Site Directed Mutagenesis And Enzymology Properties Characterization Of Mutant Strains From Aspartokinase In Corynebacterium Pekinense

Aspartokinase (AK) is the first key enzyme in biosynthetic pathway of aspartate familyamino acids, catalyzing the substrate L-aspartic acid and ATP generating aspartyl-β-phosphateand ADP, but its activity is feedback inhibited by metabolite products of branch pathways. Inthis study, on the basis of Corynebacterium pekinense AK gene cloned successfully andrecombinant plasmid pET-28a-AK achieved heterologous expression in Escherichia coli (E.coli). The residuals associated with the AK inhibitor binding sites were carried out sitesaturation mutagenesis, by high throughput screening (HTS) method to gain AK mutant strainsof high activity. Intenting to deregulate the feedback inhibition of AK in the biosyntheticpathway of aspartate family amino acids, and to detect the enzymatic propertiescharacterization of mutant strains, laying the foundation of constructing genetically engineeredbacteria of aspartate family amino acids.1. By blast sequence alignment, amino acid sequence homology of Corynebacteriumpekinense AK and Corynebacterium glutamicum AK is99%, guessing that they have similarspatial structure and regulatory mechanisms. Through multiple sequence alignment determiningCorynebacterium pekinense T361site and A362site are conserved sequences, and fullycorresponded Corynebacterium glutamicum T361site and A362site. In addition, using DS3.5software found that T361site connected to the inhibitor lysine through hydrogen bonds, A362site connected to the inhibitor lysine by a water molecule forming hydrogen bonds, and thepolar interaction made the combination of lysine and AK more stable, enhancing feedbackinhibition of lysine to AK. Therefore, the crystal structure of Corynebacterium glutamicum AK3AAW was used as a template，T361and A362were determined to conduct site saturationmutagenesis, respectively.2. Through high throughput screening method, mutant strains that the AK enzyme activityimproved significantly were carried out expanding culture, by sequencing verifying, threemutant strains were obtained, they were T361K, T361N, and A362I. Ultrasonication and thenon-denatured nickel column were utilized to purify AK, by SDS-PAGE and Western blotverifying AK, the results showed that protein molecular weight of AK was48KDa, proving thatAK protein of mutant strains expressed in E. coli successfully.3. The purified AK of wild type and mutant strains made enzyme kinetic analysis, gainingAK enzyme kinetics curves of wild-type and mutant strains and nonlinear fitting was carried out as the Hill equationV=Vm ax(Sn)/(K n+Sn), the results showing that the Vmax of T361K,T361N and A362I increased10.34times,47.99times, and34.60times compared to Vmax ofwild type(99.74U/mg). n value of wild-type2.93was greater than1, showing that AK was atypical allosteric enzyme and had positive cooperative effect. N value of T361K, T361N andA362I decreased compared to n value of wild type, indicating that the positive cooperativeeffect of the mutant strains reduced, T361N AK and A362I AK tend to be Michaelis enzymes.Compared to Km value of wild-type, Km values of T361K and A362I decreased, indicating thataffinity of T361K AK and A362I AK with substrate increased.4. AK enzymatic properties characterization of T361K, T361N, A362I and wild type wereperformed. The results showing that the optimum pH of T361K rised to8.5, compared with theoptimum pH8.0of wild-type, the optimum pH of T361N decreased to7.0, the optimum pH ofA362I was not changed. The optimum temperature of T361K went up to35℃compared to thethe optimum temperature26℃of wild type, the optimum temperature of T361N was constant,the optimum temperature of A362I increased to28℃. The half-life of T361K and wild-type was4h, the half-life of T361N extended to7h, the half-life of A362I enhanced to6h. Compared towild type, T361K and T361N show resistance against metalions and organic solvents,particularly T361K show good resistance against different concentrations of metalions. A362Ishowed good resistance against inhibitors Lys, Met, Thr+Met, Lys+Met and Thr+Lys+Met,with in the scope of the test concentrations, the feedback inhibition of lysine to AK wasderegulated, and concerted feedback inhibition of threonine and lysine on AK reduced, that isgreat significance to excessive accumulation of aspartate family amino acids.