Mutagenesis,optimization And Enzymatic Properties Of ?-glucanase Production By Chaetomium Globosum

?-glucanase,also known as dextranase?EC 3.2.1.11?,acts primarily by recognizing and cleaving?-1,6 linkages in dextran??-glucan?,breaking down long chain glucan into glucose or linear oligosaccharides.It has important application in the sugar industry,preparation of medical dextran,and prevention and treatment of dental caries,and it also has great application prospect in the fields of chemical industry and medical detection.In this paper,a new strain of?-glucanase production was successfully isolated from soil samples by comparing the size of transparent zones produced by the strain on the screening medium of blue glucan.The strain was identified as Chaetomium globosum according to its phenotype,biochemical characteristics,and rDNA analysis.The strain has been deposited in the china general microbiological culture collection center,with preservation number 15867.The strain was induced by atmospheric and room temperature plasma?ARTP?combined with ethyl methyl sulfone?EMS?mutagenesis.Irradiation distance of 2 mm,injection of gas helium,gas flow rate of 10 L·min^-1,power of 100 W,time of 120 s are the optimal conditions of ARTP mutagenesis.0.5 m and 4 h are the optimal working final concentration and mutagenesis time of EMS.A positive mutant strain with higher?-glucanase production than the wild was obtained,and the yield of?-glucanase was 49.53 U·mL^-1,which was 30.31%higher than 38.01 U·mL^-11 of the wild.The mutagenic strain was stable for several subcultures.The culture medium conditions of the enzyme-producing strain were optimized.The best carbon and nitrogen sources for enzyme production were dextran T20 and yeast extract at optimum concentrations of 20 and 10 g·L^-1,respectively.The optimum concentrations of K₂HPO₄ and MgSO₄ were 2.0 and 0.5 g·L^-1,respectively.The enzyme activity of?-glucanase reached 427.91U·mL^-11 under these optimum culture conditions for 8 days.The optimum conditions of fermentation were as follows:initial pH of 7.0,volume of 70 mL,shaking speed of 200 r·min^-1,culture temperature of 28°C and inoculation of 6%.The enzyme activity of?-glucanase under these optimum culture conditions for 8 days reached 824.73 U·mL^-1,which was 16.65-fold higher than the enzyme activity before optimization(49.53 U·mL^-1).After ammonium sulfate salting,dialysis concentration and Sephadex G75 chromatography,the specific enzyme activity reached 8350.8 U·mg^-1,with an 11.81-fold specific activity and a recovery of 18.8%.The enzyme produced by the mutant strain displayed the same optimum pH and temperature values of 5.5 and 60?,respectively,as the enzyme produced by the wild-type strain.Enzyme activity was stable at pH 4.5-7.0 and displayed sufficient thermal stability at temperatures20-50?,but a higher heat resistance of the enzyme after mutagenesis than before at 20-70?.Homologous modeling and molecular docking technology were used to study the differences of?-glucanase molecular structure before and after mutagenesis.The catalytic centers were Asp 408,Asp 409 and Asp 431 but the 578 amino acids of the mutated enzyme changed from histidine to tyrosine,which may result in minor changes in the secondary structure of?-glucanase and affect its properties.The ?-glucanase from C.globosum is an endodextranase.It can hydrolyze large molecules of glucan into different moleculars,and it has a higher catalysis to large molecules of glucan.Within0-15 minutes,the enzyme catalyzed the reaction of glucan T2000?3%?with a final concentration of2 U·ml^-1,the molecular weight of glucan decreased from 2000 kDa to 41 kDa.The enzyme with a final concentration of 1 U·ml^-1 catalyzed glucan T2000 at different concentrations.When the concentration raised to 3%,the catalytic efficiency was the highest,and the molecular weight decreased from 2000 kDa to 60.2 kDa.?-glucanase has great inhibition in growth of Streptococcus mutants and the synthesis of glucan biofilm.As the concentration of?-glucanase increased,the growth of S.mutans decreased and the synthesis of biofilm was inhibited.When the concentration of?-glucanase reached 50U·ml^-1,the rate of inhibition of S.mutans biofilm formation by?-glucanase was 71.58%,while the clearance rate was 49.07%.This enzyme has great potential in the prevention and treatment of dental plaque.