Expression of secreted His-tagged S-adenosylmethionine synthetase in the methylotrophic yeast Pichia pastoris and its characterization, one-step purification, and immobilization.
S-Adenosylmethionine synthetase (SAM synthetase) catalyzes the synthesis of S-adenosylmethionine (SAM), which plays an important role in cellular functions such as methylation, sulfuration, and polyamine synthesis. To develop a simple and effective way to enzymatically synthesize and produce SAM, a soluble form of SAM synthetase encoded by SAM2 from Saccharomyces cerevisiae was successfully produced at high level ( approximately 200 mg/L) by the recombinant methylotrophic yeast Pichia pastoris. The secreted His6-tagged SAM synthetase was purified in a single chromatography step with a yield of approximately 82% for the total activity. The specific activity of the purified synthetase was 23.84 U/mg. The recombinant SAM synthetase could be a kind of allosteric enzyme with negative regulation. The enzyme functioned optimally at a temperature of 35 degrees C and pH 8.5. The stability of the recombinant synthetase and the effectiveness of different factors in preventing the enzyme from inactivation were also studied. Additional experiments were performed in which the recombinant SAM synthetase was purified and immobilized in one step using immobilized metal-chelate affinity chromatography. The immobilized synthetase was found to be 40.4% of the free enzyme activity in catalyzing the synthesis of SAM from dl-Met and ATP.
Luo
Yunxing
Y
Key Laboratory of Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun 130023, China.
Yuan
Zhongyi
Z
Luo
Guimin
G
Zhao
Fukun
F
D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000302:isolation & purification / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology / Q000379:methods; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008716:Methionine Adenosyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012436:S-Adenosylmethionine; D000255:Adenosine Triphosphate; D008715:Methionine; D008716:Methionine Adenosyltransferase