Study On Strain Screening Of High Biotin-Producing

Biotin is an essential human nutrient. Biotin is used for many enzymes as the cofactor in the body. It plays an important role in the metabolism of carbohydrates, lipids, proteins and nucleic acid. In this dissertation, strain breeding of high biotin-producing had been studied systematically, including the determination method of biotin, strain screening of secreting biotin by growth rings method, the strain improvement by rational selection, the optimization of fermentation medium and conditions, and the identification of high biotin-producing strain.Analyzing method of biotin by oxford plate assay system was introduced. The result shows that the relative standard deviation, the coefficient of linear correlation and the average recovery were 4.98⁷.57%, 0.9933, and 101.02% respectively.Growth rings method was used to screen strain of secreting biotin in this dissertation. B-3 which was high yield and stable was got through several rounds of screening. The productivity reached 250μg/L. It was hundreds of times higher than other reported strains.Based on the biosynthetic pathway and metabolic regulation, a relational selection procedure was performed to obtain a higher biotin-producing strain. After UV mutation, ¹³⁷Csγmutation, ion beam mutation, the higher productive strain B3R6-3 was selected which could grow well on the medium with 5-（2-thenyl） pentanoic acid and 2-thenoyltrifluoroacetone. The productivity reached 939.8μg/L, which was 4 times than that of the parent strain B-3.The optimization of the fermentation progress was studied. The results showed that glycerin 40 g/L,（NH₄）₂SO₄ 10 g/L, urea 2.33 g/L, K₂HPO₄ 2.1 g/L, MgSO₄ 0.5 g/L, the volume of medium 50 mL, the inoculation amount 9%, pH7.5, culture temperature 30℃, shaking speed 200 r/min ferment for 5 days. The productivity reached 4131μg/L.Finally, the B3R6-3 was characterized with morphological and physiologic and biochemical analysis, the 16S rDNA sequence of B3R6-3 was also studied. The result proved that it was a Pseudomonas fluorescens species strain.