Purification, characterization and gene identification of a α-Neoagarooligosaccharide hydrolase from an alkaliphilic bacterium Cellvibrio sp. WU-0601
| Autor: | Kana Kashimura, Teruhiko Watanabe, Kohtaro Kirimura |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0106 biological sciences
0301 basic medicine Cellvibrio Bioengineering medicine.disease_cause 01 natural sciences Biochemistry Catalysis 03 medical and health sciences chemistry.chemical_compound 010608 biotechnology Hydrolase medicine Glycoside hydrolase Escherichia coli biology Process Chemistry and Technology Agarase Nucleic acid sequence Hydrolase Gene biology.organism_classification 030104 developmental biology chemistry Galactose biology.protein |
| Zdroj: | Journal of Molecular Catalysis B: Enzymatic. 133:S328-S336 |
| ISSN: | 1381-1177 |
| Popis: | 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 42 kDa by SDS-PAGE and 84 kDa 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 1092 bp encoding a polypeptide constituted of 364 amino acid residues, deduced to be 41 kDa, and this α-NAOS hydrolase belongs to the glycoside hydrolase (GH) family 117. In addition, 3D-modeling structure based on the nucleotide sequence of α-NAOS hydrolase from non-marine bacterium Cellvibrio sp. is shown. The α-NAOS hydrolase gene was overexpressed in Escherichia coli BL21 as the host, and the recombinant α-NAOS hydrolase was functionally active. |
| Databáze: | OpenAIRE |
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