The Research On Pyrroloquinoline Quinone Biosynthesis In Gluconobacter Oxydans

Pyrroloquinoline quinone is the third type of co-enzyme after niacin-amide, riboflavin co-enzyme, a new member of vitamins B, has important physiological functions, therefore,large-scale preparation of PQQ, has the vital significance. Due to the chemical synthetic steps,low yield, and more isomer and by-products removal need multi-step purification, and heavy metal catalyst is bad for the environment, the biological synthesis of PQQ, is the best way to large-scale preparation, therefore, this subject mainly research the oxidation of glucose coli synthesis of PQQ, fermentation condition, purification and enzymatic synthesis of molecular basis, specific as follows:1, Fermentation conditions for preparation of PQQ use Gluconobacter oxydansWith single factor test and orthogonal test method was carried out on the oxidation of glucose acid bacteria fermentation medium optimization, the results showed that the oxidation of glucose bacillus preparation of PQQ fermentation culture medium the best addition amount of glutamate 4g/L, tyrosine 4g/L, Fe SO4 5mg/L, the synthesis of PQQ is0.813 ml/L, most agreed with the theoretical prediction. Using single factor analysis method for the oxidation of glucose acid bacteria fermentation conditions of optimization, the results show that the pH value of 6.5, fermentation temperature 28 ?, the fermentation speed of 200r/min is the best fermentation conditions.2, The separation and purification of PQQ from Gluconobacter oxydans fermenting liquidPQQ structure contains three carboxyl, chromogenic acid. According to the structure characteristic, by using the method of complexation extraction for separation and purification,fermented liquid of high-speed centrifugal, put on the clear liquid in double extraction system of the solute complexation extraction, three amine as the complexing agent, n-hexane as diluent, get rich in the upper liquid of PQQ, reoccupy ammonia extraction, vacuum concentration after freeze drying, a single product. By HPLC to detect the purity can reach more than 90%. Process conditions of the method is simple, fast, easy to large-scale industrial production.3, PQQ synthetic gene from Gluconobacter oxydans cloning and expression of the cluster and enzymatic synthesisAfter bacteria fermentation in the production of PQQ attempt, we further use simple and controllable synthesis of it, cell free system. LC-MS, according to the results of PQQ is can the in vitro synthesis, it is the precursor of polypeptide PqqA. Under the action of enzymes,PqqA has experienced a series of changes, the resulting PQQ, but all kinds of enzyme reaction mechanism is not clear, needs further research. To this end, we cloned and expressed the oxidation of glucose coli synthesis PQQ gene cluster of various enzymes, and through His-tag nickel ion separation column on crude protein purification and concentration,obtained the recombinant strains, recombinant plasmid and enzyme, such as biological material, try the enzymatic synthesis of PQQ, the study has not yet achieved results.Comprehensive above, through this topic research of the PQQ, rapid detection technology, optimization of fermentation conditions and rapid purification methods, as well as the synthesis of PQQ gene recombinant plasmid and enzyme protein, for the study of enzymatic synthesis of it, lay a solid foundation. Therefore, this topic has important theoretical value and practical value.