Molecular Modification Of L-aspartate-α-decarboxylase From Corynebacterium Glutamicum By Site-directed Mutagenesis

Site-directed mutagenesis technique, an efficient tool to study relationships betweenprotein structure and function, is an effective experimental method in the field of biology andmedicine. Site-directed mutagenesis method using rational protein design is an effective wayto improve the performance and function of the enzyme, including substrate specificity,enantioselectivity, pH/temperature stability, resistance to organic solvents. In this experiment,recombinant bacterium Escherichia coli BL21(DE3)-pET24a (+)-panD was selected to beresearch strain to explore the possibility of site-directed mutagenesis techniques for positivechanges of L-aspartate α-decarboxylase (ADC) properties.ADC catalyzes the α-decarboxylation of L-aspartate in the major route of β-alanineproduction, it also can be used to chiral separate D,L-aspartic acid. β-Alanine, the onlyβ-amino acid existing in nature, is an important raw material for syntheticism of drugs andother intermediates, such as antioxidants carnosine, calcium pantothenate which is a vitaminB5supplement, bonin which is used to relieve bone dissolving. β-Alanine is also one of therecent evidence-based supplements that have entered the nutraceutical market. Currently, themethods used by the industrial production of β-alanine are mainly concentrated on chemicalconversion. The on-going trends to process improvements, cost reductions and increasingquality, safety, health and environment requirements of industrial chemical transformationshave strengthened the translation of global biocatalysis research work into industrialapplications. Enzymatic conversion method has an advantage of simple process, low energyconsumption, easy purification and less pollution. The research on catalytic properties ofADC will help promote β-alanine green synthesis process.The purified protein ADC was optimal at55°C and pH6.0with excellent stability at16-37°C and pH4-7. Under standard condition (37°C and pH6.0), the enzyme reactionproceeded through a substrate inhibition mechanism and the Kmand KIon L-aspartate were73.7μmol·L-1and11.0mmol·L-1, respectively. ADC catalyzed removal of α-carboxyl groupof L-aspartate, generating β-alanine and carbon dioxide. The gradual volatilization of carbondioxide brought a slow rise of pH as the catalytic proceeded.5g·L-1of L-aspartate could becompletely α-decarboxylated after12-h enzymatic catalysis with the initial pH6.0and finalpH7.2. Keeping reaction system pH-stat at the range of6.0-7.2is propitious to relatively highenzyme activity and conversion rate. ADC was sensitive to pH and the loss of enzyme activityin alkaline pH environment was significant. Substrate inhibition and gradual loss of activity inreaction process also limited the application of this enzyme.The site-directed mutagenesis experiments were carried out to improve the enzymeperformance according to the published three-dimensional structure of ADC protein andamino acid sequences homology analysis combined with virtual mutation by DiscoveryStudio software. Mutants Lys9Ser, Lys9Arg and Ile86Trp were selected from amino acidsrelated with combination sites in catalysis. Mutants Ile5Met and Asp20Asn were selectedfrom non-active sites according to individual amino acids missense mutation methods.Mutants ΔGly65were selected from the loop region according to amino acid deletion mutation method.Non-catalytic site mutation I5M, catalytic site mutation K9S and loop deletion mutationΔG65were of no enzyme activity, indicating that either catalytic site or non-catalytic site hadan effect on protein conformation and function. The changes of binding site mutation I86Wand non catalytic site mutation D19N on enzyme performance were not notable, suggestingthat some single point mutation on protein had no significant effect on the conformation andfunction of protein, thus meaning enzyme has a certain self-stability in structure. The catalyticsite mutation K9R changed the enzyme function, indicating the important changes in thecatalytic site might affect enzyme and substrate interaction. The catalytic site mutation hadimplications for the new development of enzyme function.