Molecular Modification Of L-aspartate-?-decarboxylase By Error Prone PCR

In this thesis,we developed a rapid method for screening of recombinant strains with high L-aspartate-?-decarboxylase activity.Test tube method,plate method,Berthelot method and paper chromatography were used and compared.It was found that paper chromatography can be used to more directly and sensitively detect?-Ala and L-Asp content than other methods.In addition,paper chromatography method is lower-cost,faster and more convenient.All these features allow this method for more effective screening of recombinant strains with high PanD activity.We established the error-prone PCR system of panD with 1.0 mmol/L Mn²⁺,0.12 mmol/L dATP and dGTP,1.0 mmol/L dCTP and dTTP.Forty eight mutants and seven mutants with null and high PanD activity,respectively,were acquired based on two-round screening from panD mutant library with around 2000 clones.The PanD activities of mutants6-83 and 6-85 were 2 fold higher than parent strain.The panD gene in 7mutants with high enzyme activity was sequenced,and mutants 6-85 and6-85 were found to have the same mutation sites.Site directed mutagenesis of panD in parent strain and mutant 6-85 was performed on the mutant sites and revealed that the mutation?A?T?at 114bp-position of the PanD gene can increase the PanD activity.We also found that the transcriptional level and cell concentration of mutant 6-85 is significantly higher than the parent strain.Sequencing of panD in 24 null mutants revealed the amino acid residue change on panD.Site-directed mutagenesis experiments were performed on 7 site of 6 mutants and revealed that both Leu¹⁰?His¹00 and Arg¹²?Leu¹22 mutations have no obvious effect on PanD activity,but Leu¹⁰?Pro¹⁰,Arg¹²?Leu¹22 and His¹¹?Arg¹11 mutations cause the loss of PanD activity.In addition,both Ser²⁵?Pro²55 and Gly²⁴?Ser²44 mutations will probably hinder the shear process of PanD.