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Directed Evolution of Atrazine Chlorohydrolase (TrzN)

Author: QiuZuo
Tutor: CuiZhongLi
School: Nanjing Agricultural College
Course: Microbiology
Keywords: atrazine chlorohydrolase(TrzN) directed evolution Error prone PCR reverse mutation site-directed mutagenesis saturation mutagenesis
CLC: TQ925
Type: Master's thesis
Year: 2011
Downloads: 40
Quote: 1
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Abstract


TrzN is a novel s-triazine hydrolase, it has similar function as AtzA with a broader substrate spectrum. However, most of the TrzN enzyme expressed was found in inclusion bodies and largely inactive. The aim of this paper is to improve soluble expression and the catalytic activity using directed evolution.The two important steps of directed evolution are the generation of molecular diversity and the building of high throughput screening method. Error prone PCR is used to generate gene diversity and vector pRBB is used to create the mutants library. The screening strategy used in this study is based on the diameter of the halos around colonies, which is formed by hydrolyzing atrazine. Larger halo represents higher catalytic activity, or increased quantity of enzyme. Therefore, after incubation at 37℃overnight, clones which show larger halo dimension/colony dimension than TrzN(wild) were selected and_verified further by measuring the enzymes activity. We got a mutant strain named MuA-55, which had 2.5 folds catalytic activity of TrzN(wild) and high soluble expression. DNA sequence analysis reveals that MuA-55 contains one amino acid transition (G190S) and two synonymous mutations that lead to higher level of Codon Usage. Ser has one hydroxyl group than Gly in sidechain, which increase the hydropHilicity of protein and improve the capacity of solubility expression, we also got a mutant strain named MuE-118,which had low catalytic activity than TrzN (wild). DNA sequence analysis reveals that MuE-118 contains one amino acid transition that same to MuA-55 and four synonymous mutations.We used the methods of reverse mutation, site-directed mutagenesis and saturation mutagenesis to explain the differences between MuA-55 and MuE-118. The result shows that the causes of the differences are both of amino acid mutations and Codon Usage change. We also reversely muted one null mutant strain MuF-212.The result shows that the main reason of inactivity should be attributed to 126th amino acid substitution(V126A), near to active center.We preliminarily compare the enzymatic properties of MuA-55, MuE-118 and TrzN(wild) to explain the differences between MuA-55 and MuE-118. The result reveals that they all exhibited highest activities at pH 4.8 and 50℃. Through comparing of their optimum pH, we found that wide range of pH adaptation of MuA-55 and MuE-118 should be attributed to 126th amino acid substitution. The Experiment of effects of metal ions on TrzN(wild) and MuA-55 activity shows that Zn2+ and Co2+ could promote the activity, Cr2+ and Ni2+ could inhibit the activity, the activity was not affected by supplementation with Li+, Mg2+ and K+To summarize, we obtained a TrzN mutant MuA-55 with increased catalytic activity and solubility expression, and wide range of pH adaptation by directed evolution.

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