ISOLATION AND CHARACTERIZATION OF A NOVEL AGAR-DEGRADING BACTERIUM, Microbacterium barkeri sp. SELA 4, FROM SOIL ENRICHED WITH LABORATORY AGAR

Abstract

An agar-degrading bacterium, designated as strain SELA 4, was isolated from agriculture soil enriched with laboratory agar. The isolated strain was Gram-positive, rod-shaped, aerobic bacterium with off-white colonies showing marked depression on Mineral Salt Agar (MSA) medium. A similarity search was done based on its 16S r DNA sequence and the analysis revealed that the strain SELA 4 shared 99% similarity with the genus Microbacterium, showing its affiliation with Microbacterium barkeri strain which is known to be famous as biocontrol agents. On the basis of results obtained after biochemical analysis, physiological and phylogenetic analysis this strain was named Microbacterium barkeri sp. SELA 4. The strain SELA 4 is able to utilize agar as the only carbon source when grown at 37oC, 5 pH and produced the enzyme, β-agarase. Highest activity of 2.04 U/ml was observed at 1.5% agar concentration in the growth media. The enzyme activity at 37oC was 2.09 U/ml and at 5 pH was 1.96 U/ml respectively. Initially, the activity was qualitatively determined by iodine clear zone assay and later confirmed by activity staining. Activity staining showed the secretion of agarase enzyme by the strain whose molecular weight was found approximately 55.6 kDa. The enzyme hydrolysis product of agar was analyzed using Thin Layer Chromatography, indicating that the agarase enzyme from SELA 4 was β agarase which is known for cleaving agarose into Neoagarohexaose as the final product.