The Study Of Chemical Composition And The Identification Of Prenyl-cleaving Enzymes On Schizophyllum Commune

Background:In nature,natural products can be derived from fungi,bacteria,plants and animals.Most of these natural products have certain biological activities?antioxidant,antifungal,antibacterial,etc.?and practical application value?medical,edible and industrial production,etc.?.Such as Lovastatin,from Aspergillus terreus with cholesterol-lowering effect;Cephalosporins C,from Crown cephalosporin with bactericidal effect;Artemisinin,from Artemisia annua Linn has the effect of treating human malaria.Fungi in nature have developed into one of the main sources of life-saving drugs and agricultural medicines.Fungi can be divided into higher fungi and common fungi as well.In addition,the extensive biological activity and practical application value of fungal-derived secondary metabolites have attracted the interests of scientific researchers to conduct extensive research on it,so that there will be a series of active drugs that are continuously reported,and promoting the development of new antibiotics.Schizophyllum commune is a famous medicinal-edible merger type higher fungus.Its medicinal value is mainly reflected in its rich polysaccharides,which can play a role in preventing chronic bacterial infections and inhibiting tumor growth.At present,this function has been widely used in the field of medicine.Previous studies reported several secondary metabolites with typical medicinal value derived from Schizophyllum commune,including Schizine A\B,which has the function of inhibiting the growth of cancer cells,and the isomer of Schizine A,Epischizine A,which has cholesterol lowering effect.The compound squalene synthesis inhibitor-Schizostatin,Ergosterol peroxide with anti-tumor activity,anti-tumor drug-Sisophila?Schizophyllan,also known as sonifilan?.In view of the relatively few natural products isolated from Schizophyllum commune,the chemical composition of them has been studied in this paper in order to obtain some compounds with novel structures or good biological activities.And excavate some new biological enzymes with unique catalysis to broaden the source of biological enzymes.Obejective:1.Under laboratory conditions,select the most suitable medium for Schizophyllum commune.2.Using Schizophyllum commune as the operating strain,look for natural products with potential application value,and conduct series of natural product isolation and structural analysis.3.Through bioinformatics,combined with biosynthesis related technologies,excavate new biological enzymes and explore their catalytic functions through enzymatic characterization.Methods:1.using different culture media to cultivate Schizophyllum commune,observeing its growth,and determining the optimal growth conditions of Schizophyllum commune in the laboratory by LC-MS analysis.2.Under the optimal conditions,a large number of Schizophyllum commune was cultivated,and the organic solvent was extracted to obtain the crude product extract.3.Using TLC and silica gel chromatography to separate the crude extract.The compound components after the crude separation are finely separated by MCI chromatography column,Sephadex LH20 column chromatography,semi-preparative liquid phase method,etc.to obtain a pure product.4.Identifing the structure of the compound by nuclear magnetic resonance hydrogen spectrum,carbon spectrum and two-dimensional spectrum?NOESY,COSY,QC,BC,etc.?.5.Using bioinformatics methods?local Blast,antismash,NCBI,etc.?to mine enzymes with DUF2236 functional domain in Schizophyllum commune.The protein expression plasmid was obtained through target gene synthesis,PCR amplification,and recombinant expression plasmid construction,and the catalytic function of the biological enzyme was proved by in vitro enzymatic characterization.Results:1.Cultivating Schizophyllum commune in a small amount on PDA,rice,ICI,and Salmonella medium.Mass spectrometry detection determines that rice is the most suitable medium for Schizophyllum commune.2.After 30 days of large-scale cultivation of Schizophyllum commune in rice medium,the organic solvent was extracted in three times,and a total of 22.0 g of crude extract was obtained.3.The total crude extract is subdivided into 12 components?Fr.1-Fr.12?by silica gel chromatography column method.According to the characteristics of the metabolic spectrum,Fr4,Fr.5 and Fr.6 are combined,named as Fr.456.And MCI column separation is performed on Fr.456,Fr.7,Fr.9 and Fr.11.4.In Fr.456,compound 1 was isolated and identified.The structure of the compound is relatively simple,and its practical application value is not high.It has been widely studied;In Fr.11,compound 2 is isolated and identified,which is currently widely used as an antioxidant In the medical field,it has the advantages of long half-life and no obvious toxic and side effects.Compound 3 was isolated and identified in Fr.7,which is a more effective squalene synthase inhibitor with cholesterol-lowering effect;In Fr.9 compound 10 was isolated and identified,and its structural difference from compound 3 was that the terminal ethylenic bond became a hydroxyl group.5.The bioinformatics method found a homologous protein Sc-S6-37-2 with high consistency and similarity to Globin family protein MpaB.The in vitro enzymatic characterization results of Sc-S6-37-2 and 3 indicate that it can catalyze the cleavage of the 3-terminal ethylenic bond to form 10.Conclusion:Under conventional laboratory conditions,this subject determined that the most suitable medium for Schizophyllum commune was rice.Four natural products were isolated from Schizophyllum commune by fermentation of fungi,extraction of metabolites,and separation of different compounds.Compound 1 was2-methoxyresorcinol with molecular formula C₇H₈O₃,and compound 2 was N-acetyl with molecular formula C₁₂H₁₄N₂O.The base tryptamine,compound 10 is2-Butenedioic acid of formula C₁₇H₂₆O₅,and compound 3 is Schizostatin of formula C₂₀H₃₀O₄.Finally,the biological enzyme Sc-S6-37-2 with the functional domain of DUF2236 derived from Schizophyllum commune not only can catalyze the cleavage of FDHMP,but also catalyze the cleavage of the 3 terminal olefin bond to 10,thus enriching the enzymes with such functions family resources have laid the foundation for the subsequent application of combinatorial biology to use this type of enzyme for the transformation of active natural products.