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海洋弧菌HN897β-琼脂糖酶基因vas1-1339异源表达及活性分析
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作者 喻飞 金兴坤 +5 位作者 雷天影 曹海航 陈锵辉 阳耀帆 李佳航 赵哲 《热带海洋学报》 CAS CSCD 北大核心 2022年第2期170-176,共7页
海洋弧菌HN897株是一株高产琼脂糖酶的Vibrio astriarenae菌,前期功能缺失实验证明其β-琼脂糖酶基因vas1-1339的缺失可显著降低弧菌HN897株的琼脂水解活性。文章在大肠杆菌中异源表达Vas1-1339基因编码蛋白,分析该基因的表达及蛋白功... 海洋弧菌HN897株是一株高产琼脂糖酶的Vibrio astriarenae菌,前期功能缺失实验证明其β-琼脂糖酶基因vas1-1339的缺失可显著降低弧菌HN897株的琼脂水解活性。文章在大肠杆菌中异源表达Vas1-1339基因编码蛋白,分析该基因的表达及蛋白功能活性。首先,构建含vas1-1339基因开放阅读框全长的表达载体pET28a-1339,利用原核表达技术在大肠杆菌中成功地异源表达了Vas1-1339琼脂糖酶;然后,利用卢戈氏碘液染色分析发现异源表达Vas1-1339琼脂糖酶的大肠杆菌能够高效水解琼脂,且异丙基硫代半乳糖苷(IPTG)最优诱导浓度为10μmol·L^(-1);免疫印迹分析发现胞内基因表达产物羧基端可能存在切割现象,推测Vas1-1339琼脂糖酶存在复杂的成熟过程。对纯化的琼脂糖酶活性分析发现海洋弧菌HN897株β-琼脂糖酶Vas1-1339能够独立发挥琼脂糖降解功能。 展开更多
关键词 β-琼脂糖酶 海洋弧菌 琼脂糖酶解 异源蛋白表达
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A Polysaccharide-Degrading Marine Bacterium Flammeovirga sp.MY04 and Its Extracellular Agarase System 被引量:5
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作者 HAN Wenjun GU Jingyan +4 位作者 YAN Qiujie LI Jungang WU Zhihong GU Qianqun LI Yuezhong 《Journal of Ocean University of China》 SCIE CAS 2012年第3期375-382,共8页
Bacteria of the genus Flammeovirga can digest complex polysaccharides (CPs), but no details have been reported regarding the CP depolymerases of these bacteria. MY04, an agarolytic marine bacterium isolated from coast... Bacteria of the genus Flammeovirga can digest complex polysaccharides (CPs), but no details have been reported regarding the CP depolymerases of these bacteria. MY04, an agarolytic marine bacterium isolated from coastal sediments, has been identified as a new member of the genus Flammeovirga. The MY04 strain is able to utilize multiple CPs as a sole carbon source and grows well on agarose, mannan, or xylan. This strain produces high concentrations of extracellular proteins (490 mg L-1 ± 18.2 mg L-1 liquid culture) that exhibit efficient and extensive degradation activities on various polysaccharides, especially agarose. These proteins have an activity of 310 U mg-1 ± 9.6 U mg-1 proteins. The extracellular agarase system (EAS) in the crude extracellular enzymes contains at least four agarose depolymerases, which are with molecular masses of approximately 30-70 kDa. The EAS is stable at a wide range of pH values (6.0-11.0), temperatures (0-50℃), and sodium chloride (NaCl) concentrations (0- 0.9 mol L-1). Two major degradation products generated from agarose by the EAS are identified to be neoagarotetraose and neoagarohexaose, suggesting that β-agarases are the major constituents of the MY04 EAS. These results suggest that the Flammeovirga strain MY04 and its polysac-charide-degradation system hold great promise in industrial applications. 展开更多
关键词 Flammeovirga polysaccharide degradation extracellular agarase system neoagaro-oligosaccharide
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