Swift Steven M, Hudgens Jeffrey W, Heselpoth Ryan D, Bales Patrick M, Nelson Daniel C
Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland, United States of America.
Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland, United States of America; National Institute of Standards and Technology, Biomolecular Measurement Division, Gaithersburg, Maryland, United States of America.
PLoS One. 2014 Nov 19;9(11):e112939. doi: 10.1371/journal.pone.0112939. eCollection 2014.
Alginate is a polysaccharide produced by certain seaweeds and bacteria that consists of mannuronic acid and guluronic acid residues. Seaweed alginate is used in food and industrial chemical processes, while the biosynthesis of bacterial alginate is associated with pathogenic Pseudomonas aeruginosa. Alginate lyases cleave this polysaccharide into short oligo-uronates and thus have the potential to be utilized for both industrial and medicinal applications. An alginate lyase gene, algMsp, from Microbulbifer sp. 6532A, was synthesized as an E.coli codon-optimized clone. The resulting 37 kDa recombinant protein, AlgMsp, was expressed, purified and characterized. The alginate lyase displayed highest activity at pH 8 and 0.2 M NaCl. Activity of the alginate lyase was greatest at 50°C; however the enzyme was not stable over time when incubated at 50°C. The alginate lyase was still highly active at 25°C and displayed little or no loss of activity after 24 hours at 25°C. The activity of AlgMsp was not dependent on the presence of divalent cations. Comparing activity of the lyase against polymannuronic acid and polyguluronic acid substrates showed a higher turnover rate for polymannuronic acid. However, AlgMSP exhibited greater catalytic efficiency with the polyguluronic acid substrate. Prolonged AlgMsp-mediated degradation of alginate produced dimer, trimer, tetramer, and pentamer oligo-uronates.
藻酸盐是由某些海藻和细菌产生的一种多糖，由甘露糖醛酸和古洛糖醛酸残基组成。海藻藻酸盐用于食品和工业化学过程，而细菌藻酸盐的生物合成与致病性铜绿假单胞菌有关。藻酸盐裂解酶将这种多糖切割成短的寡聚糖醛酸，因此有潜力用于工业和医药应用。来自微小杆菌属6532A的一个藻酸盐裂解酶基因algMsp被合成为一个经大肠杆菌密码子优化的克隆。所得到的37 kDa重组蛋白AlgMsp被表达、纯化并进行了表征。该藻酸盐裂解酶在pH 8和0.2 M NaCl条件下表现出最高活性。藻酸盐裂解酶的活性在50°C时最大；然而，当在50°C孵育时，该酶随时间不稳定。该藻酸盐裂解酶在25°C时仍具有高活性，并且在25°C下孵育24小时后活性几乎没有损失。AlgMsp的活性不依赖于二价阳离子的存在。比较该裂解酶对聚甘露糖醛酸和聚古洛糖醛酸底物的活性表明，聚甘露糖醛酸的周转速率更高。然而，AlgMSP对聚古洛糖醛酸底物表现出更高的催化效率。AlgMsp介导的藻酸盐长时间降解产生了二聚体、三聚体、四聚体和五聚体寡聚糖醛酸。
