Purification and biochemical characterization of an alkaline protease from marine bacteria Pseudoalteromonas sp. 129‐1
Purification and biochemical characterization of an alkaline protease from marine bacteria Pseudoalteromonas sp. 129‐1
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DOI：
10.1002/jobm.201500327
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发表时间：
2015-12
影响因子：
3.1
通讯作者：
Shimei Wu;Ge Liu;Dechao Zhang;Chao-Yang Li;Chaomin Sun
中科院分区：
文献类型：
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作者：
Shimei Wu;Ge Liu;Dechao Zhang;Chao-Yang Li;Chaomin Sun
采用硫酸铵沉淀法、阴离子交换色谱法和凝胶过滤法纯化了由海洋细菌假互变单胞菌sp. 129‐1产生的胞外碱性蛋白酶。该蛋白酶的纯度经SDS-PAGE证实，分子量估计为35 kDa。该蛋白酶在10 ~ 60℃、6 ~ 11的温度范围内保持了相当大的活性和稳定性，在50℃、pH = 8的温度范围内活性最佳。金属蛋白酶抑制剂EDTA对蛋白酶活性没有抑制作用，即使浓度高达15 mM，而15 mM PMSF，一种常见的丝氨酸蛋白酶抑制剂，极大地使蛋白酶失活。在表面活性剂（SDS、Tween 80和Triton X‐100）、氧化剂H2O2和商业洗涤剂的存在下，观察到蛋白酶的高稳定性。此外，蛋白酶对大多数测试的有机溶剂具有耐受性，对盐水的耐受性高达30%。有趣的是，铜绿假单胞菌PAO1的生物膜在0.01 mg ml−1的蛋白酶浓度下大大减少，在1 mg ml−1的浓度下几乎完全消失。总的来说，该蛋白酶作为洗衣剂的添加剂和无毒的抗生物膜剂显示出宝贵的价值。
An extracellular alkaline protease produced by marine bacteria strain Pseudoalteromonas sp. 129‐1 was purified by ammonium sulphate precipitation, anion exchange chromatography, and gel filtration. The purity of the protease was confirmed by SDS–PAGE and molecular mass was estimated to be 35 kDa. The protease maintained considerable activity and stability at a wide temperature range of 10–60 °C and pH range of 6–11, and optimum activity was detected at temperature of 50 °C and pH of 8. Metallo‐protease inhibitor, EDTA, had no inhibitory effect on protease activity even at concentration up to 15 mM, whereas 15 mM PMSF, a common serine protease inhibitor, greatly inactivated the protease. The high stability of the protease in the presence of surfactants (SDS, Tween 80, and Triton X‐100), oxidizing agent H2O2, and commercial detergents was observed. Moreover, the protease was tolerant to most of the tested organic solvents, and saline tolerant up to 30%. Interestingly, biofilm of Pseudomonas aeruginosa PAO1 was greatly reduced by 0.01 mg ml−1 of the protease, and nearly completely abolished with the concentration of 1 mg ml−1. Collectively, the protease showed valuable feathers as an additive in laundry detergent and non‐toxic anti‐biofilm agent.