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This research investigated the aspartate kinase (AK) of Corynebacterium pekinense (CpAK). The amino acid residue A297, which is closely related to feedback inhibition, was selected for site-directed mutagenesis, high-throughput screening, and enzyme kinetic analysis. In this research, the enzyme activity of mutant A297K AK was enhanced. The maximum reaction rate of A297K AK increased by 9.55 times compared with that of the wild-type CpAK (WT AK). In addition, Km value increased and n value decreased. The enzymologic properties are characterized as follows: optimum reaction temperature increased from 25°C to 30°C; the optimum pH remained to be 8.0; and the stability of A297K AK under the optimal conditions extended from 3.5 h to 5 h. Feedback inhibition of low concentrations of threonine and lysine and the synergistic feedback inhibition of both amino acids were successfully relieved. Introduction Aspartate kinase (AK) is the first ratelimiting enzyme [1. 2] of amino acids of the aspartate family during microbial fermentation. Feedback inhibition by the end-product of the aspartate pathway leads to low yield of amino acids, especially methionine, of the aspartate family [3]. Fermentative production of methionine remains unachievable [4], and the use of microbial fermentation to produce methionine is currently a widespread concern. The AK gene of Corynebacterium glutamicum (CgAK) is used as template for the AK gene of Corynebacterium pekinense (CpAK), which is our research object. The amino acid sequences of CpAK and CgAK are 99% similar, and these enzymes demonstrate similar feedback inhibition mechanism [5]; research development on CpAK relies on the crystal structure of CgAK [6. 7]. This study aims (1) to eliminate or weaken the synergistic feedback inhibition of end-product of the aspartate pathway by using CpAK [8], to obtain a high-dynamic CpAK, (2) to provide a reference for microbial fermentation employed to yield methionine [9], and (3) to provide insights into structural transformation of proteins by exploring the mechanism of synergistic feedback inhibition. Materials and Methods Materials Plasmid extraction kit was purchased from Sangon; PCR extraction kit was purchased from TaKaRa; Dpni digestive enzymes was purchased from Fermentas; Non-denaturing nickel column was purchased from GE; SDS was purchased from Sigma; PVDF Membrane was purchased from BIO-RAD; Denatured protein electrophoresis Marker was purchased from TaKaRa; Nondenaturing electrophoresis Marker was purchased from Life Technologies. International Conference on Biomedical and Biological Engineering (BBE 2016) © 2016. The authors Published by Atlantis Press 28 Application of Bioinformatics Methods to Determine The Mutation Site By using the CgAK crystal structure 3aaw as template, we selected one of the binding sites for threonine inhibitors, and the highly conserved residue A279 was selected for subsequent investigations following multiple sequence alignment. The inhibitors of CpAK feedback inhibition were removed through saturated site-directed mutagenesis of the A297 site of CpAK. Site-Saturation Mutagenesis The design primer Primer 5.0 was used to induce mutation during PCR amplification. The PCR products were electrophoresed on 1% agarose gel to verify whether the target gene was considerably amplified. Primers:5'-GCACGGAAAACCTTCGCNNNCTCGCCTGGC-3';5'GATAAGCCAGGCGAGNNNGCGAAGGTTTTC-3' Sequencing and High-Throughput Screening of Mutants A successful transformation A single colony was transferred into 96-well plates for activation and induction of fermentation. Screening activity was conducted to determine significantly improved mutants for sequencing. Native-PAGE, SDS-PAGE and Western Blot Verification Native-PAGE was used to verify the molecular weight of the intact protein. SDS-PAGE was employed to verify whether the structure of CpAK and CgAK is similar to a known structure and whether both structures contain α and β subunits. Western blot analysis was conducted to determine the molecular weight of the recombinant protein. Kinetic Analysis of AK Ten experimental groups were investigated in AK enzyme kinetics research. The final concentration of L-aspartic acid substrate was adjusted to 0.5, 1, 3, 5, 7, 9, 10, 12, 14, and 16 mmol/L. In each group, a control experiment (without substrate) and three parallel experiments were performed. The specific activity of AK in each group was measured and calculated. Data on kinetic parameters of AK protein were analysed before and after mutation using nonlinear analysis combined with the tendency of respective results and the Hill formula, n max n n V=V ) / ( (S K +S ) . Enzymatic Properties of AK Optimum temperature: Eight experimental groups were reacted for 30 min at 15, 20, 25, 26, 28, 35, 40, 30, 45 and 50°C. Optimum pH: Under optimum temperature, eight experimental groups were separately reacted for 30 min at pH 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0. The maximum relative enzyme activity of the experimental group is set to 100%. Thermal stability: The enzymatic activity of AK was determined by adding 5 mL of the reaction system into the enzyme solution every 1 h. Any change was recorded. The relative enzyme activity determined at 0 h in water bath was set as 100%. Effects of substrate inhibitors: The final concentrations of inhibitor in seven experimental groups were 0.2, 1, 5, and 10 mmol/L). The relative enzyme activity in the absence of inhibitor was set to 100%. Three parallel groups were prepared for each concentration. Results and Discussion Site Selection of A297 This study used the 3D crystal structure of CgAK as template. Pymol is used to identify amino acid residues near T501 and around the inhibitor (Fig. 1A). One of the highly conserved sites (i.e. A297) after multiple sequence alignment and bioinformatics analysis was selected as research object (Fig.

Exploration and Research on Relieving Feedback Inhibition for Aspartate Kinase form Corynebacterium Pekinense by using Site-Saturation Mutagenesis and High Throughput Screening