Molecular identification of an alpha-L-rhamnosidase from Bacillus sp strain GL1 as an enzyme involved in complete metabolism of gellan.

The genes (rhaA and rhaB) for two alpha-L-rhamnosidases of Bacillus sp. strain GL1, which assimilates a bacterial polysaccharide (gellan), were cloned from a genomic DNA library of the bacterium constructed in Escherichia coli, and the nucleotide sequences of the genes were determined. Gene rhaA (2661 bp) contained an open reading frame (ORF) encoding a protein (RhaA: 886 amino acids) with a molecular weight (MW) of 98280. Gene rhaB (2871 bp) contained an ORF encoding a protein (RhaB: 956 amino acids) with a MW of 106049. RhaA exhibited significant identity (41%) with alpha-L-rhamnosidase of Clostridium stercorarium, while RhaB showed slight homology with enzymes from other sources. An overexpression system for the two enzymes was constructed in E. coli, and the enzymes were purified and characterized. Both RhaA and RhaB were highly specific for rhamnosyl saccharides, including gellan disaccharide (rhamnosyl glucose) and naringin, and released rhamnose from substrates most efficiently at pH 6.5-7.0 and 40 degrees C. Bacillus sp. strain GL1 cells grown in a gellan medium produced only RhaB, indicating that RhaB plays a crucial role in the complete metabolism of gellan.