Cloning And Expression Of S.Cereviase Sam2 Gene In Actinomucor Elegans And Optimization Of Synthetic Conditions For SAM

S-adenosylmethionine was an important metabolic intermediate that provided the function of transmethylation, sulfur transformation and aminopropyl transformation. It was used in the treatment of liver disease, depression, arthritis and other diseases. Therefore, SAM had a broad application market. The article mainly included the following contents:the establishment of detection methods of SAM, cloning and expression of S.cerevisiae sam2 gene in Actinomucor elegans, optimization of synthetic conditions of recombined Actinomucor elegans for SAM.The first part mainly summarized the structure, property, physiological function and the clinical application of SAM. It also expounded the research progress of its preparation methods.The second part established the detection methods of SAM. The optimum paper-electrophoresis conditions were as follows:Na2HPO4/ citric acid buffer (0.05 mol/L, pH 7.0), sample loading quantity 10μL, voltage 300 V, time 30 min, cut the spotted paper into pieces and extracted by pH 1.0 HCl for 4 h, then determined OD254 and calculated the concentration of SAM. The method was efficient, but the detection limit was relatively high. To solve the problem, we developed HPLC method, whose optimum conditions were as follows:Inertsil ODS-SP column (5 μm,4.6×250 mm),15% methyl alcohol-85% buffer (0.6% ammonium acetate,0.01% octanesulfonate), and used methanoic acid to adjust pH to 3.0, calculated concentration of SAM through peak area. The method was sensitive and accurate, whose detection limit reached 1.0μg/L.The third part used the technique of PCR to clone S.cerevisiae sam2 gene to obtain recombined vector pCB1004-PgpdA-sam2, and transformed Actinomucor elegans protoplast under the mediate of PEG-CaCl2 to get SAM productive engineering strain. The result showed that the SAM production of screened strain improved from 0.3947 g/L to 0.7744 g/L, which increased by 96.20%.Response surface methodology was used to optimize the convertion conditions for SAM production of recombinant Actinomucor elegans. The results showed that the optimum conditions were as follows:temperature 37℃, pH6.0, glucose 80 g/L, xylene0.4%, K2HPO466.16 g/L, MgSO4 1.71 g/L, adenine 3.91 g/L and L-Met 8.37 g/L. The production of SAM improved from 0.7504 g/L to 1.1823 g/L, which increased by 57.56%.