Effect Of Codon Selection On Expression Of D-aminoacylase In TIR Region

D-valine,D-2-Amino-3-methylbutyric acid,an important material for agricultural chemicals,antibiotics and physiological active peptide,is widely used in the fields of biology,physic and so on.There are two main methods for preparing D-amino acids,one method is using enzymatic or chemical asymmetric synthesis,another method is carried out by external chiral separation after chemical racemic.N-acyl-D-amino acid amidohydrolase-catalyzed chiral preparation is the main way to produce D-type amino acids.For different substrates,D-ANase catalytic rate vary greatly from different sources,the optimum effect is generally the substrate D-Met,D-Leu D-Phe and N-acyl.But these aminoacylase catalyzed acyl derivatives of D-valine is not efficient.Using D-valine as a measure,Alcaligenes genus sources D-aminoacylase have the highest activity.The codons of D-ANase gene from Alcaligenes A-6 were substituted by the codons abundant in E.coli.And then the D-ANase gene was synthesized by the two-step method based on PCR technology.Synthetic gene and pET-32 a vector were digested with Bgl II and Xho I,ligated by T4 DNA ligase.The ligation mixture transformed into E.coli BL21(DE3)competent cell.Recombinant protein was detected by SDS-PAGE?Western-blot and activity assay.D-ANase can be expressed efficiently in E.coli.The secondary structure around the initiation codon in the translational-initiation region(TIR)of the mRNA can strongly influenced the translational efficiency in E.coli.The minimal folding free energy(?G)of a local hairpin structure was found to be linearly correlated with the relative expression level.In this study,the relationship between stability of the secondary structure surrounding the TIR and D-ANase expression levels in E.coli has been investigated.According to ?G value,we picked up three groups of different secondary structure of TIR in pET-dan,and surveying D-ANase expression levels and activity.The results show that the lower group of ?G value of TIR has better D-ANase expression levels and activity than higher group.Furthermore a firm foundation was laid for the industrial use of the D-ANase.