Study On Immobilized Glycoside Hydrolase And Preparation Of Rare Ginsenoside CK

Because of its extremely high pharmacological activity,rare ginsenoside CK has become a research hotspot and is widely used in the pharmaceutical and skin care industries.However,CK is almost absent in natural ginsenoside plants.At present,the preparation of CK mainly uses microbial or enzyme catalysis to hydrolyze the sugar chains on the original ginsenoside saponins.Among these two methods,the enzymatic method has the characteristics of high specificity,high yield,and production efficiency,and is currently the preferred method for preparing CK.However,the use of free enzymes for catalysis has the disadvantages of product pollution,difficulty in recycling,and enzyme preparations that cannot be reused.Immobilization of free enzymes on carriers to make immobilized enzymes can well solve the above problems,thereby meeting the needs of industrial production of CK.Therefore,in this paper,a fermentation enzyme solution of a strain of Aspergillus niger isolated earlier in the laboratory was fixed with resin,and Rb₁ was used as a substrate to produce rare ginsenoside CK.The main contents of this thesis are as follows:(1)finding a suitable immobilization carrier;(2)optimizing the immobilization conditions;(3)exploring the enzymatic properties of the immobilized enzyme and determining the reaction conditions of the immobilized enzyme.(4)Preliminary exploration of the technological mode of industrial production of CK.The results obtained are as follows:(1)we selected the epoxy-amino bifunctional resin HFA-001 for immobilization and its enzyme activity reached 5450 U/g;(2)the immobilization conditions were:resin carrier and mass fraction of 2%Glutaraldehyde was mixed and cross-linked for about 1.5 h at a ratio of 1:10(g/mL),then 1 g of resin was taken,10 mL of free enzyme with a protein concentration of 2.2 mg/mL was added,and mixed at a temperature of 15?-25?fixed 8 h;(3)(1)Free The optimum reaction conditions for the enzyme were pH=4.0 and 50?.The optimum pH for the reaction of the immobilized enzyme is 4.5,55?.(2)The K_m values of substrate Rb₁ of free enzyme and immobilized enzyme were 0.522 mmol/L,6.3 mmol/L,and V_maxax were 18.69mmol/min and 1.42 mmol/min,respectively.(3)The thermal stability of the immobilized enzyme is improved.(4)The stability of low-temperature storage has not improved significantly.(5)When the immobilized enzyme is reused for the second time,the enzyme activity decreases to 68.48%.With the increase of the number of uses,the enzyme activity continues to decrease.After six uses,the enzyme activity is about 25%on average;(4)Different in the inquiry Effects of organic solvents on enzyme activity,the immobilization was more resistant to ethanol than free enzyme.(5)Treatment of the immobilized enzyme with pure ethyl acetate,the enzyme activity of the immobilized enzyme was increased by nearly 1.4 times,and the reaction was performed in a 15%ethanol solution.Compared with the enzyme activity before the treatment,the enzyme activity of the immobilized enzyme was increased.(6)In the production process,we choose the batch hydrolysis mode.When the enzyme is immobilized in the column is 9g,300 mL of substrate Rb₁ with a concentration of 0.4mg/mL can be added at 45?and the flow rate is 10 mL/min.After 6 hours of reaction,ethanol and macroporous resin were added to another column,so that the ethanol concentration in the system was 15%.After reacting for another 10 hours,the maximum yield of CK recovered from the macroporous resin was 74%.