Study On Highly Synthetic S - Enzyme System Of S - Adenosine Homocysteine ​​and Its Application

S-adenosylhomocysteine (SAH) is the product of biological methylation reactions that use S-adenosylmethionine (SAM) as methyl donor. It plays an important role in many biochemical reactions involving transmethylation. SAH can also be used as a biomarker in incipient diagnosis of various pathological disorders. SAH is also an effective sedative, sleep modulator, and anticonvulsant, so its production has attracted the attention of many researchers, due to its attractive use in medicine and research.To obtain extremely thermostable S-adenosylhomocysteine hydrolase (SAHase) and lactate dehydrogenase (LDH), we amplified the genes encoding these enzymes from the hyperthermophilic bacterium Thermotoga maritima (T. maritima) and successfully cloned them into expression vector pHsh-amp to generate pHsh-sahase and pHsh-ldh, which were then transformed into E. coli to express SAHase and LDH. Recombinant SAHase was purified to homogeneity by a simple two-step procedure involving heat treatment and nickel-affinity chromatography; recombinant LDH was purified to homogeneity by heat treatment and ion-exchange chromatography on DEAE-Sepharose FF. Indeed, the recombinant SAHase and LDH were already purified to near gel electrophoretic homogeneity by heat treatments.We found that the synthetic activity of the T. maritima SAHase was strongly depended on the concentration of co factor NAD. T. maritima SAHase exhibited the strongest activity at 85℃, pH 8.0, and had a specific activity of 6.2 U/mg when 1 mM NAD was added to the reaction mixture. Nevertheless, the optimal reaction conditions shifted to 100℃ and pH 112, and its specific activity increased to 36.8 U/mg when the NAD concentration was raised to 8 mM. The enzyme kept stable for a long time and possessed activity at high level at 60℃. SDS-PAGE analysis showed that the molecular mass of the expressed recombinant SAHase was about 45 kD, which was exactly the size predicted.The optimal temperature and pH of T. maritima LDH were observed 95 ℃ and 7.0. The purified enzyme had a half-life of 2 h at 90 ℃, and exhibited better stability over a pH range from 5.5 to 8.0. The Km and Vmax values were 1.7 mmol/L,3.8×104 U/mg of protein for pyruvate, and 7.2 mmol/L and 1.1×105 U/mg for NADH, respectively. The expression of Tm-LDH in T7 system couldn’t obtain high efficiency, but it has been soluble over-expression in pHsh system and reached 340 mg/L. SDS-PAGE analysis showed that the molecular mass of the expressed recombinant LDH was about 33 kD, which was exactly the size predicted. The superior stability and productivity of Tm-LDH will lay the foundation of its industrial-scale fermentation and application in the NAD regeneration.In the present work, we sussussfully cloned, expressed and characterized extremely thermostable SAHase and LDH from the same organism T. maritima; and established an efficient cofactor regeneration system for the enzymatic synthesis of SAH by using these two thermostable enzymes. This coupling of SAHase and LDH produced a 6-fold increase in SAH production. Thus, Tm-SAHase and Tm-LDH form an efficient NAD consumption and regeneration system in the biosynthesis of SAH. These results support the utility of this method for industrial-scale enzymatic production of SAH.