Gui Yuanyuan, Gu Xiaoqian, Fu Liping, Zhang Qian, Zhang Peiyu, Li Jiang
College of Environmental Science and Engineering Qingdao University, Qingdao, China.
Marine Bioresource and Environment Research Center, First Institute of Oceanography, Ministry of Natural Resources, Qingdao, China.
Front Microbiol. 2021 Mar 12;12:631039. doi: 10.3389/fmicb.2021.631039. eCollection 2021.
The complete genome of sp. NJDZ03, which was isolated from the surface of Antarctic macroalgae, was analyzed by next-generation sequencing, and a putative carrageenase gene was obtained. was cloned and expressed in BL21(DE3). After purification by Ni-NTA chromatography, the recombinant Car3206 protein was characterized and the antioxidant activity of the degraded product was investigated. The results showed that the recombinant plasmid pet-30a- was highly efficiently expressed in BL21(DE3). The purified recombinant Car3206 showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 45 kDa. The optimum temperature of the recombinant Car3206 was 55°C, and it maintain 60-94% of its initial activity for 4-12 h at 55°C. It also kept almost 70% of the initial activity at 30°C, and more than 40% of the initial activity at 10°C. These results show that recombinant Car3206 had good low temperature resistance and thermal stability properties. The optimum pH of recombinant Car3206 was 7.0. Car3206 was activated by Na, K, and Ca, but was significantly inhibited by Cu and Cr. Thin-layer chromatographic analysis indicated that Car3206 degraded carrageenan generating disaccharides as the only products. The antioxidant capacity of the degraded disaccharides was investigated and the results showed that different concentrations of the disaccharides had similar scavenging effects as vitamin C on , •OH, and DPPH•. To our knowledge, this is the first report about details of the biochemical characteristics of a carrageenase isolated from an Antarctic strain. The unique characteristics of Car3206, including its low temperature resistance, thermal stability, and product unity, suggest that this enzyme may be an interesting candidate for industrial processes.
从南极大型藻类表面分离得到的 sp. NJDZ03 的全基因组，通过下一代测序进行分析，并获得了一个假定的卡拉胶酶基因。该基因被克隆并在 BL21(DE3) 中表达。经镍-氮三乙酸层析纯化后，对重组 Car3206 蛋白进行了表征，并研究了降解产物的抗氧化活性。结果表明，重组质粒 pet-30a- 在 BL21(DE3) 中高效表达。纯化后的重组 Car3206 在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上显示出一条单一的条带，表观分子量为 45 kDa。重组 Car3206 的最适温度为 55°C，在 55°C 下 4 - 12 小时内保持其初始活性的 60 - 94%。在 30°C 时也保持近 70% 的初始活性，在 10°C 时保持超过 40% 的初始活性。这些结果表明重组 Car3206 具有良好的低温耐受性和热稳定性。重组 Car3206 的最适 pH 为 7.0。Car3206 被 Na、K 和 Ca 激活，但被 Cu 和 Cr 显著抑制。薄层色谱分析表明，Car3206 降解卡拉胶产生二糖作为唯一产物。研究了降解二糖的抗氧化能力，结果表明不同浓度的二糖对 、•OH 和 DPPH• 的清除作用与维生素 C 相似。据我们所知，这是关于从南极 菌株分离得到的卡拉胶酶生化特性细节的首次报道。Car3206 的独特特性，包括其低温耐受性、热稳定性和产物单一性，表明这种酶可能是工业生产中一个有吸引力的候选酶。
