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首页> 外文期刊>Molecular biotechnology >Substitutions of Thr-103-Ile and Trp-138-Gly in Amidase from Pseudomonas aeruginosa Are Responsible for Altered Kinetic Properties and Enzyme Instability
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Substitutions of Thr-103-Ile and Trp-138-Gly in Amidase from Pseudomonas aeruginosa Are Responsible for Altered Kinetic Properties and Enzyme Instability

机译：铜绿假单胞菌酰胺酶中Thr-103-Ile和Trp-138-Gly的取代负责动力学特性和酶的不稳定性

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摘要

Pseudomonas aeruginosa PhI is a mutant strain derived from strain A13. The strain AI3 is able to use acetanilide as a carbon source through a mutation (TI031) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thrl03lle and Trpl38Gly) by direct sequencing of PCR-amplified mutant gene from strain Phi and confirmed by sequencing the cloned PCR- amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wi.ld-type and mutant amidase genes (WI38G, TI031-WI38G, and T1031) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed sev- eral differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T1031) amidase acted only on p-nitrophenylacetamide and phenyl acetamide. On the other hand, the T1031 enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermo- stable than the wild-type enzyme, as the mutant (W138G and W138G- T1031) enzymes exhibited tl/2 values of 7.0 and 1.5 min at 55°C, respectively. The double substitution T1031 and W138G on the amidase mol- ecule was responsible for increased instabi.liby due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its Mr value and monoclonal antibodies reacted differently with the active and inactive T1031-W138G amidase.
机译：铜绿假单胞菌PhI是衍生自菌株A13的突变菌株。菌株AI3能够通过amiE基因中的一个编码脂肪族酰胺酶（EC 3.5.1.4）的突变（TI031）将乙酰胺用作碳源。通过对来自菌株Phi的PCR扩增的突变基因进行直接测序，已经鉴定了amiE基因中的突变（Thr03lle和Trp138Gly），并且通过对克隆的PCR扩增基因进行测序来确认。使用定点诱变来改变Gly 138位的野生型酰胺酶基因。将wi.ld和突变型酰胺酶基因（WI38G，TI031-WI38G和T1031）克隆到表达载体中，并通过在环氧活化的琼脂糖6B-乙酰胺/苯乙酰胺上的亲和层析，然后进行凝胶过滤层析，纯化这些酶。与野生型酶相比，改变后的酰胺酶显示出动力学特性的一些差异，即底物特异性，对尿素的敏感性，最佳pH和酶稳定性。 W138G酶作用于乙酰胺，丙烯酰胺，苯乙酰胺和对硝基苯乙酰胺，而双突变体（W138G和T1031）酰胺酶仅作用于对硝基苯乙酰胺和苯基乙酰胺。另一方面，T1031酶作用于对硝基乙酰苯胺和乙酰胺。改变的酶的热稳定性表明它们比野生型酶热稳定性差，因为突变型（W138G和W138G-T1031）酶在55℃下的t1 / 2值分别为7.0和1.5分钟。酰胺酶分子上的双取代T1031和W138G造成了不稳定的增加，这是由于单克隆抗体检测到的酶分子构象变化所致。酰胺酶的这种构象变化不会改变其Mr值，单克隆抗体与活性和非活性T1031-W138G酰胺酶的反应也不同。

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