Study On The Production Of Pyrroloquinoline Quinone By Fermentation

Pyrroloquinoline quinone（PQQ）is a cofactor for dehydrogenases bound on the cell membrane of some bacteria.These dehydrogenases are usually alcohol dehydrogenase（methanol dehydrogenase and alcohol dehydrogenase）and glucose dehydrogenase.At present,the use of chemical methods to produce PQQ faces the problems of complex steps and high costs.In contrast,microbial fermentation methods can make the product PQQ easily separated from the culture medium and have the advantages of low cost.Therefore,the synthesis of PQQ by microbial fermentation methods suffers More and more widespread attention,and has a broad potential application market.This article first designed and improved three different methods of detecting PQQ,and clarified the scope of its application.The improved redox cycle method and high performance liquid chromatography（HPLC method）were used to screen PQQ high-yielding bacteria from soil samples,and the obtained high-yielding strains were identified.Secondly,the fermentation conditions and fermentation medium composition were optimized at the shake flask level through the single factor method,and the concentration of each component of the fermentation medium was optimized by the response surface method,and the optimal medium was determined.Finally,the feed strategy and p H control method were used to increase the PQQ production at the level of the 5 L fermentor.The primary contents and results of the research are described as follows:（1）The research designed and improved the three detection methods of PQQ,namely redox cycle method,spectroscopy and HPLC method,and analyzed and compared the detection limit,precision and recovery rate of the three methods.The application range of the three detection methods is clarified.The redox cycle method has high detection sensitivity,but the linear range is small,so it is suitable for the rough quantification of samples.In this study,it is used as the primary screening method for PQQ producing bacteria;the spectrometric method is fast and simple.,But it is easily interfered by other light-absorbing substances in the sample to be tested,so it is suitable for the detection of purified PQQ samples;HPLC method has high precision and good reproducibility,and can be used for qualitative and quantitative analysis of PQQ.In this study,it is used as PQQ Re-screening method for producing bacteria.（2）A high-yielding strain of PQQ was screened from the soil by oxidation-reduction cycle method and HPLC method.After five days of shake flask culture,the yield of PQQ could reach 20.6 mg·L^-1.The strain was identified as the genus of methylotrophic bacteria Methylopila sp.and named It is Methylopila sp.Z1.The effects of carbon sources,nitrogen sources and inorganic metal ions on the growth of strain Z1and the synthesis of PQQ were studied at the shake flask level.It was determined that methanol was the sole carbon source and ammonium sulfate was the sole nitrogen source,and the yield of PQQ was the highest.The Plackett-Burman experimental design,the steepest climbing experimental design and the response surface method were used to optimize the concentration of all the components in the fermentation medium,and the significant influence components were disodium hydrogen phosphate,ammonium sulfate and ferric citrate.The optimized optimal concentrations are 8 g·L^-1,1.62 g·L^-1 and 157.2 mg·L^-1,respectively.The strain Methylopila sp.Z1 can produce up to PQQ in the optimized medium 29.7 mg·L^-1,an increase of 44%compared to the initial conditions.（3）The effect of initial methanol concentration on cell growth and PQQ synthesis was studied on a 5 L fermentor.It was found that when the initial methanol concentration was a medium concentration of 25 g·L^-1,the highest PQQ yield was 44.6mg·L^-1.Feeding carbon source methanol was used to increase the production of PQQ.The feeding method was studied,and the results showed that constant-rate flow and medium concentration methanol was better than one-time supplementary quantitative methanol,and constant-rate flow was 5 g·h^-1.The methanol can increase the yield of PQQ to 70.2 mg·L^-1.The p H control strategy is adopted.The initial medium p H is 7.3.When the p H drops to 6.2,the p H of the medium is maintained at 6.2 by adding ammonia water.At this time,the PQQ synthesis rate is the fastest,and the PQQ production is increased to 86.2 mg·L^-1,Compared to the initial conditions increased by about 140%.