| Cellulose is the most abundant polysaccharide in nature and also the cheapest renewable resources.The key of cellulose exploitation is to get low-cost cellulase with high enzyme activity.Cellulase divided into microcrystalline cellulase,CMCase and ?-glucosidase by their different catalytic functions,high cellulase activity can be detested only when their ratio were appropriate.Sporotrichum thermophilic is a kind of thermophilic filamentous fungi which can produce mixed cellulase enzymes.For its high cellulose degrading speed,strain breeding still has an important significance for enlarge the range of cellulase producing microorganisms.This research optimized the cultivate condition of Sporotrichum thermophilic 2441 through orthogonal test,and enzymatic characteristics was discussed as well.By using strain breeding and protoplast fusion technologies,the cellulase activity was enhanced,which enables 2441 to be used on industrial cellulase production and straw degradation.The main research and results are as follow.(1)The experiment was designed through L9(34)orthogonal array to optimize the cultivate condition and culture media of Sporotrichum thermophilic 2441.The optimized medium component is as follow,microcrystalline cellulose 15.0g/L,bran 1.5 g/L,yeast extract 1.0g/L,glucose 2.0g/L,(NH4)2SO4 2.0g/L,KC1 1.0g/L,K2HPO41.0g/L,FeSO4 0.01g/L,MgSO4 1.0g/L,MnSO4 0.05g/L,ZnSO4 0.002g/L.The optimized fermentation condition is 45?,pH5.0 170 r/min and 5d cultivate time.The microcrystalline cellulase,CMCase and ?-glucosidase activity after optimized improved by 24.275%,18.350%and 23.449%respectively.(2)The optimum temperature of microcrystalline cellulase,CMCase and ?-glucosidase are 60?,65? and 70? respectively.The optimum pH are all 5.5.The enzyme activity will be lowered if the temperature is higher than 70 ? or in an alkaline condition.(3)UV and NTG were used for screening high cellulose activity mutants U-2-06 and N-3-16.The microcrystalline cellulase,CMCase and ?-glucosidase activity of U-2-06 improved by 16.221%,27.363%and 51.023%respectively.The microcrystalline cellulase,CMCase and ?-glucosidase activity of N-3-16 improved by 18.490%,25.099%and 45.293%respectively.(4)It is observed that the cellulase activity of Sporotrichum thermophilic 2441 can be repressed by glucose.NTG was used for screening glucose resistant mutant NA-1-01.The microcrystalline cellulase,CMCase and ?-glucosidase activity of NA-1-O1 improved by 13.203%,47.176%and 119.517%respectively,(5)NTG was used for screening fungicidin resistant mutant NB-1-04.The microcrystalline cellulase,CMCase and J3-glucosidase activity of NB-1-04 improved by 14.319%,26.407%and 49.199%respectively.(6)UV was used for screening mesophilic mutant UC-1-03.The microcrystalline cellulase,CMCase and ?-glucosidase activity of UC-1-03 improved by 11.411%,29.484%and 54.759%respectively when being cultured at 32 ? compared with 2441 cultured at 45 ?.(7)Fusing the protoplasts of U-2-06,N-3-16,anti-glucose mutant NA-1-01 and anti-fungicidin mutant NB-1-04,After selection,fusion named F-1-16 with anti-glucose effect,anti-fungicidin capacity and high enzyme activity was obtained.The microcrystalline cellulase,CMCase and ?-glucosidase activity of F-1-16 increased 48.260%,60.026%and 179.515%respectively than the initial strain 2441.Passage test indicated that the heritability of the fusion was steady.Innovation points of the research:(1)After optimizing the experiment,the cellulase productivity of Sporotrichum thermophilic 2441 has increased obviously.(2)The glucose resistant effect has been detested during the cellulase synthesis of Sporotrichum thermophilic 2441 and the mutant with anti-glucose resistant ability NA-1-01 was scrreened,its enzyme activity has greatly increased.(3)According to the results,we found that Sporotrichum thermophilic 2441 can also grow at 28 ?,but only mycelium can been seen and there were no spores.By using UV,UC-1-03,a mutant with a high cellulase productivity and low producing temperature,was obtained.(4)By using multi-parent protoplast fusion,a fusion named F-1-16 with high enzyme activity,anti-glucose effect,anti-fungicidin capacity and high genetic ability was obtained.These strains are practically meaningful to improve the productivity of cellulase,lower the costs and enlarge the application range of microorganisms.Due to the limited time,protoplast fusion was done only once in this research.The enzymatic activity might be improved in a higher level if progressive fusion is used.Meanwhile,whether the gene sequences of fusion F-1-16,mesotherm mutant UC-1-03 and initial strain 2441 are different is still necessary for further research. |
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