Identification of a marine agarolytic pseudoalteromonas isolate and characterization of its extracellular agarase

Abstract

The phenotypic and agarolytic features of an unidentified marine bacteria that was isolated from the southern Pacific coast was investigated. The strain was gram negative, obligately aerobic, and polarly flagellated. On the basis of several phenotypic characters and a phylogenetic analysis of the genes coding for the 16S rRNA, this strain was identified as Pseudoalteromonas antarctica strain N-1. In solid agar, this isolate produced a diffusible agarase that caused agar softening around the colonies. An extracellular agarase was purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography on DEAE-cellulose. The purified protein was determined to be homogeneous on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it had a molecular mass of 33 kDa. The enzyme hydrolyzed the beta-1,4-glycosydic linkages of agar, yielding neoagarotetraose and neoagarohexaose as the main products, and exhibited maximal activity at pH 7. The enzyme was stable at temperatures up to 30 degreesC, and its activity was not affected by salt concentrations up to 0.5 M NaCl.

FIG. 1

Dendrogram of the relatedness of strain N-1 with several Pseudoalteromonas species based on the 16S rDNA sequences. The unrooted tree was constructed by neighbor-joining analysis. +, Branch found by parsimony; ∗, branch found by maximum likelihood (P < 0.01). Percentages are indicated by bootstraps (500 replicates for neighbor-joining analysis; 100 replicates for parsimony).

FIG. 2

Growth and agarase activity of P. antarctica N-1.

FIG. 3

Chromatography of agarase from P. antarctica N-1 on Sephadex G75.

FIG. 4

SDS-PAGE of purified agarase from P. antarctica N-1. Lane 1, molecular mass standards; lane 2, purified agarase (ca. 10 μg).

FIG. 5

(A) Effect of pH on the activity of the purified agarase. The activity was determined at a pH between 3.6 and 10.0 using the following buffers: 100 mM sodium acetate (pH 3.6 to 5.0 [□]), 20 mM morpholineethanesulfonic acid (pH 5.0 to 6.0 [■]), 20 mM sodium phosphate (pH 6.0 to 7.6 [○]), Tris-HCl (7.5 to 9.0 [▵]), and 50 mM glycine-NaOH (pH 9.0 to 10 [•]). (B) Effect of temperature on the stability of agarase from P. antarctica N-1. The enzyme was incubated in 50 mM sodium phosphate at pH 6.5, and the residual activity was determined at 30°C.

FIG. 6

Hydrolysis products of agar by agarase from P. antarctica N-1. Digested agar (20 μl, 3% [wt/vol]) was injected into a Polyspher CH-Na column (Merck) equilibrated with deionized water at 0.3 ml/min as described in Materials and Methods. The oligosaccharides were detected by determining the refractive index with a detector (Gilson, Middleton, Wis.). The positions of the neoagarohexaose (NH), neoagarotetraose (NT), neoagarobiose (NA), and galactose (G) standards are indicated.

FIG. 7

(A) ¹³C NMR spectrum of the hydrolysis products of unsubstituted agar by agarase from P. antarctica N-1. (B) Oligosaccharides released by agarase.