Inulinase Producing Strains Screening And The Optimization Of Its Enzyme Production And Inulin Ethanol Fermentation Condition

Object: In this study inulinase strains was isolated from Jerusalem artichoke roots soil in Xinjiang Shihezi area, being screened and purified, and through 60 Co mutation increased the enzyme activity.By screening a strain of high inulinase strains of surface optimization, response to the conditions of enzyme production. Combined with the beer yeast for ethanol fermentation, after 60 Co mutation, high inulinase activity was determined, and Jerusalem artichoke ethanol fermentation optimized the culture condition, so as to establish the theoretical basis of ethanol production from Jerusalem artichoke.Methods:(1) acquisition of Jerusalem artichoke roots soil, were cultured by using selective culturing, inulinase strains were isolated and purified by plate method and limited dilution method.(2) The strains isolated were the determination of enzyme activity, and selected a inulinase and strains were 60 Co mutation breeding, which was a high-yielding inulinase strains.(3) According to designing experiment, the enzyme production conditions were optimized. The Plackett-Burman was designed based on the single factor experiment(PB) test, Plackett-Burman(PB) according to the experimental results, the effects of different factors, using the steepest ascent experiment to determine the significant influence factor of center point, with the steepest ascent experiment results as Central Composite Design(CCD) response surface experimental design, response surface optimization experiment. Statistical data was processed and mapped by using Design-Expert8.0 software and Origin8.0 software.Results and Conclusion:1. Helianthus tuberosus were successfully isolated from saline alkali soil of area of Xinjiang Shihezi. By using the limited dilution and streaking’ methods single colony were selected, obtaining 12 strains producing the inulinase, which their enzyme activity was measured. One of them have a higher activity. The enzyme activity reached 17.2U/m L, which was named as G-60. The strain G-60 as starting strain of inulinase, 60 Co mutagenesis, which can improve the ability of inulinase.2. Selected 0Gy, 8000 Gy, 1000 Gy and 12000 Gy dose of 60 Co, respectively, strain G-60 was irradiatted mutagenesis. After 60 Co irradiation induced mutation, 100 ul mutated spore suspension was obtained. Positive mutantions were selected by appropriate dilution coating plate, and the activity of enzyme was measured after subcultured which the production capacity reaches the 31.1U/m L and it is 1.8 times the mutation of 17.2U/m L, named G-60’.3. Strain G- 60’optimization of enzyme producting conditions, on the basis of the Plackett- Burman(PB) experiment, using the steepest climbing a hill, determining the Central Composite Design(CCD) response surface Design test center, three factors of the inulin concentration, peptone and fluid volume affecting significantly the response surface optimization experiment was carried out, established strains G-60’ inulinase from the enzyme activity of quadratic polynomial regression model,through the response surface method, the validity and reliability of the model were analyzed and the interaction between various factors, obtained from the strain G-60’ inulinase optimizing process conditions for enzyme activity: inulin 66.5 g/L, peptone 29.1 g/L, and liquid volume of 49.4m L. Under the process conditions, strain G-60’inulinase from the enzymatic activity of 67.8U / m L, and optimization before inulinase from the strain G-60’ enzyme activity 31.1 U/m L compared increased 2.18 times.4. Strain G- 60’as the enzyme producting strains, beer yeast was the ethanol fermentation strains, which can conduct inulin ethanol fermentation. The fermentation conditions were optimized through the Plackett- Burman(PB) experimental Design and Central Composite Design(CCD) response surface experiment Design, making the concentration of ethanol and utilization rate of inulin reach maximum. By Plackett- Burman(PB) screening for concentration of inulin, fluid production and fermentation time were significant factors influencing the utilization ratio of ethanol fermentation and inulin. The utilization rate of inulin and ethanol concentration multiple quadratic polynomial mathematical regression model were established by using Central Composite Design(CCD) response surface optimization. The effectiveness of the model and the interaction between different affecting factors were analyzed, and the regression equations were obtained by searching the best parameters and using the rate of inulin inulin in ethanol fermentation of inulin to: 175.4g/L, medium volume 102.5m L, fermentation time 75.0h. Under these conditions, the utilization rate of inulin can reach 92.2%; when the inulin concentration, liquid volume in flask and fermentation time were 175.1g/L, 102.5m L and 75.7h,respectively, ethanol concentration could reach the maximum value 81.3g/L. Combined with the actual experimental conditions to select the inulin concentration 175.0g/L, the volume of liquid 100.0m L, fermentation time 75.0h, the multiple regression model had been tested for two tests, and the test results close to the predicted value, indicating that the response surface model can better predict inulin utilization rate and ethanol concentration prediction in inulin ethanol fermentation.