Study On The Expression And Application Of Recombinant Amino Acid Decarboxylase

Amino acid decarboxylation is a kind of amino acid metabolism which can transform amino acid to biogenic amino or small molecule amino acid. Biogenic amine and small molecule amino acid were widely used in medical, food, health products, feed and so on. With the development of molecular biology, enzymatic method has been paid more and more attention. Compaired with chemical synthesis method, enzymatic methods is a valuable process for its rich source substrate, high conversion ratio, mild reaction conditions, cheap cost of production and non-pollution.In this paper the targetgenes (tyrosine decarboxylase, aspartate a-decarboxylase, arginine decarboxylase) were recombinant expressed in E. coli BL21 (DE3) via pET-28a or pET-Duet-1. Use the recombinant enzymes to biotransform L-Tyrosine, L-Aspartate and L-Arginine to tyramine, β-alanine and agmatine.Specifically the following:1. Recombinant expression of tyrosine decarboxylase.The reaction was optimal at pH 5.5 and 40℃. In 10 mL acetate buffer solution, the conversion rate of 0.9 g L-tyrosine can reache 99%.5L fermentation tank was used to explore the conditions of industrialization. The cell growth curve in the fermentation tank was drawed, and the optimal induce time is determined:OD600 was 5.5. By detectingthe plasmid loss rate, we make sure that using pET-Duet 1 as the carrier is more stabler than using pET-28a.2. The two kinds L-aspartate-a-decarboxylase genes (from wild fungi and from synthesized after rare codon optimization) were cloned into pETDuet-1, and expressed in E. coli BL21 (DE3). These engineered strains werw named DM2417 and DM2418 respectively. The recombinant enzymes can biotransform L-aspartic acid to (3-alanine. The reactionswere optimal at pH 7.0 and 37℃. The comparative study of this two engineering strains showed engineering bacterium DM2417 had a higher activity than DM2418.For forming functional L-aspartate-a-decarboxylase, the pro-enzyme protein had to undergo self-cleavage.Our hypothesis is that the DM2418 had a higher expression rate. The expression speed was too fast to make sure all the recombined protein can be formed to the right configuration.3. The arginine decarboxylase gene (adiA) was cloned fromE. coli K-12 MG1655 and expressed in E. coli BL21 (DE3) via pET-28a. The agmatine enzyme was removed by ammonium sulfate precipitation method. The optimized aADC reaction condition was pH 9.0 and 50℃, and stable pH was good at forming agmatine.Compared with the engineering bacterium DM204 which the garget gene is speADC(another gene of ADC in E. coli K-12 MG1655), the aADChad higher temperatures tolerance, wider pH tolerance and higher conversion rate.