| Resveratrol in Polygonum cuspidatum is a ?-glycoside,which can be hydrolyzed to resveratrol by ?-glucosidase.?-glucosidase is mainly from fungi in microorganisms,such as Trichoderma,Aspergillus and so on.The enzyme activity is low and the stability range of temperature and pH is narrow,so that the price of ?-glucosidase is high,Limiting its use in industry.Since 99% of the microorganisms in nature are non-culturable,it is difficult to find ?-glucosidase with high enzymatic activity and good enzymatic properties by the traditional method.However,the macro genome technology can directly extract the total DNA in the habitat,without the need for microbial isolation and culture,so that the microbial non-culturable resources in nature can be fully utilized.In this study,the recombinant Escherichia coli(E.coli BL21(DE3)-p ET32a-bgl2238)screened and constructed from the Heilongjiang maize soil was constructed by the method of Box-Behnken optimization.The optimum medium ratio was glycerol 9.32 g / L,yeast extract powder 12 g /L,tryptone 19.13 g /L,NaCl 8g /L,K2HPO4.3H2 O 19.13 g /L,KH2PO4 2g / L,lemon Acid high iron by 0.2g / L and trace element mother liquor 6m L / L.The optimum fermentation conditions were as follows: fermentation temperature 37 ?,initial pH 8.0,3% inoculation amount,25 mL liquid loading,and IPTG final concentration of 0.5 mM.The optimum fermentation conditions were as follows: fermentation temperature 37 ?,initial pH 8.0.When the recombinant Escherichia coli Bgl2238 was fermented in a 250 m L Erlenmeyer flask,the enzyme activity of Bgl2238 was 2910 U / L under the optimum fermentation medium and culture conditions,which was 61.77% higher than that in the initial culture medium.In this paper,the process of degradation of polydatin from polygonum cupidatum was studied.The optimum enzymolysis process was as follows: the amount of enzyme was 1: 4,the ratio of material to liquid was 1:25,the time of enzymolysis was 12 h,Temperature 44 ?,speed 120 rpm,the degradation of polydatin rate can reach 95%.The content of resveratrol in polygonum cuspidatum was 6.87% by HPLC,which is extracted with 95% ethanol.The content of resveratrolwas 6 times higher than that of undigestion with Bgl2238.In order to further reduce the cost of Bgl2238 on the degradation of Polygdatin,Bgl2238 was used to immobilizing,and the stability of Bgl2238 can be improved.The preparation conditions and immobilization process of Bgl2238 immobilized were optimized by single factor method: the concentration of chitosan was 2.0%,the concentration of acetic acid was 1.5%,the concentration of NaOH was 15%,the concentration of methanol was 20%,the concentration of glutaraldehyde 0.5%,the best crosslinking time is 1h,the optimum crosslinking temperature is 20 ?,the best adsorption time is 4h,Bgl2238 crude solution is diluted 2 times.The recovery rate of enzyme activity of Bgl2238 immobilized was 87%,and the activity was 859.65 mU / g.The optimum temperature for immobilized Bgl2238 is 50 ° C,which is 6 ° C higher than the optimum temperature of free Bgl2238,and the temperature stability and pH stability are improved.In order to investigate the repeated properties of immobilized Bgl2238,when the immobilized Bgl2238 consecutive acted on polydatin,the degradation rate of polydatin was still more than 70%,which proved that the reusability of immobilized Bgl2238 was very good. |
PDF Full Text Request