The Enzymatic Production Of S-adenosyl-L-methionine

S-adenosyl-L-methionine (SAM), discovered by Cantoni in 1953, is a natural molecule which is ubiquitous in virtually all organisms, involved in more than 40 biochemical reactions, playing a significant role in synthesis of proteins, nucleic acids, neurotransmitters, phospholipids and vitamins. As an important metabolic intermediate, SAM is of vital importance for the organisms’ metabolic activity. It is synthesized by the action of S-adenosyl-L-methionine synthetase (EC 2.5.1.6) in the presence of L-Met and ATP. At present, SAM gives good performance in clinical medicine due to its high application value, and has been applied mainly in pharmaceutical industry involving the therapies for liver, nervous system, osteoarthritis, sexual dysfunction and cancer. Nowadays, three methods-microbiological fermentation, chemical synthesis and enzymatic synthesis-are mainly being adopted in the production of SAM. The method of chemical synthesis has some shortcomings, such as the difficulties in separation and purification and the high costs; as a prevailing method in production of SAM, microbiological fermentation is applied by using yeast cells, while with the disadvantages of a complicated process of separation and purification and a long production cycle; compared with the other two methods, enzymatic synthesis with advantages of a short reaction period, a high conversion rate, simple process of separation and purification and environmental friendliness, has become a hotspot of research recently. However, SAM synthetase has a small quantity and a low enzyme activity. Under the circumstances, the cloning of SAM synthetase which are over-expressed and with a high enzyme activity is the key to the production of SAM. This dissertation focuses on the overexpression of SAM synthetase, with SAM synthetase genes MetK and MATTt cloned with Escherichia Coli’s and Thermus thermophilus’s genomes as the templates, with plasmid pET22b (+) as the transfer vector plasmid, in the host Escherichia Coli BL21 (DE3) to achieve high-yielding SAM recombinant bacteria, as well as on the research in its culture conditions, reaction conditions and enzymatic property; the immobilization on SAM synthetase gene MATTt derived from Thermus thermophiles was studied, and the enzymatic properties of SAM synthetase was characterized by immobilizing it to carbon nanotubes; the crystallization and structure analysis of enzymes derived from Thermus thermophiles were carried out, facilitating the preliminary reveal on its thermostability mechanism, in this way providing us with valuable references on the production of SAM by adopting the method of enzymatic synthesis. This dissertation is mainly about the following research contents:1. SAM synthetase gene MetK was amplified by using Escherichia Coli chromosome DNA as templates. We integrated the previous genes to the transfer vectors pET22b (+), and constructed genetically engineered bacteria where SAM synthetase can be over-expressed by using T7 as a strong promoter for transcription and using DE3 as an expression strain. As a result, in Escherichia Coli we obtained the overexpression of SAM synthetase. The enzyme activity of metK was 33.42 U/L (compared to the volume of fermentation solution), which was 123.70 times that of empty plasmid.The culture conditions of SAM synthetase gene MetK were optimized: lactose as optimum carbon and urea as the best nitrogen source; 0.25‰ as the optimum additive of inducer IPTG, OD value of 0.5 as induction occasion,6 hours as induction time. Reaction conditions of the synthetase were optimized: 70℃ as the optimum reaction temperature; Tris-HCl at pH 8.0 as the optimum buffer; Mg2+ as the optimum ion; 4 g/L as the optimum amount when catalyzing ATP and L-Met of lOmmol/L; ATP as the inhibitive substrate. Thermostability of the synthetase was studied:with a little alteration in enzyme activity when stored at 70℃ in 5 hours, about 15% of enzyme activity remaining when stored up to 15 hours, till to 25 hours about 5% of enzyme activity remaining, while no enzyme activity remaining after 30 hours. The enzymatic reaction kinetics showed us that the Vmax was 0.07 mM/min, the Km of ATP was 4.13 mM, and the L-Met was 2.20 mM, respectively.2. pET22b-MATTt, the recombinant plasmid which contains SAM synthetase gene derived from Thermus thermophiles, was constructed. We obtained the overexpression of SAM synthetase gene MATTt in Escherichia Coli, and purified the synthetase through Ni-chelating affinity chromatography. As for MATTt, the calculated specific activity and enzyme activity were 120.5 U/g and 12.70 U/L, respectively.The enzymatic properties of MATTt were studied. The enzymatic reaction kinetics showed us that the Vmax was 0.84 μol/min/mg, the Km of ATP was 4.19 mM, and the L-Met was 1.2 mM, respectively. This Km is similar to that of SAM synthetase TkMAT derived from Thermococcus Kodakarensis. The most outstanding feature was its superior stability. According to the circular dichroism spectra, the enzyme MATTt remained stable at pH values of from 5 to 10. In terms of thermostability, the enzyme activity remained unchanged at 70℃ within 24 hours and still remained 60% within 36 hours.The optimum reaction conditions for MATTt were studied. MATTt exhibited catalytic activity in a wide range of temperatures. Its catalytic activity rose with the increase of temperature, peaking at 80℃. MATTt remained active in the range of pH 6 to 11 and the optimum pH was 8.0. MATTt showed a significant preference to Zn2+The SAM synthetase MATTt derived from Thermus thermophiles was immobilized to catalyze the synthesis of SAM, by applying functionally transformed multi-wall carbon nanotubes (MWCNTs) as carriers. We characterized the enzymatic properties of the immobilized enzyme whose activity remained 80% after 4 batches.3. After several crystallization conditions for MATTt protein were achieved, we acquired its crystal structure which could be used for X-ray diffraction, and the crystal structure was determined by the method of molecular replacement. Comparing the structures of MATTt and metK, we found that the main difference was related to the previously reported "disordered loop". In contrast to the most of structures, the loop region of MATTt had a good density and all the amino acids in this region could be visualized. From the crystal structures, there were obvious conformation differences between the loop on MATTt and the counterparts on integrate or defective metK, which suggested that this loop was the leading factor of the thermostability differences between MATTt and metK.