In-Silico Screening Of Anti-Mildew Agents From Plant Alkaloid

Natural resources are the material foundation of modern industry.Lignocellulose,the most abundant renewable biomass resource,supports the prosperity of many modern industries including wood processing,forestry chemicals,textile,papermaking,packaging and express delivery.However,lignocellulose raw materials and products are prone to mildew and decay in warm and humid environments,which seriously reduces their value in use.Although serveral measures have been taken,e.g.the improvement of storage conditions and the use of chemical antifungal agents;however,there are still problems such as the significant increase of costs for process control and the potantial environmental safety risks.To date,many kinds of plant alkaloids have been studied as promising mold inhibitors,but there is lack of direct research work on fumdamentals.In this study,the method of computational simulation was used to screen plant alkaloids with anti-fungal activity.The docking of plant alkaloids with the enzymes required for mold growth was carried out to evaluate the binding sites,interaction force and binding energy between them.Experimental work was also performed to verify the in-silico prediction.The computer domain protein structure models of T.reesei cellulase CBHI,CBHII,EGI,and EGII simulated were docked with 16 small-molecule plant alkaloids.The results of the two simulation programs Autodock 4 and Vina operation are generally consistent,The larger the size of the alkaloid molecule,the stronger the binding energy and the stronger the inhibition of cellulase activity.Among the 16 plant alkaloids obtained by computer simulation,the plant alkaloids with the strongest inhibition on cellulase are Sol,Res and Cam,and the plant alkaloids ranked in the middle are Ber,Vin,Cro,Pip,Col,Atr,Mat,Sec,Cyt,the weakest are Gen,The,Tri,Sta.The reliability of the computer simulation results was verified by the experiment of inhibition of cellulase by plant alkaloids.Matrine,berberine,genistein and atropine sulfate with better water solubility were selected.The models of T.reesei amylase and chitinase were constructed by homologous modeling,and the models were evaluated and optimized.The catalytic activity regions of T.reesei amylase and chitinase were predicted.Then six different hydrolases from T.reesei,including ?-amylase,?-glucosidase,endo-chitinase,epoxide hydrolase,endo-mannanase and endo-xylanase were docked with alkaloids using Autodock Vina,almost all alkaloids could bind with the active pocket in any of these hydrolases.Specifically,berberine,matrine,genistein,atropine sulfate and stachydrine were screened out through experiments due to their inhibitory effects onthe growth of T.reesei.Among them,berberine showed the strongestinhibition.The addition of berberine up to 0.02 M into the culture medium prevented the growth of spores and hyphae of T.reesei within 72 hours.Three strains of fungi were isolated from bamboo parasites,which were identified to be Arthrinium sp.,Aspergillus sp.and Stagonospora sp.The cellulase models of Stagonospora sp.and Arthrinium sp.were constructed using the method of homology modeling.The models were refined with 3DRefine dynamic optimization.After the optimized models ANEG,SEG and AEG were used to dock with 16 plant alkaloids.The docking results showed similar relationships between the binding energy and the molecular size of ligands as compared with the T.reesei hydrolases.The subsequent experiments verified the inhibitory effects of matrine,berberine and cytisine on the growth of bamboo rot fungi.Using a two-step separation process of ammonium sulfate salting out and acetone precipitation,the activity of peroxidase can be isolated from A.triandra fresh fruits.The peroxidase was studied to catalyze the oxidative polymerization of isoeugenol.The activity of peroxidase was indentified from the characterization of polymerization products.The addition of A.triandra peroxidase in plant alkaloids strengthened the inhibition of matrine and atropine sulfate on cellulase activity.In another word,the enzyme,A.triandra peroxidase,has the potential use to improve the activity of anti-mildew agent from plant alkaloids.