Screening And Study Of Riboflavin Biosynthesis Inhibitor Producing Strain

Riboflavin (riboflavin) serves as the precursor of FMN and FAD. Human and animals must obtain riboflavin from dietary except for small synthesis by intestinal bacteria, while many microorganisms, such as gram-negative pathogenic bacteria and pathogenic yeasts, rely on biosynthesis by themselves because of the deficiency of an effective intake system. Therefore, the riboflavin biosynthesis can be a new target for antibacterial material screening. This study aimed to screen strains from soil that can inhibit the biosynthesis of riboflavin by an in vivo screening, and then investigate the inhibition performance in vitro. In this research the strain was also identified, and fermentation medium and fermentation condition were optimized. The results were as follows.(1)Using Candida famata as the indicator bacteria which can produce riboflavin, strain Q210 that can inhibit riboflavin biosynthesis was isolated by screening of agar block method, followed by screening of cylinder-plate method and the shake flask test. The inhibition rate of riboflavin synthesis to per biomass of Candida famata was 57.63%.(2) In vitro enzyme reaction system of riboflavin biosynthesis of Candida famata was constructed. Then in vitro confirmation experiment of strain Q210 inhibition was carried on. The results showed that the inhibition rate of riboflavin biosynthesis was 93.62%. The conditions of enzyme reaction system were optimized. The results indicated that the optimum temperature was 29℃and the optimum reaction time was 30 min. When stored at -30℃for 45 days, the activity of crude enzyme liquid was still 91.75%.(3)According to physiological characteristics, biochemical characteristics and 16S rDNA sequence analysis, strain Q210 was identified as Bacillus subtilis.(4)The physical and chemical properties of active substance of strain Q210 had been studied. The results showed that the active substance was water-soluble substance produced in cell. It has stability at 20℃to 90℃or pH 2 to pH 12 range. When stored at 4℃for 10 days, the activity almost did not change. Proteinase K experiment demonstrated active substance was multi-peptides or protein material.(5)The fermentation condition and foundation fermentation medium had been studied. The results showed the optimum foundation fermentation medium was modified alkaline PDA medium and the optimum fermentation time was 4 days. Orthogonal test experiment showed the best culture conditions were 60 h of strain age, 40 mL of the medium volume in the 250 mL Erlenmeyer flask, pH 8.0 of the fermentation medium, and 2% of the inoculum amount.