S-腺苷甲硫氨酸合成酶在毕赤酵母中的分泌表达

S-腺苷-L-甲硫氨酸（SAM）是甲硫氨酸（Met）的活性形式。生物体内，SAM由S-腺苷甲硫氨酸合成酶（SAMS）EC2．5．1．6]催化L．甲硫氨酸（L．Met）和A11）反应而合成。酿酒酵母细胞中有2种SAM合成酶的同工酶，即SAMS1和SAMS2，分别由基因saml和sam2编码，二者氨基酸序列的同源性高达92％。在过量L-Met存在下，saml的转录受到抑制；已有酶促法合成研究显示sam2编码的合成酶没有产物抑制现象。笔者将sam2基因连接于毕赤酵母分泌型表达载体pPIC9K，并通过同源重组整合到酵母染色体上，以实现SAM合成酶的分泌性表达，为开发和建立酶促法生产SAM工艺奠定基础。

Secreted Expression of S-adenosy-L-methionine Synthetase in Pichia pastoris

Objective] The research aimed to study the secreted expression of S-adenosyl-L-methionine synthetase (SAMS) in Pichia pastoris. Method] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was amplified by PCR and inserted into the secreted expression vector pPIC9K to get recombinant plasmid. The recombinant plasmid pPIC9K-sam2 was integrated into Pichia pastoris GS115 genome by electroporation and induced by methanol. The activity of the recombinant enzyme was measured using high-performance liquid chromatography (HPLC) by determining the production of S-adenosy-L-methionine (SAM) with the enzyme secreted. Result] The molecular weight of the expression protein identified by SDS-PAGE was about 50 kD, being larger than the theoretical molecular mass of SAMS, which might be due to the glycosylation in the process of secretion. Methanol-induction as well as preliminary purification could enhance the enzyme activity, especially the latter, after which the specific activity of SAMS was improved to 61.48 U/mg. Conclusion] SAMS with biological activity was secreted successfully in Pichia pastoris GS115 for the first time. And it is the start for the genetic engineering strains to open up prospects for industrial production.