Font Size: a A A

Molecule-Directed Evolution And Site-Directed Mutagenesis Of D-Hydantoinase

Posted on:2007-05-18Degree:MasterType:Thesis
Country:ChinaCandidate:R KongFull Text:PDF
GTID:2120360185450998Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
D-amino acids are widely used as intermediates for the synthesis of semisynthetic antibiotics, peptide hormones, pyrethroids and pesticides etc. Nowadays, the production of D-amino acids mostly relies on enzymatic conversion in the industrial field. The process is that 5'-monosubstituted hydantoin is firstly transferred into the relevant intermediate, N-carbamy-D-amino acid by D-hydantoinase and the resulting N-carbamy-D-amino acid is further converted to the corresponding D-amino acid by N-carbamyolase or inorganic acid. Therefore, D-hydantoinase is regarded as a key catalyst in the bioconversion of D-amino acids.Indeed, all of enzymes are sensible by pH, temperature, oxidant and so on, resulting in decreasing or losing their activity. To explore the better character of D-hydantoinase-producing strain, a method of the molecule-directed evolution in vitro was undertaken. The random mutation of D-hydantoinase gene was introduced by Error- prone PCR. Based on conditional tests, three pairs of ions were chosen, i.e. 1.5mmol/L Mn2+ 3mmol/L Mg2+; 0.3mmol/L Mn2++ 3mmol/L Mg2+; 0.5mmol/L Mn2+ +2.5mmol/L Mg2+. PCR products were separately cloned into vector pET-3a and constructs were transformed into E.coli BL21 (DE3). The positive mutant was screened on a 96-well microtiter plate by using enzyme activity. Two mutants with higher activity, E.coli BL21/ pE-HDT41 (A) and E.coli BL21/ pE-HDT45 (B), were achieved in about 1000 colony. It was shown from the enzymatic activity that the strain A was 2.7-fold and 20-fold as well as the strain (B) was 2.0-fold and 15-fold, compared with the starting recombinant strain (E.coli BL21/ pE-HDT) and wild-type strain (Pseudomonas putida YZ-26) with p-hydroxyophenylhydantoin as the substrate respectively. DNA sequence analysis indicates that the enzyme gene in the strain (A) belongs to the internal mutation by changing the Glu codon (GAA→GAG) and the Asp codon (GAT→GAC ), whilst the gene in...
Keywords/Search Tags:D-hydantoinase, directed evolution in vitro, error-prone PCR, site-directed mutagenesis, enzymatic activity
PDF Full Text Request
Related items