Directed Evolution Of Atrazine Chlorohydrolase And Cause Analysis Of AtGSTZ Inclusion Body Ablation

Directed evolution is a general term used to describe using various techniques for gene manipulation and selection of desirable RNA, protein and expression element. Over the last three decades, directed evolution has emerged as a powerful technology platform in protein engineering. This technology has been advanced considerably by the availability of molecular biology tools and emerging high-throughput screening technologies. These methodologies have simplified the experimental processes and facilitated the identification of mutants with even small improvements in desired function. In this paper, we first used directed evolution on Atrazine chlorohydrolase (AtzA).Atrazine chlorohydrolase has attracted widespread interests as it catalyzes the toxic atrazine to non-toxic hydroxyatrazine and can be used in the biodegradation of atrazine. For this purpose, directed evolution was performed on AtzA to obtain favorable mutants with higher enzyme activity, thus providing materials for atrazine biodegradation. Unfortunately, AtzA was expressed as inclusion body in E. coli, which limits the application of E. coli in screening. To overcome this bottleneck, a Haematococcus pluvialis-based method was applied to screen AtzA mutants from a random mutagenesis library. Eight mutants with enhanced enzyme activity were successfully obtained and their values of specific activity and kinetic parameter were measured. All of them showed2.7-to5.0-fold increase in specific activity and2.5-to4.1-fold increase in catalytic efficiency (kcat/Km) compared with the wild-type. Sequencing revealed that the two mutants that increased the most (10-7,21-1), contained single substitution at Val12and Leu395, respectively, while several improved mutants contained substitutions at four sites of Met315, His399, Asn429and Val466simultaneously, indicating that these residues contribute to the enzyme activity of AtzA. The modeled three-dimensional structure showed that AtzA was comprised of a typical (β/α)8domain of the amidohydrolase superfamily and a dual β-sheet domain. Ligand binding prediction showed that the iron ion and5ligand-binding residues were located in β-barrel core of the (β/α)8domain. The distribution of the substitution sites and hydrogen bond analysis revealed that some substitution sites involved in hydrogen formation in the (β/α)8-neighbouring β-sheet had great impact on the AtzA function.When overexpressed in Escherichia coli (E. coli), the recombinant proteins usually aggregate into inactive inclusion bodies. This bottleneck seriously restricts the application of the technique in commercial production of protein for bio-pharmaceutical interest, and also sets up a considerable barrier in mutant screening and determination of enzyme activity of AtzA. In order to explore the mechanism underlying the inclusion body formation and its relation with the protein structure, and to be able to have a better understanding of the Glutathione S-transferase (GST) which can be also used to degrade the atrazine in the environment, mechamism of inclusion body were explored by Arabidopsis thaliana Zeta Class Glutathione iS-transferase(AtGSTZ) whose crystal structure had been analyzed. Error-prone PCR (EP-PCR) and DNA shuffling were performed on4insoluble mutants (ISMs)(S73F, S73L, S73K, and S73R) of Arabidopsis thaliana Zeta Class Glutathione S-transferase. Eleven soluble mutants (SMs) were screened out from the random mutagenesis library and their values of specific activity and kinetic parameter were also determined. Among them,5were derived from S73R,3from S73K,2from S73L and1from S73F. All these mutations were amino acid substitution, while no deletion and duplication was observed. The solubility of the multi-site and single-site substitution mutants reached as high as80%and63%, respectively. The comparison of mutants and wild-type AtGSTZ in three-dimension structure revealed that changes of protein structure, such as the formation of "crack" or "indentation" around redisue73, and surface charge distribution, are two important reasons for the formation or ablation of inclusion bodies in AtGSTZ. Furthermore, kinetic analysis indicated that the residue164was also involved in substrate binding, while residue140or residue160was involved in enzymatic catalysis.