Separation And Purification Of ?-mannanase From Bacillus Licheniformis HDYM-04 And Study Of Its Enzymatic Properties And Structure

?-mannanase is a kind of hydrolytic enzyme can hydrolysis any beta-1.4glycosidic bond hydrolysis,due to the ?-mannanaseuesd in food,paper making,textile and other industries has an important role,So the separation and purification of?-mannanase and enzymology properties research has caused widely public concern,In order to obtain high purity of ?-mannanase,For the separation and purification of?-mannanase has a lot of reports and has set up a crystal now,But the pure enzyme only get from the fungus,The ?-mannanase is lessly originate from bacterial,so the thesis chose the preservation strains selected the Bacillus licheniformis HDYM-04,?-mannanase putified by the means of acetone precipitation,anion exchange column chromatography,gel filtration chromatography and other methods for the combination of pure enzyme and study its enzymology properties,it will be study the pured enzyme by round two chromatography,its secondary structure,through studying the change of secondary structure under different conditions to master its change rule;For beta mannase applications,this experiment to dye decolorization test of purified enzyme,use the dye degradation test for the possibility of the strains on the production application to provide theoretical basis and experimental basis.This study first selection with ammonium sulfate precipitation and acetone precipitation crude enzyme extract compared in order to improve the enzyme activity,the extraction yield through comparison of acetone precipitation enzyme activity extraction yield is much higher than 99.13% ammonium sulfate fractional precipitation,acetone precipitation and it has the advantages of easy to recycle,extracted rapidly,and then to flow through the DEAE Sepharose fast flow and the method of combining with G-75 gel permeation chromatography to puirfied ?-mannanase step by step,with?-mannanase activity as the test indexes,to get ?-mannanase pure enzyme,finally purification ratio is reach 19.32,much higher than has been reported means,The pure samples by sds-page electrophoresis to test its degree of purity,the molecular weight of40 kda probably,than contrast with the sequence from B.licheniformis the?-mannanase consistency of 93.6%,so it can be identified as a ?-mannanase.This experiment apply with the circular dichroism spectroscopy research of its secondary structure,its mainly constitute with beta folding and corner,different temperature and pH effect on the ?-mannanase secondary structure,the results show that the influence of different temperature on the beta mannase is not obvious;Under different pH conditions,as the alkaline is gaining higher,the enzyme is reduce constantly,beta-fold is decreases and the corner is higher,which can be presumed,the corner to play an important role in maintaining the enzyme activity.it can be presumed to its constitute of the beta-fold,the structure is relatively stable.This experiment to study the ?-mannanase dye degradation effect,the selection of27 dyes decolorizing system full wavelength scanning experiment was carried out.Measured the ?-mannanase have strong degradation ability with azo-dye,the methyl orange,Congo red dye and phenol red decolorization rate can reach more than100% in 12 h,effect of decolorization of triaromatic methane is less obvious,including water soluble aniline blue and malachite green dye within 12 h decoloring effect is about 90%,decoloring effect is obvious,crystal violet within 12 h decolorization rate of more than 50%,other dye effect is not significant.This research through the ?-mannanase purification conditions optimization,improve the purification of ?-mannanase multiples,thus reach downstream purification time,reduce the production cost of purpose,and by the test of dye degradation,to explore the possibility of the strain on the production application,lay the foundation for the industrialization application.