Breeding Of Efficient Isoquercetin Producing Strains By Heavy Ion Mutagenesis And Optimization Of Transformation Conditions

Because of its good quality of pharmacological effects,isoquercetin has caused widespread concern in the medical field.However,the content of isoquercetin is very low in natural source,and the extraction is so difficult that its promotion and use were severely affected.The method of bio-enzymatic hydrolysis from rutin to isoquercetin is simple and isoquercetin can be produced in large scale.In recent years,this method is one of the most effective approaches to produce isoquercetin.Bacillus litoralis C44,which was screened by our laboratory in previous work,is capable of degrading rutin to produce isoquercetin specially.After C44 ¹²C⁶⁺ ion mutagenesis breeding,we obatined 330 mutants that grew well under the condition of pH 9.0 and 32 ?.13 strains were obtained by quantitative TLC screening,the ?-L-rhamnosidase conversion efficiency of the strains were higher than that of the original strain,among which M5B15 strain had the highest transformation activity and the best genetic stability.Under the same conditions of 3 g/L of substrate conversing for 24 h,the conversion rate of the strain M5B15 was 171.3% of the original strain C44.The molar conversion efficiency of the mutant strain M5B15 was 155% of that of the original strain C44 by HPLC.The medium composition and fermentation conditions of the mutant strain M5B15 were optimized.The OD600 values of the cells were 2.5±0.12 and 16.4±0.39,respectively,and the cell concentration was improved by 5.6 times.Which were effectively improved the unit time biomass of the bacterial,reducing the cost of fermentation.The transformation system was further optimized based on the optimized conversion conditions.The results showed that the minimum time for the substrate to be completely transformed into the product was 10 h under the concentration of 3 g/L rutin and the wet cell concentration was 100 g/L in pH 9.0 glycogen-sodium hydroxide conversion system,shortened by 14 h,shorter than the original strain 38 h;By HPLC analysis,the substrate conversion rate was 2.27 times higher than that before optimization,4.62 times higher than that of the original strain,which were effectively improving the substrate conversion efficiency.Under the condition of ultrasonic power of 25 kW and phacoemulsification time of 30 min,soybean lecithin could produce good inclusion with rutin at a molar ratio of 1: 1 at a concentration of 10 g/L,and the relative transformation rate of the strain after adding soybean lecithin increased by 12.6%.The optimum reaction conditions were as follows: substrate concentration 10 g/L,cell concentration 350 g/L,transformation time 24 h.Further study showed that the concentration of substrate was 20 g/L glucose and 10 mmol/L Zn2+ in the transformation system,and the substrate transformation rate increased by 18.0%.In this paper,¹²C⁶⁺ heavy ion mutagenesis of isoquercetin-producing strain C44 was carried out,and a highly active mutant M5B15 was screened.When the substrate concentration was 3 g/L,the cell transformation time was greatly shortened and the molar conversion efficiency was significantly improved.The results showed that the optimization of the medium composition and fermentation conditions of the mutant strain M5B15 effectively increased the per unit time biomass and reduced the fermentation cost.Through the optimization of the transformation system,the conversion time was 10 h at substrate concentration of 3 g/L,and the substrate conversion efficiency was improved significantly.The solubility of the substrate was increased from 3 g/L to 10 g/L,and the conversion system was expanded to 1 L,which laid the foundation for the large scale production of isoquercetin.