The Research On Pyrroloquinoline Quinone Biosynthesis In Acinetobacter Baumannii

Pyrroloquinoline Quinone（PQQ）is a novel oxidoreductase coenzyme widely distributed in bacteria,animals,plants and food.Its unique biological function has great research significance and economic value in the fields of new food medicine.In this study,a strain of using methanol as a carbon source for fermenting Pyrroloquinoline Quinonefrom the soilwas screenedand identified.Its fermentation characteristics of biosynthesis Pyrroloquinoline Quinone were studied.The separation and purification process of pyrroloquinoline quinone and its anti-oxidation application were studied at the same time.The study is briefly described as follows:I.By means of morphological observation,physiological and biochemical identification and molecular biological identification,the strain GGZ190116 was identified as Acinetobacter baumannii.The growth kinetic curve and yield kinetic curve study show that the growth kinetic curve is basically consistent with the yield kinetic curve trend.When the strain grew to48 h,it entered a stable growth period and the PQQ yield was maximized.II.The yield of PQQ synthesized by strain GGZ190116 was studied by optimizing the medium components（carbon source,nitrogen source,inorganic salt）and fermentation conditions.The results showed that the optimal medium composition was divided into:methanol 5 g/L,mixed nitrogen source（ammonium sulfate:Glu:Tyr=2:1:1）5 g/L,calcium chloride 0.1 g/L.The optimized PQQ yield increased from 2.02 mg/L to 37.13 mg/L,which was 18 times higher than the initial fermentation yield.The optimal fermentation conditions were:culture temperature 30℃,pH 6.5,inoculum size 0.5%.PQQ yield after optimization of fermentation conditions was 31.02 mg/L,which was 15 times higher than the initial fermentation yield.III.The results of complex extraction,selective chromatography and recrystallization process indicate that the optimal extraction system for PQQ complex extraction and separation is n-butanol,n-octanol,trioctylamine and ammonia solution.optimal extraction The conditions are:material ratio is 4,temperature is 30℃,extraction times are 5.PQQ concentration is up to 26.32 mg/L after extraction.The optimal conditions for the separation and purification of PQQ by polyamide resin were as follows:material ratio was 5,eluent concentration was 50%,eluent volume was 8 times of column volume.PQQ concentration was 3.71 g/mL after separation and purification.The best crystallization method for PQQ crystallization refining process is ethanol aqueous system,the purity after crystallization is98%.the best crystallization condition is:crude concentration 8 g/L,crystallization at 16℃for 24 h.High-purity PQQ was prepared by the best process for the fermentation broth.HPLC-DAD analysis showed that the purity of PQQ reached 99.5%.The mass spectrum of the product,¹H-NMR and¹³C-NMR identified the crystalline product as the reduced PQQ dipotassium salt（PQQH₂K₂）.IV.Taking vitamin C as a positive control,the scavenging ability of reduced pyrroquinoline quinone to ABTS free radicals was compared.The results show that the reaction rate of nitrogen free radicals in the reduced state PQQ increases rapidly after 0-3 min,and the equilibrium reaches equilibrium within 10 min.The scavenging ability increases with the increase of mass concentration.The scavenging rate is linear with the PQQ molar concentration of the reduced state,and the IC₅₀0 value of the reduced pyrroquinoline quinone to remove ABTS free radical is 86.86 nmol/mL,which is two-thirds of vitamin C.At the same molar concentration,the reduced pyrroquinoline quinone has a higher ability to scavenge ABTS radicals than vitamin C 1.5 times.When the clearance rate reaches the highest,the PQQ removal rate of the reduced state is 6.55%higher than that of vitamin C,and the clearance rate of 5 min can reach over 90%.The removal rate of ABTS free radicals by the original PQQ and vitamin C decreases with the increase of heating temperature and heating time within a certain range.In the same heating temperature or heating time,the rate of decrease of the PQQ clearance rate of the reduced state is lower than that of the vitamin C.The thermal stability of the vitamin C is lower than that of the reduced state PQQ.