Extraction And Purification Of Crude Protein From Bacillus Subtilis Against Pathogenic Fungus

Bacillus subtilis isolated from soil,which has strong inhibitory against plant pathogenic fungi.In order to know more about Bacillus subtilis,some experiments were proformed,such as bacteriostasis and antibacterial mechanism of crude proteins from Bacillus subtilis were researched,and the crude proteins extracted according to different saturability of(NH4)2SO4.In order to identify the antimicrobial,the antimicrobial substances were isolated and purified by Polyacrylamide gel electrophoresis and preliminary analysis the purified protein.The main results were summarized as follows:1.Bacterial cell suspension,fermentation liquid with and without bacterial cells of B.subtilis,all has inhibiton to 6 kinds of plant pathogenic fungi.Besides,the inhibition of Bacterial cell suspension and fermentation liquid without bacterial cells have reached 60%.2.Observing bacteria-growth curve,cultured microbial 42 h,and measured optical density value,growth curve tends to be smooth,the bacteria in the logarithmic and stable growth period at this point,and it is suitable for extracting proteins.3.B.subtilis crude proteins all have inhibition to 6 kinds of plant pathogenic fungi.Crude proteins extracted by different saturability of(NH4)2SO4 have different inhibitions,and the extracellular crude proteins more stability than intracellular crude proteins.The crude proteins have obvious destructive effect to C.gloeosporioides mycelia,and the mycelial cells malformation,expand and twisted.B.subtilis crude proteins to C.gloeosporioides conidial germination and germ-tube elongation have good inhibition.20-40% saturability of(NH4)2SO4 of extracellular protein with the concentration was 0.11mg/mL,respectively inhibition were 65.70% and 51.65%;40-60%saturability of(NH4)2SO4of intracellular protein with the concentration was 0.11mg/mL,respectively inhibition were 30.78% and48.69%.The inhibition of B.subtilis extracellular crude proteins better than intracellular crude proteins.4.The thermal stability of B.subtilis extracellular crude proteins better than intracellular crude proteins,and the inhibition rate decreases with the increase of temperature.When the temperature reached at 80 ?,extracellular crude proteins still have inhibition,but the inhibition rate only reached 10.19%,the inhibition of extracellular crude proteins only 7.10%,when the temperature reached 100?,thecrude proteins have no inhibitions.The antimicrobial activity of crude proteins changes little from pH4 to 9.B.subtilis crude proteins after deal with different proteinases,bacteriostatic rate contrast with control group were significant difference(P=0.05).To extracellular protein,after deal with trysin,pepsin and proteinase K,the inhibition rate relative to the control were 74.36%?58.87%?43.96%,respectively;To intracellular protein,after deal with trysin,pepsin and proteinase K,the inhibition rate relative to the control were 40.88%,35.67%,45.94%,respectively.The results showed that,the extract of crude protein of antimicrobial substance is mixed composition,it contains part of protein.5.20-40% saturability of(NH4)2SO4 of extracellular crude protein and 40-60%saturability of(NH4)2SO4 of intracellular crude protein were inoculated in apples,The results showed,the test of inoculation after applying crude proteins,extracellular crude proteins control effect can reached 100% without diluted;the control effect was61.9% until diluted 100 times.the test of inoculation before applying crude proteins,the control effect of extracellular crude protein was 55%.and the control effect of intracellular crude protein was 29.17%.The test results showed,the control effection of extracellular crude protein was better than intracellular crude protein,The result indicated that the mode of action of B.subitilis was mainly protective.Control Fusartum graminearum of field trials showed that,B.subtilis crude proteins have protective effect to Fusartum graminearum.6.The result of PAGE-Polyacrylamide gel electrophoresis showed,the crude protein contains 6 kinds of protein.Antibacterial results showed,only the sixth stripe has the bacteriostatic action.The antimicrobial protein also have inhibition on Fusartum graminearum,Rhizoctonia cerealis,Rhizoctonia solani,Fusarium oxysporum f.sp.vasinfectum,Sclerotinia sclerotiorum.The antimicrobial protein have obvious destructive effect to C.gloeosporioides mycelia,and the mycelial cells malformation,expand and twisted.7.The thermal stability results shows that,the antimicrobial activity have little influences under 20~60?for 30 min,and some antimicrobial activity have loss after suffer under 80 ? for 30 min,but it also have some antimicrobial activity,so the proteins have heat resistance.The antimicrobial activity of antimicrobial proteins changes little from pH4 to 9.Ultraviolet irradiation and the inbitor,organic solvent treatment has no effect on the bacteriostatic activity.The results showed the antimicrobial mycoprotein have good stability.8.Analysis the sixth protein stripe by mass spectrum,Peak marked with number showed it has corresponding peak matching;the peak intensity less than 5% marked with light color.Database test showed that,detecting bacteria all library in NCBI by Mascot data analysis and the calculation score over 65 points were reliable.The score of 2 kinds of protein more than 65,Respectively they were Hypothetical protein BSU35980 gi|16080651,with a relative molecular mass of 11079 Da and isoelectric point(pI)of 4.78,the matching ratio was 68%,and serine hydroxymethyltransferase gi|16080743,with a relative molecular mass of 45575 Da and isoelectric point(pI)of5.56,the matching ratio was 67%.