Hejazi M A, Kleinegris D, Wijffels R H
Food and Bioprocess Engineering Group, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands.
Biotechnol Bioeng. 2004 Dec 5;88(5):593-600. doi: 10.1002/bit.20238.
We show that it is possible to extract beta-carotene selectively from Dunaliella salina in two-phase bioreactors. The cells continue to produce beta-carotene and the extracted part is substituted by newly produced molecules. This process is called "milking." We performed several experiments to understand the exact mechanism of the extraction process. The results show that direct contact between the cells and the biocompatible organic solvent was not a requirement for the extraction but it accelerated the extraction. Electron microscopy photographs showed an undulated shape of the cell membrane and a space between the cell and the chloroplast membranes in the cells growing in the presence of dodecane (a biocompatible solvent). Extra-chloroplast beta-carotene globules located in the space between the cell and the chloroplast membranes were observed in these cells as well. It was shown that dodecane was taken up by the cells. The concentration of dodecane in the cells was about 13 pg.cll(-1). It can be concluded that dodecane uptake by the cells is responsible for the morphological changes in the cells and leads to more activity in the cell membrane. The results suggest two possible modes of extraction. One of the mechanisms is transport of the globules from the chloroplast to the space between the cell and the chloroplast membranes and subsequently from there to the outside by exocytosis. Another possible mode for the extraction could be release of beta-carotene from the globules as a result of alterations in the membrane in response to the uptake of dodecane. beta-Carotene molecules diffuse from the chloroplast to the space between the cell and the chloroplast membranes and from there to the medium either by diffusion or by exocytosis after accumulation in the vesicles.
我们证明，在两相生物反应器中从盐生杜氏藻中选择性提取β-胡萝卜素是可行的。细胞继续产生β-胡萝卜素，被提取的部分会被新产生的分子所取代。这个过程被称为“挤奶”。我们进行了多项实验以了解提取过程的确切机制。结果表明，细胞与生物相容性有机溶剂的直接接触并非提取的必要条件，但它会加速提取过程。电子显微镜照片显示，在存在十二烷（一种生物相容性溶剂）的情况下生长的细胞中，细胞膜呈波浪状，细胞与叶绿体膜之间存在间隙。在这些细胞中还观察到位于细胞与叶绿体膜之间间隙中的叶绿体外部β-胡萝卜素小球。结果表明细胞摄取了十二烷。细胞中十二烷的浓度约为13 pg·cell⁻¹。可以得出结论，细胞对十二烷的摄取导致了细胞形态的变化，并使细胞膜的活性增强。结果提示了两种可能的提取模式。其中一种机制是小球从叶绿体转运到细胞与叶绿体膜之间的间隙，随后通过胞吐作用从那里转运到细胞外。另一种可能的提取模式可能是由于膜因摄取十二烷而发生改变，导致β-胡萝卜素从小球中释放出来。β-胡萝卜素分子在叶绿体中积累后，通过扩散或在囊泡中积累后通过胞吐作用从叶绿体扩散到细胞与叶绿体膜之间的间隙，再从那里扩散到培养基中。
