Abstract
Methionine adenosyltransferase (MAT, EC2.5.1.6) catalyzes the synthesis of S-adenosylmethionine (SAM) using l-methionine and adenosine triphosphate (ATP) as substrates. The mutant MAT pDS16 was obtained through DNA shuffling previously in our lab. Overexpression of pDS16 in Pichia pastoris led to about 65 % increase of MAT activity and SAM accumulation, compared with the strain overexpressing Saccharomyces cerevisiae MAT gene SAM2. Different strategies were tested to facilitate the expression and purification of pDS16. However, addition of the hexahistidine tag to pDS16 was shown to decrease the enzyme activity, and the yeast α-factor signal sequence could not effectivley direct the secretion of pDS16. The intracellular pDS16 was purified by a simple two-step procedure combining an ion exchange and hydrophobic interaction chromatography. Protein purity was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 93 %, with the specific activity of 1.828 U/mg. Two-dimensional electrophoresis revealed pI of ∼5.5. The purified enzyme followed Michaelis kinetics with a Km of 1.72 and 0.85 mM, and Vmax of 1.54 and 1.15 μmol/min/mg for ATP and L-methionine, respectively. pDS16 exhibited optimal activity at pH 8.5 and 45 °C with the requirement of divalent cation Mg2+ and was slightly stimulated by the monovalent cation K+. It showed an improved thermostability, about 50 % of the enzyme activity was retained even after preincubation at 50 °C for 2 h.
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Acknowledgments
We are grateful to National Basic Research Program (973 Program 2013CB733600), National Natural Science Foundation of China (20976050), and Program for New Century Excellent Talents in University (NCET-11-0639) for the financial supports to this research.
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Yao, G., Qin, X., Chu, J. et al. Expression, Purification, and Characterization of a Recombinant Methionine Adenosyltransferase pDS16 in Pichia pastoris . Appl Biochem Biotechnol 172, 1241–1253 (2014). https://doi.org/10.1007/s12010-013-0594-3
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DOI: https://doi.org/10.1007/s12010-013-0594-3