Clonging, Expression And Properties Of Aromatic Aminotransferase From Lactobacillus Paracasei

Phenyllactic acid (PLA) is a novel antimicrobial compound against a variety offungal pathogens and bacteria. Aromatic aminotransferase transaminated phenylalanineto phenylpyruvic acid (PPA) and PPA further reduced to PLA. Aromaticaminotransferase is the rate-limiting enzyme for PLA formation in the lactic acidbacteria.We designed the primers according to the Aromatic aminotransferase gene of L.casei str. Zhang. The genomic DNA of L. paracasei W2was used as the template toamplify the Aromatic aminotransferase gene, the gene was then cloned into pMD19-Tvector. The results showed that the gene sequence is1164bp. Compared with Aromaticaminotransferase from L. casei str. Zhang, the homologous rates of gene sequences were100%.The structure and characteristics of Aromatic aminotransferase from L. paracaseiW2were analyzed and predicted, in order to guide the experimental research for itsbiological function and application. The hydrohobicity analysis, phospholyration sitesprediction, secondary and tertiary structure prediction, multiple sequences alignmentsand the phylogenetic tree were carried out to the sequence by bioinformatics sofewarepackage. The results showed that the gene coding for387amino acids, molecu1arweight of Aromatic aminotransferase was predicted to be41961.2Da and its isoelectricpoint was6.03. The coding protein was demonstrated to have a complete conserveddomain, conserved functional sites and two trans-membrane regions in the deducedamino acid sequence. Aromatic aminotransferase gene from Lactobacillus paracaseihas high homology with Aromatic aminotransferase gene from Lactobacillus casei.The gene was ligated to the expressed plasmid and transferred into E. coliBL21(DE3), the specific fusion protein was expressed with IPTG. The molecular weightof Aromatic aminotransferase was42kD shown by SDS-PAGE. The activity ofAromatic aminotransferase was determined at pH6.8,50℃, and thermal stability was well under50℃. Fe3+and Cu2+showed a strong inhibitory effect on Aromaticaminotransferase, whereas Mn2+, Ca2+, Mg2+and Zn2+stimulated the activity ofaminotransferase. The Km value for Aromatic aminotransferase was0.0734mol/L andthe Vmax was1.976μmol/min using Glu. The Km value for Aromatic aminotransferasewas0.2038mol/L and the Vmax was1.145μmol/min using Asp.