米根霉胞内氨基酸的高效液相色谱测定及其代谢途径分析  

Determination of Intracellular Amino Acids of Rhizopus Oryzae by High Performance Liquid Chromatography and Its Application to Metabolizing Pathway

作　　者：史纯珍[1] 张红漫[1,2] 黄和[1] 李霜[1] 宋萍[1] 何四龙[1] 

机构地区：[1]南京工业大学生物与制药工程学院,南京210009 [2]南京工业大学理学院,南京210009

出　　处：《分析化学》2012年第4期574-578,共5页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(Nos.21176124;20936002);国家科技支撑计划(No.2011BAD23B03)资助项目

摘　　要：建立了米根霉胞内代谢物提取方法定量评价体系,用100%甲醇作为提取剂获得对米根霉胞内能荷物质最佳的提取效果。用HPLC法建立了米根霉产富马酸体系胞内18种氨基酸的含量测定方法。采用异硫氰酸苯酯(PITC)作为柱前衍生化试剂,所有氨基酸在28min内洗脱完毕。以Sepax AA柱(250mm×4.6mm,5!m)为分析柱,流速1.0mL/min,检测波长254nm,以0.1mol/L醋酸钠(pH 6.5)-乙腈(93∶7,V/V)为流动相A,乙腈-水(80∶20,V/V)为流动相B,梯度洗脱。18种氨基酸在1.87～45.3mg/L范围内线性关系良好,相关系数大于0.991;加标回收率在96.3%～102.7%之间;相对标准偏差在1.6%～2.9%之间。运用本方法测定不同发酵时间米根霉胞内氨基酸的含量,得到其在发酵过程中的变化趋势,并结合氨基酸代谢途径分析了该发酵条件下富马酸产量偏低的原因。A quantitative evaluation system of intracellular metabolite extraction techniques for Rhizopus oryzae mtabolomics was established. The best performance for intracellular energy carrier extraction, in terms of efficacy, was achieved with 100% cold methanol. A method for quantifying 18 amino acids in cells of R. oryzae by HPLC was developed and validated. Isothiocyanates was used as the pre-column derivatization reagent. All amino acids were successfully separated within 28 min by using a Sepax AA column （250 mm × 4. 6 mm, 5 μm）, with a flow rate of 1. 0 mL/min and the detection at 254 nm, a gradient elution of mobile phase A （0. 1 mol/L sodium acetate （pH 6. 5）- acetonitrile （93： 7, V/V）） and mobile phase B （acetonitrile-water（80 ： 20, V/V））. A linear correlation coefficient （higher than 0. 991） was observed in the range of 1.87-45.3 mg/L. The recoveries were 96.3%-102.7%, with the RSD of 1.6%-2.9%. The method was used to analyze the content of amino acids in Rhizopus. oryzae cells at different fermentation times, and the variation trend of amino acids content was obtained. Combining with the metabolic pathways of amino acids, the reason for the low yield of fumaric acid under this fermentation condition was discussed.

关 键 词：高效液相色谱法 柱前衍生化 氨基酸 米根霉 

分 类 号：TQ920.1[轻工技术与工程—发酵工程] O657.72[理学—分析化学]