Cloning and characterization of a novel amidase from Paracoccus sp M-1, showing aryl acylamidase and acyl transferase activities
Cloning and characterization of a novel amidase from Paracoccus sp M-1, showing aryl acylamidase and acyl transferase activities
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副球菌 M-1 中新型酰胺酶的克隆和表征，显示出芳基酰胺酶和酰基转移酶活性
DOI：
10.1007/s00253-011-3704-6
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发表时间：
2012-05-01
影响因子：
5
通讯作者：
Li, Shunpeng
中科院分区：
文献类型：
--
作者：
Shen, Weiliang;Chen, Honghong;Li, Shunpeng
从Paracoccus sp.克隆了一个新的酰胺酶基因，命名为pamH。M-1。定点突变和生物信息学分析表明，PamH蛋白属于酰胺酶标志酶家族。在大肠杆菌中表达、纯化和鉴定了PAMH。测得PAMH的分子质量为52 kDa，等电点为5.13。PAMH在45A℃、pH 8.0时酶活最高，在pH 5.0-10.0范围内稳定。PAMH酶表现出酰胺酶活性、芳基酰胺酶活性和酰基转移酶活性，使其能够在非常广泛的底物谱上发挥作用。PAMH对芳香族和短链脂肪族酰胺(苯甲酰胺和丙酰胺)有较高的活性，对氨基酸酰胺有中等活性，对脲酶活性较弱。在所考察的苯胺中，只有丙醇是PAMH的良好底物。丙醇的k(CAT)和K(M)分别为2.8mU·S(-1)和158mU·M，催化效率(k(CAT)/K(M))为0.018 mU·M-1 S(-1)。此外，PAMH对于酰胺和苯胺底物，包括乙酰胺、丙胺和4-硝基乙酰苯胺，都能催化酰基转移反应为羟胺，其中异丁酰胺的反应速率最高。这些特性使PAMH成为环境修复的极佳候选者和生物合成新型酰胺的重要酶。
A novel amidase gene, designated pamh, was cloned from Paracoccus sp. M-1. Site-directed mutagenesis and bioinformatic analysis showed that the PamH protein belonged to the amidase signature enzyme family. PamH was expressed in Escherichia coli, purified, and characterized. The molecular mass of PamH was determined to be 52 kDa with an isoelectric point of 5.13. PamH displayed its highest enzymatic activity at 45A degrees C and at pH 8.0 and was stable within a pH range of 5.0-10.0. The PamH enzyme exhibited amidase activity, aryl acylamidase activity, and acyl transferase activity, allowing it to function across a very broad substrate spectrum. PamH was highly active on aromatic and short-chain aliphatic amides (benzamide and propionamide), moderately active on amino acid amides, and possessed weak urease activity. Of the anilides examined, only propanil was a good substrate for PamH. For propanil, the k (cat) and K (m) were 2.8 s(-1) and 158 mu M, respectively, and the catalytic efficiency value (k (cat)/K (m)) was 0.018 mu M-1 s(-1). In addition, PamH was able to catalyze the acyl transfer reaction to hydroxylamine for both amide and anilide substrates, including acetamide, propanil, and 4-nitroacetanilide; the highest reaction rate was shown with isobutyramide. These characteristics make PamH an excellent candidate for environmental remediation and an important enzyme for the biosynthesis of novel amides.