Gene cloning, expression and characterization of a neoagarotetraose-producing β-agarase from the marine bacterium Agarivorans sp. HZ105
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A β-agarase gene hz2 with 2,868 bp was cloned from the marine agarolytic bacterium Agarivorans sp. HZ105. It encoded a mature agarase HZ2 of 102,393 Da (920 amino acids). Based on the amino acid sequence similarity, agarase HZ2 was assigned to the glycoside hydrolase family 50. The β-agarase shared a gene sequence identity of 98.6% with the reported but much less characterized β-agarase agaB from Vibrio sp. JT0107. Its recombinant agarase rHZ2 was produced in E. coli cells and purified to homogeneity. The agarase rHZ2 degraded agarose and neoagarooligosaccharides with degrees of polymerization above four, to yield neoagarotetraose as the dominant product, which was different from β-agarase agaB of Vibrio sp. JT0107. The agarose hydrolysis pattern suggested that rHZ2 was an endo-type β-agarase. Beta-mercaptoethanol (90 mM) and dithiothreitol (9 mM) increased the agarase activity of rHZ2 by 72.9% and 17.3% respectively, while SDS (9 mM) inhibited the activity completely. The agarase activity was independent of Na+, K+, Mg2+ and Ca2+. The maximal enzyme activity was observed at 40°C and pH 7. The kinetic parameters K m, V max, K cat, and K cat/K m values toward agarose of agarase rHZ2 were 5.9 mg ml−1, 235 U mg−1, 401 s−1 and 6.8 × 105 M−1 s−1, respectively. Agarase rHZ2 could have a potential application in the production of bioactive neoagarotetraose.
KeywordsAgarivorans sp. HZ105 Agarase Cloning Expression Characterization Neoagarotetraose
This work was supported by the National Natural Science Foundation of China (No. 41076106), Guangdong Natural Science Foundation (No. S2011030005257), the Science & Technology Project of Guangdong Province (No. 2009B030803051) and the Key Science and Technology Innovation Project for University by the Department of Education of Guangdong Province (No.CXZD1124).
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