Characteirzation And Construction The Engineered Strain Secreted D-aminoacylase From Alcaligenes A-6

D-valine, D-2-Amino-3-methylbutyric acid, an important material for agriculturalchemicals, 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 isusing enzymatic or chemical asymmetric synthesis, another method is carried out by externalchiral separation after chemical racemic. N-acyl-D-amino acid amidohydrolase-catalyzedchiral 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 generallythe substrate D-Met, D-Leu D-Phe and N-acyl. But these aminoacylase catalyzed acylderivatives of D-valine is not efficient. Using D-valine as a measure, Alcaligenes genussources D-aminoacylase have the highest activity.The codons of D-ANase gene from Alcaligenes A-6were substituted by the codonsabundant in E. coli. And then the D-ANase gene was synthesized by the two-step methodbased on PCR technology. Synthetic gene and pET-32a vector were digested with Bgl II andXho I, ligated by T4DNA ligase. The ligation mixture transformed into E.coli BL21(DE3)competent cell.Recombinant protein was detected by SDS-PAGE、Western-blot and activityassay. D-ANase can be expressed efficiently in E. coli and the expressed protein content canreach to64%of the total bacterial protein content. In addition, the fermentation activity canachieve96U/mL. After removing the fusion tag, the expressed protein content can reach to53%of the total bacterial protein content, the codon optimized D-ANase expressed directlyby T7promoter got up to52U/mL in the fermentation activity, and the codon un-optimizedD-ANase only got39U/mL in the fermentation activity.The secondary structure around the initiation codon in the translational-initiationregion(TIR) of the mRNA can strongly influenced the translational efficiency in E. coli.Theminimal folding free energy (ΔG) of a local hairpin structure was found to be linearlycorrelated with the relative expression level. In this study, the relationship between stabilityof the secondary structure surrounding the TIR and D-ANase expression levels in E. coli has been investigated. According to ΔG value, we picked up three groups of different secondarystructure of TIR in pET-dan, and surveying D-ANase expression levels and activity. Theresults show that the lower group of ΔG value of TIR has better D-ANase expression levelsand activity than higher group.Further enzymatic properties studies showed that, D-ANase from Alcaligenes A-6actspreferentially on N-acyl derivatives of D-Ala, D-Met, D-Phe, D-Leu, D-Trp and D-Val.Whenthe N-acetyl-DL-valine concentration is10mmol/L, the reaction temperature is55℃, pH isfrom7.0to8.0, the D-ANase have the highest catalytic efficiency. After immobilizationtreatment, the D-ANase have the broader ph and thermal stability. After ten consecutivebatches split N-acetyl-DL-valine, the enzyme activity maintain rate is78%. Furthermore afirm foundation was laid for the industrial use of the D-ANase.