Transformation Of Rare Ginsenoside CK By β-glucosidase From Aspergillus Niger And Its Immobilization Characteristics

Ginsenosides are the main active components isolated from ginseng.Compared with common saponins,rare saponins CK usually show good anti-tumor,improve immunity,enhance nervous system function and other pharmacological activities,but rare saponins do not exist in nature.The source of rare saponins is limited,the structure of is similar and difficult to separate,and the structure is complex and difficult to synthesize.Therefore,in this thesis,the enzyme conversion method with mild conditions,low pollution,high purity and specificity was used to obtain rare saponin CK,and high-yieldingβ-glucosidase strains were screened,and the conditions for enzyme production and conversion were optimized.The main results of the study are as follows:First,the strain Wu-16,which can rapidly transform and produce rare ginsenoside CK,was obtained by screening.ITS sequence analysis and observation of strain morphology confirmed that Wu-16 was Aspergillus niger.The results of Wu-16 enzyme production medium composition and enzyme production optimization showed that the medium composition was 2%bran,0.2%（NH₄）₂SO₄ and 0.5%KH₂PO₄,and the culture conditions were:inoculum volume 3%,30℃,150 rpm and p H=5.5,the highest enzyme activity was 0.71 U/m L.As an inducer,ginsenoside Rb1 has the most obvious promoting effect on the production ofβ-glucosidase.When Rb1 was 5 g/L,the enzyme activity was 1.287 U/m L.The enzyme activity was increased by 40.89%after adding Fe³⁺.In addition,after the initial separation and purification ofβ-glucosidase,G-25 demineralization column was used to remove ammonium sulfate.For each 1 m L of crude enzyme solution purified,the first 3 m L of enzyme fractions were collected.The results of protein molecular weight determination showed that the main molecular weights were 120,60,35 and 30 k Da.Secondly,in order to improve the conversion rate of ginsenoside Rb1 catalyzed by the enzyme,the enzymatic reaction conditions were optimized.The results of single factor optimization were that the reaction time was 48 hours,p H=3,temperature was 55°C,and the ratio ofβ-glucosidase to substrate was0.8.In addition,when the reaction time was 48 hours,the response surface optimization result was that the temperature was 55.03°C,the ratio of enzyme to substrate was 0.85,and p H=3.4,the yields of F2 and CK were 50.11%and 33.50%,respectively.On this basis,0.5 m M Co²⁺significantly increased the production of rare ginsenoside CK with a yield of 56.10%.The free enzyme stability experiment indicating that the free enzyme has poor stability under various harsh conditions.Finally,the MOFs co-precipitation method was used to immobilize the enzyme.When the metal and organic ligands were Cu²⁺and terephthalic acid,respectively,a large amount of precipitation could be produced in a short time.The results showed that when the ratio of metal to organic ligand was 2:1 and the co-precipitation time was 0.5 h,the encapsulation rate reached 71.43%and the encapsulation capacity was171.78 mg/g.Structural analysis,in which SEM showed that the morphology did not change and the porosity decreased after fixation.XRD,IR and TGA analysis showed that the original MOF structure and properties were not changed before and after immobilization,which also verified that the enzyme was successfully immobilized.Under adverse environments such as high temperature,different p H,the presence of chemical denaturants,and long-term storage,the stability is greatly improved,which solves the problem of poor stability of free enzymes.The immobilized enzyme was able to catalyze ginsenoside Rb1directionally,and it was completely converted into the rare saponin CK with a conversion rate of 49.46%.The immobilized enzyme has repeat stability,and the enzyme activity remains 53%after 8 cycles of application.Therefore,theβ-glucosidase obtained from the fermentation of the safe strain Wu-16 can be used to catalyze the conversion of the common saponin Rb1 into the rare saponin CK with good anticancer efficacy.The cost of using enzymes is reduced,and this method is a simple,convenient and fast way to produce rare saponin CK.