微生物发酵液中脂肽类生物表面活性剂的测定

Determination of Lipopeptide Surfactants in Cell-Free Broth

综合文献报道的17类脂肽的氨基酸组成的实测结果,其中12类脂肽分子中含有一个谷氨酸或谷氨酰胺残基。基于此事实提出了通过测定氨基酸来测定微生物发酵液中脂肽类生物表面活性剂的方法。将枯草杆菌发酵液离心除菌体,测得表面张力为26mN/m,用氯仿/甲醇萃取,除去肽和蛋白,盐酸水洗,得到分析用生物表面活性剂样品。用TLC方法定性为一种脂肽,用红外光谱法确认属于环脂肽类。用氨基酸定量分析法测得该脂肽含有谷氨酸或谷氨酰胺、天冬氨酸或天冬酰胺、缬氨酸及亮氨酸残基,由谷氨酸浓度得到发酵液中脂肽浓度为2 82×10-4mol/L。实验确定了有机溶剂萃取和酸洗次数及酸水解时间等操作参数。该方法简便易行,取样量少(10mL发酵液),测定结果准确,平均回收率94 1%,标准偏差1 21,相对偏差1 28%。图3表4参29。

As for the amino acid composition, 12 of 17 classes of the lipopeptides (LPs) published in literature have in their molecules one glutamic acid or glutamine residue. Based on this experimental fact, a method of determining the LP(s) in fermentation broth by the way of amino acid analyses is suggested. The broth with cultivated bacteria Bacillus substilis, centrifugalized to remove the bacterial cells and having surface tension 26 mN/m, is extracted by chloroform/methanol mixture for separating from peptides and proteins and washed by diluted HCl for hydrolysis to give biosurfactant samples for analysis. The biosurfactant is determined qualitatively as a lipopeptide (LP) by using TLC technique and is identified IR-spectrophotometrically as a cyclic LP. By using amino acid analyses, this LP is shown to contain Glu or Gln, Asp or Asn, Leu, and Val residues and the concentration of Glu residue, or the concentration of the LP, in the cell-free broth is of 2.82×10^(-4) mol/L. The times of organic solvent extraction and of acid washing and the duration of acidic hydrolysis needed are established. The suggested method is simple and easy, requires a little amount (10 mL) of broth samples and gives accurate determinations with a recovery rate 94.1%, a standard deviation 1.21 and a relative error 1.28%.