番红菌素中碳甲基转移酶(SfcF)的表达纯化及酶活性的检测  

The Expression, Purification and Activity Detect of SfcF, a C-Methyltransferase in Safracin

作　　者：郭建平[1] 唐满成[2] 李凯[1] 

机构地区：[1]东华大学化学化工与生物工程学院,上海201620 [2]中国科学院上海有机化学研究所生命有机化学国家重点研究室,上海200032

出　　处：《东华大学学报（自然科学版）》2013年第2期202-206,213,共6页Journal of Donghua University(Natural Science)

摘　　要：研究了番红菌素生物合成过程中的一个碳甲基转移酶(SfcF)的酶活反应时间曲线和动力学参数,并证明了其催化功能.通过镍柱亲和层析纯化得到碳甲基转移酶,用聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定其分子量大小后,以酪氨酸(Tyr)为底物,采用高效液相色谱法(HPLC)检测碳甲基转移酶的生物催化活性并分析得到其酶动力学数据.在碳甲基转移酶的生物催化作用下,可实现酪氨酸羟基邻位碳的甲基化,得到3甲基酪氨酸.通过时间曲线的测定,发现反应时间在50min以内,酪氨酸浓度低于400μmol/L时,底物的转化率与时间呈线性变化.通过碳甲基转移酶的活性检测和动力学常数的测定,为深入研究番红菌素的生物合成机制奠定了基础,并为其结构类似物ET-743(成药)的合成研究提供有益参考.Curves of enzyme reaction time and kinetics parameters of the C-methyltransferase （SfcF） involved in the biosynthetic pathway of safracin was investigated as well as its catalysis function was verified. The protein SfcF was purified by Ni-NTA affinity chromatography and examined by Sodium dodecyl sulfate--Polyacrylamide gel electrophoresis （SDS-PAGE）. Then catalysis activity and kinetics of SfcF was measured by using high performance liquid chromatography （HPLC）. The results showed that the methyltransferase SfcF could catalyze the transformation of L-tyrosine to 3-methyltyrosine under tested conditions. The efficiency of the substrate was linear within 50 minutes while the concentration of substrate was under 400 /~mol/L. The studies provide great information for further researches on biosynthetic of not only safracin but also the synthetic of ET-743, an antitumor drug.

关 键 词：生物催化 番红菌素 甲基转移酶 酪氨酸 3-甲基酪氨酸 

分 类 号：Q555.1[生物学—生物化学]