Overexpression of Pseudomonas putida catechol 2,3-dioxygenase with high specific activity by genetically engineered Escherichia coli.
Overexpression of Pseudomonas putida catechol 2,3-dioxygenase with high specific activity by genetically engineered Escherichia coli.
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基因工程大肠杆菌过表达具有高比活性的恶臭假单胞菌儿茶酚 2,3-双加氧酶。
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发表时间：
1995
期刊：
影响因子：
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通讯作者：
M. Nozaki
中科院分区：
文献类型：
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作者：
T. Kobayashi;T. Ishida;K. Horiike;Y. Takahara;N. Numao;A. Nakazawa;T. Nakazawa;M. Nozaki
从恶臭假单胞菌mt-2的tol质粒中克隆了编码邻苯二酚2，3-双加氧酶(间苯二酚-2，3-双加氧酶)的XylE基因，该基因在大肠杆菌W3110中的表达水平约为总可溶性蛋白的15%。在粗提物中的总铁中，45%是在酶上。与恶臭假单胞菌Mt-2相比，大肠杆菌结晶酶具有更高的铁含量(3.7m o l/m o l酶)和更高的比活力(536U/mg)。然而，两种酶在理化性质、蛋白质化学性质和动力学性质上没有差异。在0.36~2.8微米的浓度范围内，酶的M(R)(136,000+/-5,000)和斯托克斯半径(4.26 nm)没有变化，说明该天然酶既没有解离成亚基，也没有在此范围内聚合。在pH 7.5和25℃时，邻苯二酚和氧气的催化中心活性和Km值分别为278 S-1，1.87和7.45微米。该酶具有广泛的底物专一性。在底物中，4-甲基儿茶酚和4-氯邻苯二酚的特异性常数比邻苯二酚高约200微米-1.s~(-1)。丙酮和苯酚衍生物对邻苯二酚有竞争性抑制作用。比活力与铁含量之间的关系不是线性的，这表明部分缺铁的酶发生了构象变化。
The cloned xylE gene encoding catechol 2,3-dioxygenase (metapyrocatechase) from TOL plasmid in Pseudomonas putida mt-2 has been expressed in Escherichia coli W3110 to a level of approximately 15% of the total soluble protein. Of the total iron in the crude extract, 45% was on the enzyme. The crystallized enzyme from E. coli had higher iron content (3.7 mol/mol enzyme) and specific activity (536 U/mg) than the enzyme from P. putida mt-2. However, no differences were observed in physicochemical, protein-chemical, and kinetic properties between the two enzymes. The enzyme was a homotetramer, and no changes were observed in the values of M(r) (136,000 +/- 5,000) and Stokes radius (4.26 nm) in the concentration range from 0.36 nM to 2.8 microM, indicating that the native enzyme neither dissociated into subunits nor polymerized in this range. The catalytic center activity and the Km values for catechol and dioxygen were 278 s-1, 1.87 and 7.45 microM, respectively, at pH 7.5 and 25 degrees C. The enzyme showed a broad substrate specificity. Among substrates, 4-methylcatechol and 4-chlorocatechol showed specificity constants (approximately 200 microM-1.s-1) higher than that for catechol. Acetone and phenol derivatives competitively inhibited the activity against catechol. The relationship between specific activity and iron content was not linear, suggesting some conformational changes in the partially iron-depleted enzyme.