Rational Modification Of Aspartase And Its Application In β-aminobutyric Acid Synthesis

β-Aminobutyric acid(BABA)is a four-carbon non-protein amino acid,which is widely used in anticancer drugs,AIDS drugs,penicillene antibiotics and plant initiators.There are few domestic researches on the biological preparation of β-aminobutyric acid.The current preparation methods mainly include chemical methods and enzymatic methods.However,its efficient,economical and environmentally friendly production still faces challenges.Aspartase is the only β-aminobutyric acid producing enzyme reported so far.Aspartase(AspB)exhibits higher enzyme activity in alkaline environment and high temperature environment,but for biological enzymatic industrial production,the reaction conditions are harsh,and the reaction environment of strong alkali is bound to bring high cost.In this paper,through rational design and modification of the surface potential distribution of aspartase,a mutant enzyme with higher activity and optimal pH change was obtained,which was efficiently expressed in Escherichia coli BL21(DE3),and the substrate crotonic acid was transformed toβ-Aminobutyric acid.(1)In this study,starting from the existing research on the aspartase gene that can synthesize β-aminobutyric acid,the aspartase gene derived from YM55-1 was synthesized and constructed on the expression vector pET21a of Escherichia coli BL21(DE3).After heterologous expression,it was verified by SDS-PAGE that the protein was successfully and efficiently expressed.After purification of the obtained protein,it was used for temperature and pH enzymatic testing.It was found that the optimum temperature of the enzyme was 60℃ and the optimum pH was 9.0.It shows that the reaction conditions were harsh,unsuitable for cell transformation and resource consuming,so it is necessary to modify to change its optimum pH.(2)Taking the above-mentioned aspartase as the object,it is designed rationally with ROSETTA software.The structure of the enzyme was analyzed by Supercharge,and the basic amino acids on the enzyme surface were mutated to acidic amino acids,and 10 mutation points were designed:L12D,K19E,N87E,N125D,S133D,Y166E,Q169E,Q262E,Q449D,and N451E.Using the synthetic gene as a template,the first round of mutation of a single site and the second round of mutations of double sites are added up to obtain the final mutant enzyme K19E-N87E-N125D-S133D-Q262E-N451E,and the resulting mutants are constructed into plasmid pET21a and heterologous expression in E.coli BL21.(3)Purify the expressed enzymes of each mutant strain,perform enzyme properties determination and molecular dynamics analysis,compare the changes in temperature,pH,and kinetic parameters of the original enzyme and the mutant enzyme,and determine the effects of metal ions and other factors on the enzyme activity influences.The results showed that the optimal pH of the six-site mutant K19E-N87E-N125D-S133D-Q262E-N451E was shifted to 8.0,and the pH stability and thermal stability of the enzyme were good.Cu2+has an obvious inhibitory effect on the enzyme activity,while Mg2+has a slight promotion effect on the enzyme activity.(4)Optimize the whole cell transformation conditions of recombinant strain E.coli BL21/pET21a-K19E-N87E-N125D-S133D-Q262E-N451E and the original strain,and carry out the whole cell transformation of 1 L system in a 5 L fermentor,adding 500 g of substrate croton acid.The β-aminobutyric acid products were 556.1 g·L-1 and 393.3 g·L-1,respectively,and the recombinant strain were 1.41 times the original strain.