Isolation And Identification Of Endophytic Fungi From Polygonum Cuspidatum And Studies On The Transformation Of Polydatin

The traditional Chinese medicine Polygonum cuspidatum is rich in polydat in,and using biotransformation technology to convert it into resveratrol is an effective way to prepare natural resveratrol products.However,the common extraction methods are more limited and cause great environmental pollution.The microbial transformation method can be used to transform polydatin to obtain resveratrol.In this paper,we screened the endophytic fungi that can efficiently transform polydatin to obtain resveratrol from Polygonum cuspidatum,and studied the culture conditions,the conversion conditions of?-glucosidase and the enzymatic properties.The research results are as follows:(1)Using a selective culture plate with polydatin as the sole carbon source,four well-growing endophytic fungi were isolated and screened from Polygonum cuspidatum,and they were numbered HZ001,HZ002,HZ003 and HZ004;the four strains of cells and fermentation broth were investigated The performance of transforming polydatin and the identification of strains by morphology and molecular biology.Bacterial cell biotransformation test results show that HZ001 and HZ004 can effectively transform polydatin to produce resveratrol,and the molar conversion yield can reach 100%,while resveratrol is not detected in HZ002 and HZ003 bacterial cell transformation systems The biotransformation results of fermentation broth showed that the molar conversion yields of HZ001,HZ003,and HZ004 fermentation broths converted from polydatin to resveratrol were55%,40%,and 38%,respectively.The results show that HZ001 and HZ004 have the strongest performance in transforming polydatin to produce resveratrol,which mainly depends on intracellular enzymes.The results of strain identification showed that HZ001 and HZ002 were classified as Aspergillus aculeatus and Penicillium georgiense,respectively,and HZ003 and HZ004 were both classified as Aspergillus flavus.(2)The single factor test method was used to investigate the effect of transformation conditions on the transformation of Polygonum cuspidatum endophytic fungus Aspergillus aculeatus HZ001 to produce resveratrol.The results showed that the p H of the PBS(phosphate)buffer was 6.5,the substrate concentration was 0.1 g/L,the temperature was 30°C,the cell dosage was 1 g,the conversion time was 48 h,and the conversion rate could reach95%.(3)The single factor and response surface method were used to study the medium composition and culture conditions of Aspergillus aculeatus HZ001 strain producing?-glucosidase.The results of single factor experiments showed that the nitrogen source was sodium nitrate with a concentration of 0.2%;the carbon source was sucrose with a concentration of 2.5%;the initial p H was 7,the temperature was 28°C,the cell dosage was8%,the liquid volume was 125 m L,and the culture time was 96 h.The rotation speed is 150r/min,and the experimental result is 931 U/m L;the response surface test results show that the temperature is 29.12?,the initial p H is 6.92,the shaker rotation speed is 148.36 r/min,and its theoretical value is 1134.4 U/m L;for the convenience of the experiment,Selecting a temperature of 29°C,an initial p H of 7,and a shaker speed of 148 r/min,the enzyme activity was measured to be 1025.4 U/m L;the response surface optimization was compared with the single factor experiment results,and the optimization effect reached 23%.(4)Using salicin and polydatin as substrates,the enzymatic properties of?-glucosidase produced by the endophytic fungus Aspergillus aculeatus HZ001 from Polygonum cuspidatum were investigated.When polydatin is used as the substrate,the inhibitory effects of Mg²⁺and Ca²⁺are the most obvious under the conditions of the optimum temperature of60?and the optimum p H of 5.7.The four inhibitors of SDS,mercaptoethanol,dimethyl sulfoxide and EDTA have The enzyme activity has an inhibitory effect.The Michaelis constant Km value of the catalytic reaction is 6.993 mmol/L,and the Vmax value is 22.988?mol/(m L?h).When salicin is used as the substrate,the optimum temperature is 60?and the maximum Under the condition of suitable p H 4.8,Fe²⁺can promote the enzyme catalysis,and Co²⁺plasma obviously inhibits the enzyme catalysis.The most obvious inhibitory effect of EDTA is only about 10%.The Km value of the catalytic reaction is 2.571 mmol/L,and the Vmax value is 0.594?mol/(m L?h)...