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首页> 外文期刊>Biotechnology Progress >Expression of Secreted His-Tagged S-adenosylmethionine Synthetase in the Methylotrophic Yeast Pichia pastoris and Its Characterization,One-Step Purification,and Immobilization
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Expression of Secreted His-Tagged S-adenosylmethionine Synthetase in the Methylotrophic Yeast Pichia pastoris and Its Characterization,One-Step Purification,and Immobilization

机译：分泌的His-标记的S-腺苷甲硫氨酸合成酶在甲基营养酵母巴斯德毕赤酵母中的表达及其表征，一步纯化和固定化

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摘要

S-Adenosylmethionine synthetase(SAM synthetase)catalyzes the synthesis of S-adenosylme-thionine(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(~200 mg/L)by the recombinant methylotrophic yeast Pichia pastoris.The secreted His_6-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 °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.
机译：S-腺苷甲硫氨酸合成酶（SAM）合成酶催化S-腺苷甲硫氨酸（SAM）的合成，在甲基化，硫化和多胺合成等细胞功能中起着重要作用。为酶促合成和合成简单有效的方法提供了一种途径。重组酵母菌毕赤酵母成功地高产（〜200 mg / L），由酿酒酵母（Saccharomyces cerevisiae）的SAM2编码的SAM合成酶的可溶性形式成功生产。分泌的His_6-tagged SAM合成酶在单个色谱步骤中进行纯化纯化后的合成酶的比活为23.84 U / mg，比重为82％，重组SAM合成酶可能是一种负调控的变构酶，该酶在35°C的温度下具有最佳的功能。 C和pH值8.5。还研究了重组合成酶的稳定性以及不同因素在防止酶失活方面的有效性。进行了最终的实验，其中使用固定的金属螯合物亲和色谱法一步一步纯化并固定了重组SAM合成酶，发现该固定的合成酶占催化DL-Met和SAM合成SAM中游离酶活性的40.4％。 ATP。

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