Cloning,Expression Of Aromatic Amino Acid Ammonia Lyases And Its Application In Synthesis Of Drugs

Aromatic amino acid ammonia lyase include phenylalanine ammonia lyase(PAL),tyrosine ammonia lyase(TAL)and histidine ammonia lyase(HAL).Under natural conditions,the aromatic amino acid ammonia lyase undergoes a deamination reaction to hydrolyze the amino group of the aromatic amino acid and a reversible reaction may also occur under certain conditions.Phenylalanine,histidine and their analogs can be used as intermediates in various drugs,such as melphalan,immunosuppressants and some antiviral drugs.Herein,a novel histidine ammonia lyase(named as SbHAL)discovered from Streptomyces bottropensis by a genome mining method was cloned and characterized and more applicable substrate for the Anabaena variabilis PAL(AvPAL)was found.Homology model of SbHAL was constructed using Discovery Studio 2.5,then modeled SbHAL docked with coenzyme and histidine.We got the 3D structure of SbHAL and validated the catalytic tetrad.A systematic study of all factors influencing the SbHAL purified protein catalyst activity such as temperature,pH and the effects of metal ions were determined.The optimum enzyme activity was achieved at 30℃and pH 10 and it was shown that 0.1 mM Na+ and Fe2+ obviously promoted the activity of the enzyme,0.1 mM of Zn2+ had a strong inhibitory effect on the activity of the enzyme.Substrate specificity studies showed that this histidine ammonia lyase SbHAL also had high activity on various histidine analogues,and had potential significance and application value for the preparation of antiviral drugs and immunosuppressants.The applicable substrate for the Anabaena variabilis PAL(AvPAL)was further expanded and modified the amino group of phenylalanine and its analogues for the first time.The experimental results showed that after modified the amino group of phenylalanine and its analogues,the enzyme still had a high activity,which provided a possibility for the synthesis of drugs by new ways.