Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp. strain V134

Abstract

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40 degrees C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.

FIG. 1.

Schematic representation of the agaV region in V134 and the nucleotide sequence between orf507 and agaV. The translation start codons of orf507 and agaV are shown in uppercase letters. The putative −35 and −10 promoter elements and the ribosome binding site (RBS) of agaV are underlined.

FIG. 2.

Schematic representation of the domain organization in AgaV. The numbers refer to the positions of the amino acids of the predicted protein.

FIG. 3.

Effects of temperature (A) and pH (B) on the activity of the purified recombinant AgaV. The effect of temperature on the enzyme activity was determined under standard assay conditions at temperatures ranging between 10 and 90°C. The effect of pH on the enzyme activity was determined under standard assay conditions but in three different buffers: 50 mM citric acid-sodium phosphate (▪), 50 mM KH₂PO₄-NaOH (▴), and 50 mM glycine-NaOH (⧫).

FIG. 4.

TLC analysis of the products of agarose hydrolysis by AgaV. Hydrolysis reactions were conducted at 40°C in 50 mM sodium phosphate buffer (pH 7.0) containing 0.25% substrate. Samples were taken at the indicated incubation times and analyzed by TLC as described in Materials and Methods. ST, neoagarooligosaccharide standards.

FIG. 5.

Sequences of the N-terminal 50 amino acids of the captured secretion proteins. Predicted signal peptides are underlined.