Effects Of Heat And Ethanol On Cortex-lytic Enzyme Activity And Structure Of Bacillus Subtilis Spores

Ethanol and heat combined treatment(60-80 ?)can kill spores,and heat can affect the enzyme activity,high concentration of ethanol to passivation enzyme.Therefore,it is speculated that the reason why the synergistic action of heat and ethanol can kill the spores may be that it passivates the cortex-lytic enzyme.Therefore,in this paper,the effects of heat,ethanol and heat combined ethanol on the activity of cortex-lytic enzyme of Bacillus Subtilis were studied,and the structure changes of cortex-lytic enzyme were studied by ultraviolet absorption spectrum,fluorescence spectrum and Fourier transform infrared spectrum,so as to explore the mechanism of heat-combined ethanol killing spores.The main research results are as follows:Heat treatment on bacillus subtilis spores in the cortex-lytic enzymes,found that the cortex-lytic best enzyme activity is 40?,80 ? make enzyme inactivation.Ultraviolet absorption spectra showed that cortex-lytic enzymes showed an absorption peak around 280 nm,and the absorption intensity was different,which was related to the changes of Tip residue and Tyr residue in cortex-lytic enzyme.The fluorescence spectrum shows that the emission peak appears redshift with the increase of temperature,and fluorescence thermal quenching occurs.When the temperature is 40?,the low level redshift,tertiary structure is relatively stable.The redshift when the temperature is 80 ?,the biggest and the most unstable tertiary structure.In the infrared spectrum of the heat treatment cortex-lytic enzyme,the cortex-lytic enzyme was mainly composed of ?-angle,followed by ?-folding,a-helix and random curl.When the temperature range in the 4-30 ?,and p-folding,a-helix with the rise of temperature is almost the same,secondary structure content change is small,secondary structure is relatively stable.When the temperature is 40 'C,the(3-folding content increasing,the enzyme activity under the condition of the best.When the temperature is 80?,the ?-folding content is the lowest,enzyme activity basic inactivation,secondary structure is the most unstable.Therefore,it is inferred that the active center of cortex-lytic enzyme may be located in the ?-fold structure.The lower the enzyme activity,the greater the change of the ?-fold structure in the secondary structure and the more unstable the secondary structure.Ethanol on bacillus subtilis spores in the cortex-lytic enzymes treatment found that with the increase of ethanol concentration,cortex-lytic enzyme's ?-corner content rise,cut down the content of a-helix content and ?-folded more apparent,random coil content change is small,ethanol made the cortex-lytic enzyme secondary structure change in a certain degree.The maximum emission wavelength of fluorescence is red shift of 4 nm,indicating a change in the tertiary structure of cortex-lytic enzyme.The change of the second and third order structure of cortex-lytic enzyme is closely related to the change of enzyme activity,but it is not linear.Sulfhydryl changes indicate that new disulfide bonds are formed by free radicals on the surface of cortex-lytic enzyme.Without ethanol processing cortex-lytic enzyme endothermic peak of 72.72?,the ethanol concentration processed cortex-lytic enzyme,wide absorption fengfeng form,volatile.Ethanol in combination with heat treatment on bacillus subtilis spores in the cortex-lytic enzyme activity and structure affect found that cortex-lytic enzyme in 30%and 75%ethanol solution of the optimal temperature of 30?.The higher the temperature,the faster the enzyme activity decreases and the more obvious the passivation effect.When the temperature is 80?,the cortex-lytic enzyme basic inactivation.Fluorescence spectra showed that the cortex-lytic enzyme of 30%and 75%ethanol was significantly redshifted and the tertiary structure of cortex-lytic enzyme was changed,indicating that the ethanol co-heat treatment could make cortex-lytic enzyme inactivated and change the tertiary structure of cortex-lytic enzyme.The changes of the content of a-helix,?-folding,?-turning Angle and random crimp content in the secondary structure indicate that the changes of the secondary structure of cortex-lytic enzyme treated with different ethanol concentration and temperature lead to the changes of enzyme activity.The results show that both heat treatment and ethanol treatment could change the secondary and tertiary structure of cortex-lytic enzyme,but could not inactivate the enzyme activity rapidly,while ethanol combined heat treatment could rapidly inactivate cortex-lytic enzyme and change the secondary and tertiary structure of cortex-lytic enzyme.The mechanism of ethanol synergistic heat treatment to kill spores was revealed,which is beneficial to its application in food industry.