文献类型：期刊文献

中文题名：重组毕赤酵母发酵生产S-腺苷甲硫氨酸培养条件优化

英文题名：Optimization on culture conditions for producing S-adenosyl-L-methionine by recombinant Pichia pastoris

作者：朱志钢[1];储炬[1];胡晓清[1];庄英萍[1];王永红[1];张嗣良[1];袁中一[2]

机构：[1]华东理工大学生物反应器工程国家重点实验室国家生化工程技术研究中心,上海200237;[2]中国科学院上海生命科学研究院生物化学和细胞生物学研究所,上海200031

年份：2006

卷号：36

期号：3

起止页码：5

中文期刊名：工业微生物

外文期刊名：Industrial Microbiology

收录：北大核心:【北大核心2004】；CSCD:【CSCD2011_2012】；

基金：国家高技术研究发展计划"863计划"(No.2002AA217021;2002AA2Z3451)

语种：中文

中文关键词：毕赤酵母;高密度发酵;甲硫氨酸;S-腺苷甲硫氨酸

外文关键词：Pichia pastoris; high density fermentation; L-methionine; S-adenosyl-L-methionine

摘要：甲醇营养型毕赤酵母生产S-腺苷甲硫氨酸(SAM)是通过其表达的SAM合成酶催化L-甲硫氨酸(L-Met)和ATP反应而合成的。本文采用全合成培养基,在摇瓶上进行了培养条件的优化,确定了接种量、pH、PTM1、PO43-等初步条件。并根据SAM生物合成的特征,重点对其碳、氮源的影响作了进一步的分析优化。结果表明:当CaSO40.465g/L,K2SO49.10g/L,MgSO4.7H2O 7.45g/L,PO43-0.5mol/L情况下,生长阶段,甘油4%、硫酸铵4.00g/L为最佳;诱导表达阶段,L-Met1.0g/L,甘油与甲醇比例为0.5、硫酸铵8.00g/L为最佳。优化后,SAM产量诱导4d后达1.48g/L,诱导5d后可达1.70g/L(131mg/g干细胞),L-Met的转化率可达40.65%,既利于工艺放大又便于产品的分离纯化。
The biosynthesis process of S-adenosyl- L-methionine （SAM） is composed of the SAM synthetase expressed in Pichia pastoris to catalyze the reaction from the L-methionine （L-Met） and ATP to form SAM. Experiments were conducted in shake flasks with synthetic media. Firstly, several basic fermentation conditions were designed, such as inoculation volume, pH, trace dements （PTM1） and PO4^3- ; secondly, according to the property of the SAM biosynthesis, the further analysis of the carbon source and nitrogen source were carried out here. The optimum conditions for SAM accumulation are as follows： CaSO4 0. 465g/ L,K2SO4 9.10g/L, MgSO4·7H2O 7.45g/L, PO4^3- 0.5rnol/L, during the growth phase, the concentration of glycerol and （NH4）2SO4 should be controlled at 4 % and 4.00g/L respectively. In the induction phase, L-Met 1.0g/L, the ratio of the glycerol and methanol is 0.5 and the concentration of the （NH4）2SO4 is 8.00g/L. Under the optimum conditions, the yield of the SAM in the shake flasks reached to 1.48g/L, after four days induction and on the fifth day, it could increase to 1.70g/L （ 131mg/g DCW）, the yield coeffident of L-Met to SAM reached to 40.65 %. The above results will be applicable to the scale up of SAM production at bioreactors level as well as convenient to the subsequent separation and purification of SAM.