50, 35, 25, and 15 kDa); lane 2, purified enzyme. The arrow indicates the position of purified oligoalginate lyase. (B) Native gradient PAGE. Lane 1, molecular mass standards thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and bovine serum albumin (67 kDa); lane 2, purified enzyme. The arrow indicates the position of purified oligoalginate lyase.

FIG. 4

Effects of pH (A) and temperature (B) on activity of oligoalginate lyase. Experiments were carried out at 30°C using alginate trisaccharide (10 μg) and purified enzyme (1.8 mU). (A) To determine the effect of pH, reactions were performed at 30°C for 30 min in the following 50 mM buffers; sodium acetate (□), potassium phosphate (○), and Tris-HCl (▵). Activity at pH 8.0 in Tris-HCl buffer was set at 100%. (B) To determine the optimal temperature, reactions were performed for 30 min at various temperatures in 50 mM Tris-HCl buffer, pH 8.0. Activity at 37°C was set at 100%.

FIG. 5

Substrate specificity of oligoalginate lyase. (A) Various substrates (0.5%) indicated on the top were incubated with oligoalginate lyase (1.8 mU) at 30°C, and products formed after 0- and 30-min incubations were analyzed by TLC. Tri, alginate trisaccharide; di, alginate disaccharide. (B) Alginate (0.5%) was incubated with oligoalginate lyase (1.8 mU) at 30°C for several hours, followed by TLC analysis. Lane 1, alginate trisaccharide (10 μg); lane 2, alginate disaccharide (10 μg). Tri, di, and mono indicate the tri-, di-, and monosaccharides from alginate, respectively.

FIG. 6

Mode of action of oligoalginate lyase. Alginate trisaccharide (10 μg) was incubated with oligoalginate lyase (1.8 mU) for 30 min at 30°C, and the products were analyzed on TLC plates (lanes 3 in panels A and B) by staining with sulfuric acid (A) or TBA (B). Lane 1, alginate disaccharide (10 μg); lane 2, alginate trisaccharide (10 μg); lane 4, alginate disaccharide (10 μg) prepared by acid hydrolysis; lane 5, alginate trisaccharide (10 μg) prepared by acid hydrolysis. Tri, Di, and Mono indicate the unsaturated tri-, di-, and monosaccharides from alginate, respectively.