Preparation,separation Of Carotenoids And Primary Study On Their Biosynthesis

Carotenoids are a class of terpene pigments which exist extensively in natural organism. The fact that carotenoids play an impotant role in prevention of cancer and chronic disease and that they exhibit significant tumor suppression activity has boosted interest in their pharmaceutical potential.It is difficult to extract and synthesize carotenoids chemically, while there is less variety of carotenoids in microorganism biosynthesis. It makes application of carotenoids limited. It has been possible to produce different carotenoids in E.coli. However, there are many problems such as the lack of precursor and the limitation of bearing capacity of thallus. Photosynthetic bacteria which produce chain carotenoids can be used for gene engineering. The genes of plant carotenoids cyclase are introduced into photosynthetic bacteria and expressed to catalyze chain carotenoids into cyclic carotenoids.The crude lutein was extracted from resin of marigold by using saponification and refrigeration crystallization, etc. The content was 50%-60%, while the yield was 70%-80%. The purified lutein was gained by using silica gel, its content could reach 98% with the support of analytic HPLC.One of the primary carotenoids was separated from the metabolin of Rhodobacter sphaeroides which could produce carotenoids itself. Model plasmid was introduced into Rhodobacter sphaeroides by means of electrotransformation.Total RNA which was suitable for RT-PCR techniques was isolated from marigold petal with Rneasy Plant Mini Kit. The cDNA was gained through reverse transcription. The core regions of BCH(carotene p-hydroxylase) and LYCB (lycopene P-cyclase) were cloned with the primers designed from the conserved amino acid motifs of the known plants.