Molecular Identification of Oligoalginate Lyase of Sphingomonas sp. Strain A1 as One of the Enzymes Required for Complete Depolymerization of Alginate

Abstract

Alginate is a linear polysaccharide composed of ␤-D-mannuronate and the C 5 epimer ␣-L-guluronate, arranged in three different ways: poly-␤-D-mannuronate (polyM), poly-␣-L-guluronate (polyG), and heteropolymeric (polyMG) region in which there is a random arrangement of the monomers (4). Alginate produced by brown seaweeds is not acetylated and is widely used in food and pharmaceutical industries because of the ability of the polymer to chelate metal ions and form a highly viscous solution (18). The acetylated form of alginate is synthesized by certain bacteria, such as mucoid cells of Pseudomonas aeruginosa and Azotobacter vinelandii. P. aeruginosa causes serious chronic pulmonary infections in the lungs of patients with cystic fibrosis (CF) (2), and alginate produced by the bacterial cells seems to play a crucial role in the adherence of the bacterium to target cells (21). The alginate functions as a biofilm and decreases the effect of antimicrobial agents by repressing the penetration of the agent into the biofilm, thus making it difficult to treat biofilm-dependent bacterial infectious diseases (5).A bacterium, Sphingomonas sp. strain A1, incorporates alginate into the cells through a pit formed on the cell surface (8), and an ABC (ATP-binding cassette) transporter specific to the macromolecule is responsible for transport of alginate (16) (Fig. 1). The incorporated alginate is depolymerized by three types of cytoplasmic alginate-depolymerizing enzymes (alginate lyases A1-I [66 kDa], A1-II [25 kDa], and A1-III [40 kDa]) formed from a common precursor protein through the consecutive processes of posttranslational modification (Fig. 1) (17). A1-I is autoprocessed to give rise A1-II and A1-III (9), and these alginate lyases cleave glycosidic bonds endolytically in the alginate molecule by ␤-elimination reaction. Briefly, A1-I is active on acetylated and nonacetylated alginates. A1-II prefers polyG and nonacetylated alginate produced by brown seaweeds. A1-III efficiently liquefies polyM and acetylated alginates produced by mucoid cells of P. aeruginosa derived from the lungs of CF patients (17, 32); this property may be useful as a clinical agent for the therapy of CF and other infectious diseases caused by P. aeruginosa.Three alginate lyases (A1-I, A1-II, and A1-III) can produce di-and trisaccharides from alginate as major final products (6, 32), thus implying that the cells of Sphingomonas sp. strain A1 have an additional enzyme responsible for the degradation of alginate oligosaccharides to the constituent monosaccharides. To construct the complete metabolic pathway for alginate in Sphingomonas sp. strain A1, we have purified and characterized the enzyme (oligoalginate lyase) catalyzing the degradation of alginate oligosaccharides and determined its primary structure by cloning the gene. MATERIALS AND METHODSMaterials. Sodium alginate (average molecular weight, 25,700; polymerization degree, more than 100, viscosity, 1,000 cp) from Eisenia bicyclis and DEAEcellulose were purchased from Nacalai Tesque Co. Ltd.,...

Growth Promotion of Tomato by Application of Immobilized Arthrobacter woluwensis ED in Alginate Beads

“…토양에 도입된 alginate bead는 alginase (alginate lyase EC 4.2.2.3)를 분비하는 미생물에 의해 분해될 수 있는데, alginase 를 생성하는 미생물은 매우 다양하고 토양에도 많이 존재한다 (Hashimoto et al, 2000). 따라서 고정화에 사용된 alginate bead 는 토양에서 서서히 분해되면서 고정화된 세균을 방출시키고 환 경에 악영향을 미치지 않을 것이며 (Bashan et al, 2002), 또한 DGGE 분석 결과 alginate bead에서 방출된 A. woluwensis ED 는 토양 고유 세균군집에 별다른 영향을 미치지 않는 것으로 추 정된다 (Fig.…”

Alginate Lyase from Indonesian Bacillus megaterium S245 Shows Activities Toward Polymannuronate and Polyguluronate

“…strain A1. Hashimoto, Miyake, Momma, Kawai and Murata (2000) also reported alginate lyase from soil bacteria Sphingomonas sp. strain A1 which has activities both on polymannuronate and polyguluronate.…”

Superchannel of Bacteria: Biological Significance and New Horizons

“…6,7) The three endotype alginate lyases originate from a single gene aly; i.e., A1-I is autocatalytically processed into A1-II and A1-III, specific to polyG and polyM respectively, 7) and release unsaturated di-, tri-, and tetrasaccharides from alginate. All unsaturated oligosaccharides thus produced are then degraded by an exotype lyase into constituent monosaccharides, which are nonenzymatically converted to -keto acids.…”

“…2). 6,7) In case of alginate engulfment, dynamic structural and functional fluctuations leading to the formation of the pit occur on the cell surface of strain A1. This indicates that substantial structural and functional fluctuations occur frequently on the cell surface in other than extreme cases Cells were cultured in the presence of alginate for (upper, from left) 0, 7, and 10 h, and (lower, left) 24 h. The magnified picture of the pit (lower, middle, and right) indicates the disappearance and rearrangement of pleat molecules and substantial accumulation of alginate gel particles.…”