Purification, Characterization and Gene Identification of a α-Neoagarooligosaccharide Hydrolase from an Alkaliphilic Bacterium Cellvibrio sp. WU-0601

Publication date: Available online 14 February 2017 Source:Journal of Molecular Catalysis B: Enzymatic Author(s): Teruhiko Watanabe, Kana Kashimura, Kohtaro Kirimura Purification, enzymatic characterization and gene identification of the neoagarooligosaccharide hydrolase (EC 3.2.1.159, α-NAOS hydrolase) intracellularly produced by agar-degrading Cellvibrio sp. WU-0601, an alkaliphilic bacterium isolated from soil, were performed. α-NAOS hydrolase activity was detected only in the cell-free extract, not but culture filtrate, and α-NAOS hydrolase was purified from the cell-free extract through four purification steps. The molecular weight of α-NAOS hydrolase is estimated to be 42kDa by SDS-PAGE and 84kDa by gel filtration, indicating that α-NAOS hydrolase is a dimeric enzyme. By detailed analysis, α-NAOS hydrolase catalyzed the hydrolysis reaction cleaving specifically α-1,3 linkage of neoagarobiose (NA2) containing residues of 3,6-anhydro-l-galactose (L-AHG) and D-galactose. α-NAOS hydrolase hydrolyzed not only NA2 but also neoagarotetraose and neoagarohexaose as substrates, and released L-AHG and d-galactose or each corresponding agarooligosaccharide. The optimum reaction pH and temperature of α-NAOS hydrolase were 6.0 and 25°C, respectively. The identified nucleotide sequence of the α-NAOS hydrolase clone revealed that the open reading frame of α-NAOS hydrolase is 1092bp encoding a polypeptide constituted of 364 amino acid residues, deduced to be 41kDa, and ...
Source: Journal of Molecular Catalysis B: Enzymatic - Category: Biochemistry Source Type: research