Study On Biosynthesis Of Piperonal

Piperonal is one of the most important and attractive spices in the world,and is widely used in food,medicine,agriculture,cosmetics industries.So far,the production of piperonal is mainly by chemical synthesis.With the pursuit of green healthy products,the research on the preparation of piperonal by biological methods has drawn more and more attention.In this paper,the screening of the piperonal-producing strains,the synthesis mechanism of piperonal,the optimization of culture medium and culture conditions and the preliminary purification of the target protein were studied.The detailed research results are as follows:?1?Screening and identification of the piperonal-producing strain.A total of 1004 strains were screened by enrichment culture from 25 soil samples.A strain named ZMT-1 was found to produce the highest amount of piperonal and was isolated by a high-throughput screening method?24-well plate assay?by color reaction using 2,4-dinitrophenylhydrazine.Gas chromatography-mass spectrometry analysis was used for the qualitative analysis of the product.The quantification analysis of piperonal was carried out by high performance liquid chromatography,and the yield of piperonal was 49.8 mg·L-1.This strain was identified as Serratia liquefaciens on the basis of its morphological,physiological,and biochemical characteristics as well as its16 S rDNA sequences analysis,and has been preserved in China Center for Type Culture Collection?CCTCC NO: M2016170?.?2?Investigation on the location of enzyme capable of synthesizing piperonal.Intracellular enzymes,periplasmic enzymes and extracellular enzymes were extracted from strain ZMT-1 and were used to determine their ability to produce piperonal.The results indicated that the piperonal-producing enzyme is extracellular enzyme.?3?Optimation of fermentation medium and culture conditions.The fermentation medium compositions were optimized by the single factor experiment,which indicated that the selected carbon,nitrogen sources and metal ion were glucose,complex nitrogen source?yeast extract and NH₄NO₃?and Mg²⁺,respectively.In addition,NH₄NO₃ and K₂HPO₄·3H₂O were found to have significant effects on piperonal production by the Plackett-Burman design,and the final medium composition was determined by combining with response surface analysis: glucose 10 g·L-1,yeast extract 0.75 g·L-1,NH₄NO₃ 1.41 g·L-1,MgSO₄·7H₂O 1 g·L-1,KH₂PO₄ 2 g·L-1,K₂HPO₄·3H₂O 0.5 g·L-1,Na Cl 0.25 g·L-1.The optimal fermentation conditions in flasks were initial medium pH of 7.0,150 m L culture in a 500 m L flask,5% 8% of inoculum,30°C of temperature,10 h 12 h of seedling age,and 20 h 24 h of fermentation time.The optimized enzyme activity was 4.4 U·mL-1,which was 25.8 times higher than that before optimization?0.17 U·mL-1?.After cultivation for 48 h in 3 L fermentor,the final enzyme activity was 5.1 U·mL-1,which was increased by 1.13 times compared with shake flask fermentation.?4?Investigation of crude enzyme properties and preliminary purification of the target protein.The results indicated that the optimal reaction temperature of the crude enzyme was 30°C and the optimal reaction pH was 5.0.The target protein was preliminarily purified by ammonium sulfate precipitation,desalination using HiPrepTM 26/10 desalination column and HiTrapTM Q HP anion exchange chromatography,and SDS-PAGE showed that the protein about 50 kDa is probably the target protein.MALDI-TOF/TOF mass spectrometry was used to identify this protein,and the result indicated that the protein had the highest peptide matching rate with the translation elongation factor?Tu?in Pseudomonas,but the matching rate is only 8%,suggesting that the protein may be an unknown protein.