Biosynthesis Of Pyruvate And L-tyrosine By Muti-enzyme Coupled Reaction System

Pyruvate is widely used in pharmaceuticals, agricultural chemicals and food industries. The methods of synthesis of pyruvate can be divided into chemical synthesis way, fermentation way and biological enzymatic way. 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 (racemic as well as L-lactate are much cheaper than pyruvate) and non-pollution.L-tyrosine is one of essential amino acids, which is used as a dietary supplement and has a promise as a valuable precursor compound for various industrial and pharmaceutical applications. According to the current literature reports, the methods of preparation of L-tyrosine can be divided into extraction way, chemical synthesis way, fermentation way and enzymatic conversion way. In contrast to chemical synthesis way, biotechnological methods can produce L-tyrosine from biomass under environmentally friendly and near carbon-free conditions. L-tyrosine can be obtained by enzymatic reaction of Tyrosine phenol-lyase on phenol, pyruvate, and ammonia. Using enzymatic synthesis replace hydrolysis method is one of research hots in amino acid industry in recently years.In this paper the target genes (lactate oxidase, catalase, tyrosine phenol-lyase) were synthesized after rare codon optimization and expressed in E. coli BL21 (DE3) via pETDuet-1. Use the recombinant enzymes to enzymatic synthesis of pyruvate and L-tyrosine.Specifically the following:1 The engineered strain of E. coli carrying the plasmid pET-Duet-lod was constructed, named DM-2415, and the reaction conditions were optimized. Results are shown below.(1) Recombinant lactate oxidase was constructed, which can catalyze lactate and oxygen to pyruvate and hydrogen peroxide. The optimized reaction conditions were pH 9.0 and 50℃.(2) The enzyme activity of complete cells was 1.93 times of crude enzyme, however with time going, the accumulation of pyruvate with crude enzyme was higher than complete cells’.(3) Reducing agent (Na2SO3, Na2S2O3 and Vc) and catalase was used to decompose hydrogen peroxide. Compared with crude enzyme, Na2SO3 can depress LOD’s activity. The accumulation of pyruvate of crude enzyme with Vc and Na2S2O3 was higher in 2-7 hours, but decrease sharply at 8 hour. The accumulation of pyruvate of crude enzyme with catalase was always higher. In conclusion, catalase was the best to accumulation pyruvate.2 To solve the problem that pyruvate and hydrogen peroxide generate acrtic acid and carbon dioxide spontaneously, a recombinant E. coli strain [pET-Duet -od-cat/ BL21(DE3)]), named DM-2416 was constructed, which can co-express LOD and catalase (CAT) with pET-Duet-1 vector. The strain was used for a whole-cell biotransformation system on converting DL-lactate to pyruvate. Results are shown below:(1) The gene lod and cat were inserted into the multiple cloning site 1 (MCS 1) and multiple cloning site 2 (MCS 2) of pET-Duet 1 vector, and were expressed in E. coli BL21 (DE3) via pETDuet-1. According to Gelpro32 gray analysis software, about 75% LOD and 65% CAT were solube protein.(2) According to orthogonal test, the optimized fermentation conditions were:pH 8.5,28℃,220 rpm, add 20 mL fermented liquid to 250 mL conical flask.(3) The reaction was optimal at pH 8.5 and 50℃,220 rpm,0.1 g/mL cells,1 mM EDTA and 100 mM phosphate buffer (pH 8.5) in 250 mL erlenmeyer flask. The maximum utilization of DL-lactate reached 74.4%. The catalase expressed by DM-2416 was adequate to decompose hydrogen peroxide generated by lactate oxidase. The catalase in DM-2416 not only can solve the problem that To solve the problem that pyruvate was decomposed by hydrogen peroxide, but also the oxygen, the product of catalase, can promote lactate oxidase to generate pyruvate.3 A muti-enzyme coupled system was built to perpare L-tyrosine by useing E. coli DM-2416 and E. coli DM-940 ([pET-Duet-lod-cat/BL21(DE3)], which can produce tyrosine phenol-lyase, TPL). Using this system, L-tyrosine was efficiently produced from lactate, phenol and ammonium chloride. An optimum reaction condition of the coupled enzymatic system is as follows:reaction temperature 50℃, pH 8.5, the ratio of lactate oxidase and tyrosine phenol-lyase was 5:1 (ratio of cells quantity), the ratio of substrates was 1:1.2. When the concentration of L-lactate was 10 g/L, feeding 0.2 g phenol at every 3 h and feeding 6 times, the yield of L-tyrosine was up to 72.95%.4 Aminoacylases, the metalloenzymes, have been widely used as catalysts for kinetic resolution of N-acyl-L-amino acids. In this study, we reformed ACY1 by computer molecular simulation and molecular biotechnologies. The 53 amino acids in N-end from bovine ACY1 (bACYl) was removed, but remaining the active site and substrate binding site, the new enzyme was named XINEW. Several factors affecting enzyme activity, such as pH, pH dependence, temperature and thermal stability, substrate specificity and Kcat/Km were all examined. When compared with bACY 1, XINEW showed higher resolution rate on N-acyl-L-amino acids with big R group, but lower rate on N-acyl-L-aromatic amino acids with small R group, suggesting the important role of substrate-binding pocket size. Autodock results further indicated that the size of substrate-binding pocket may affect the resolution rate on N-acyl-L-aromatic amino acids.