The production process of pyrroloquinoline quinone (PQQ) mainly includes two methods: biosynthesis and chemical synthesis.
Biosynthesis
Biosynthesis is one of the main production methods of PQQ. In bacteria, PQQ is biosynthesized through the precursor peptide PqqA translated by ribosomes. The specific steps are as follows:
Precursor peptide PqqA: In the first step of PqqA modification, glutamic acid and tyrosine in PqqA are cross-linked by the free radical SAM enzyme PqE with the help of PqD. Then, the protease releases the Glu-Tyr molecule from the peptide backbone. Oxidation reaction: PqqB oxidizes the 2nd and 3rd positions on the tyrosine ring to form quinone, which quickly turns into AHQQ and completes the pyridine ring. Subsequently, PqqC forms the final pyrrole ring. Chemical synthesis
The chemical synthesis method involves multiple chemical reaction steps, and the specific steps are as follows: Raw material treatment: 2-methoxy-5-nitroaniline hydrochloride is used as the raw material, and the compound is obtained by alkali treatment. Formylation reaction: Compound 1 is subjected to formylation reaction under the catalysis of ionic liquid to obtain compound.
Reduction reaction: Compound 2 is reduced with sodium borohydride to obtain compound.
Diazotization reaction: Compound 3 is subjected to diazotization and reacted with HBF4 to obtain compound.
Amide exchange reaction: Compound 4 reacts with ethyl 2-methylacetoacetate to obtain compound 5, which is then treated with formic acid to obtain compound.
Esterification reaction: Compound 6 is subjected to amide exchange under the catalysis of ionic liquid to obtain compound 7, which is then reacted with dimethyl 2-oxopentaconedioate to obtain compound.
Hydrolysis reaction: Compound 8 is subjected to hydrogen chloride under the action of Cu(OAc)2·H2O to obtain compound 9, which is then alkaline hydrolyzed to obtain the final product PQQ.
Separation and purification method
After chemical synthesis, PQQ needs to be separated and purified. The specific steps include:
pH adjustment: The pH of the fermentation broth containing PQQ is adjusted to acidic with hydrochloric acid.
Adding flocculant: Adding flocculant precipitates protein and metal ions.
Standing and settling: After standing for a period of time, filter aids are added and impurities are removed in combination with the filter aids.
Ultrafiltration and nanofiltration: After ultrafiltration and nanofiltration, concentration is performed.
Extraction and stripping: The concentrate is purified by extraction and stripping.
Water washing and decolorization: After water washing, activated carbon decolorization, filtration, water washing again and suction filtration.
Drying: Finally, drying is performed to obtain the PQQ finished product.