Chen Haiqin

Evaluation of methylations and external/internal standard quantification of lipids using gas chromatography-mass spectrometry

In the present work, we evaluated different widely accepted methyl derivatization procedures which are almost obligatory in gas chromatography analysis of fatty acids (FAs), both acid- and base-catalyzed ones. We determined their derivatization efficiency and rapidity and found that combined base–acid methylation, the NaOH–BF3–MeOH method, out-performed all other methods. The analyses of FAME standards and samples were performed on a conventional 30 m × 0.25 mm Wax column, with a single run of 51 min. To accelerate the analysis while maintaining resolution, a more polar 0.1 mm ID column was used to analyze 52 FAMEs. This way, we were able to obtain results within one-third of the analysis time with almost equal separation, if not better. To further evaluate the quantification methods that are performed with or without internal standards, we constructed new calibration curves by removing a known quantity from the original calibration curves, and used them to recalculate the concentration of the known removed quantity. Comparison of the calculated and actual concentration values revealed that, when performed carefully, the external standard method could achieve results that were comparable to results obtained with the internal standard method, and thus is recommended for daily analysis.

Studies of the Cloning, Expression and Function Analysis for Δ6-II Desaturase from Mortierella alpina

产油微生物高山被孢霉中Δ6脱饱和酶是决定ω3/ω6脂肪酸代谢流的关键酶,该酶对不同底物的催化特性直接决定该菌体内脂肪酸的流向。在已完成基因组测序的高山被孢霉ATCC32222中经注释发现它存在两种Δ6脱饱和酶（Δ6-Ⅰ和Δ6-Ⅱ）,选择对其中的Δ6-Ⅱ脱饱和酶进行克隆、表达和功能鉴定。首先以p YES2/NT C质粒为骨架构建了Δ6-Ⅱ脱饱和酶基因（FADS6-Ⅱ）的表达载体（p YES2/NT C-FADS6-Ⅱ）,并转化至酿酒酵母中进行诱导表达,进一步通过在重组菌培养基中添加Δ6脱饱和酶的底物来考察Δ6-Ⅱ脱饱和酶对各底物的偏好作用。实验结果表明,在分别添加0.5 mmol/L亚油酸（LA）和0.5 mmol/Lα-亚麻酸（ALA）时,Δ6-Ⅱ脱饱和酶对LA的转化率为42.94%,对ALA没有催化作用。而当底物添加方式改为同时添加LA和ALA时（分别为0.25 mmol/L）,Δ6-Ⅱ脱饱和酶对LA的转化率为37.12%,对ALA仍没有催化作用。该实验结果为高山被孢霉中Δ6-Ⅱ脱饱和酶的催化功能研究提供了理论依据。