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Kinetics and function of two mutants of CLA hydrase from Lactobacillus plantarum p-8.
FEMS Microbiology Letters ( IF 2.2 ) Pub Date : 2020-06-19 , DOI: 10.1093/femsle/fnaa087
Wei Zhao 1 , Lili Zhao 1 , Tongtong Zhao 1 , Chao Zhi 2 , Meiqi Liu 1 , Abdul Jamil Khan 1 , Qiuhua Bao 3 , Heping Zhang 3 , Yuzhen Wang 1 , Feng Zhang 1 , Guofen Zhao 1
Affiliation  

In this work, the gene of conjugated linoleic acids hydrase (CLA-HY) was cloned from the L. plantarum p-8, and the protein of CLA-HY was expressed in the E. coli BL21. GC-MS was employed to verify that the purified CLA-HY can convert Linoleic Acid (LA) into 10-hydroxy-cis-12-octadecenoic acid (10-HOE) in the presence of FAD. And the optimal pH and temperature for maximizing CLA-HY catalytic activity were discovered as 6.0 and 35 °C, respectively. In addition, the catalytic ability of CLA-HY can be inhibited by plenty of cations such as Mg2+, Mn2+, Zn2+, Cu2+, Fe2+, Fe3+, Ni2+ and Ca2+. Finally, the Km, Vmax, Kcat and Kcat/Km of CLA-HY were determined as 7.62 mM, 2.59 mM h−1, 8.33 × 103 h−1 and 1.09 × 103 mM−1 h−1, respectively. Moreover, both M76 and G74 residues played significant roles on catalyzing LA to 10-HOE using the site-directed mutation technology and molecular simulations.

中文翻译:

植物乳杆菌p-8的两个CLA水合突变体的动力学和功能。

在这项工作中,从植物乳杆菌p -8克隆了共轭亚油酸水合酶(CLA-HY)的基因,并在大肠杆菌BL21中表达了CLA-HY的蛋白质。使用GC-MS验证纯化的CLA-HY在FAD存在下能否将亚油酸(LA)转化为10-羟基-顺式-12-十八烯酸(10-HOE)。最大化CLA-HY催化活性的最佳pH和温度分别为6.0和35°C。另外,Mg 2 +,Mn 2 +,Zn 2 +,Cu 2 +,Fe 2 +,Fe 3+,Ni 2+和Ca 2+等大量阳离子可抑制CLA-HY的催化能力。。最后,K个,V最大,K和K/ KCLA-HY的确定为7.62毫米,2.59毫米高-1,8.33×10 3 ħ -1和1.09×10 3毫米-1 ħ - 1。此外,使用定点突变技术和分子模拟,M76和G74残基均在催化LA生成10-HOE方面发挥重要作用。
更新日期:2020-06-19
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