| Cellulose is the largest amount product of photosynthesis in theearth. The recycle of this part of carbon is beneficial to persistentlyproviding economical material for agriculture, stockbreeding, fermen-tation and chemistry industry, and to preventing environment pollutionto construct a good ecosystem. We have lots of cellulose, huge wasteexists as well; meanwhile with the development of economy, there is arush need for energy. So it is necessary to find out a high yield cellu-lase producing microorganism to translate biomass to liquid energy.Cellulase includes three groups of proteins: EG(endogulcanase :EC3,2,1,4),CBH(cellubiohydrolase EC3,2,1,94)and CB (cellobiaseorβ-glucosidase :EC3,2,1,21). Only by cooperation, cellulose can behydrolyzed to glucose. The well accepted hydrolyzing pattern basingon fungi, like Trichoderma that hydrolyzes catenarian cellulose bythree groups of cellulase cooperation, is that the enzymes hydrolyzecellulose by three procedures in which procedure needs small peptides.Tkoningii, a kind of fungi 3.2774, stored by BeiJing Microbe Re-searching Institute, which was a kind of Trichoderma, had been ob-served in its morphological changes. The results showed that betweenthe second and the third day the green spores coming out, in the thirdday spores begined to mature and release and in the fourth day the hy-pha turned old. And the study had been carry out which about the op-timum ration of corn stalk and bran in the solid ferment medium andthe optimum inhibitors and concentrations of Triton-X100, Deoxycho-late Sodium, Triton-X100 plus Deoxycholate Sodium, which rangedfrom 0 to 0.5%.It turned out that the optimum ratio of corn stalk andbran is 9:1, and the best one of inhibitor is 0.2% of Triton-X100.Different mutagen create different mutation raito, but the inherita-ble character changed casually. What kinds of mutation were profitablefor Trichoderma kongii was studied here. Spores cultivated for threedays were made suspensive with the concentration of 106/ml, and themutation conditions are like this:NTG final concentration is 1.5mg/ml,30℃.Mutation time is from 5min, 15min, 30min, and 60min to 90min.Wash them under the centrifuging condition 3 times and screen bydouble layer plate medium. Although mutants were red, blue or gray,none of them had the zone of hydrolysis. They were all negative muta-tion.Then laser mutation was carried on. With a type of HN-1700 He-Nelaser machine (wavelength 632.8nm), the spore suspension was treatedwith different laser energy and time. The laser energy was from 10mW/cm2, 20m W/cm2 to30mW/cm2 and irradiating time was from20min,30min,40min,50min, 60min,70min,80min to 90min.Underthe dark light cultivation, four colonies including number8, 18, 17 and21 were selected for the first time. And basing the result, number 8 and18 were picked up for second selection.Mutant strain8, 18 and the wild strain were compared in the fer-menting condition including the original PH and buffer system. For theformer, the original PH ranged from natural, 6.0, 4.0 to 4.8; the laterdid from citric acid buffer, acetic buffer to phosphate buffer. It showedthat no matter what condition was, mutant 8 and 18 had higher cellu-lase activity. And under the condition of PH4.8, which was especiallywell for three strains to produce cellulase, the cellulase activity of mu-tant 8 were double times of wild strain and mutant 18 were more thanfour times of it during the second day.3 kinds of buffer systems: Citric Acid Buffer System, Acetic BufferSystem,Phosphate Buffer System. All of them are with the concen-tration of 0.05mol/L.The result indicated that CBS was better than ACS and PBS. In thesecond day, with CBS, mutant 18 and 8 CMC enzyme activity werehigher than wild strain, correspondently the percentage increases were160.26%,87.585%;At the same time, in FPA, the percentage increaseswere correspondently 288.89%,181.97%.Complex mutagenesis. With the spores concentration of 106/ml, dif-ferent dose of ultraviolet and LiCl were given. Under the ultravioletlight of 15W, 20cm away from it, the spores were given different irra-diating time like 0min,60min and 90min; at the same time the concen-tration of LiCl from 0.1%,0.3% to 0.5%were given and cultivated indark light. Then seven mutants were gotten including A-1, I-2, J-2,L-10, M-4, E-1, C-9. During the second day, the CMC enzyme activityof mutant E-1 was increased by 4.32IU/g,and the percentage increaseswas 350.5%; FPA, during the second day, was increased by0.214IU/g,and the percentage increases was 87.65%。The method for protoplast mutation had also been studied. In the process ofmaking protoplast, four main points including the fermenting time of hypha,the ratio of cellulase, lysozyme and snailase, and the concentration ofthe three, hydrolyzed time and temperature. With the previouslyknowledge of protoplast generation, a group of orthogonal test havingfour factors and four levels had been carried out and the result wascounted by regeneration rate. Through visual analysis and rangeanalysis, it indicated that the best conditions for the highest regenera-tion rate of protoplast were like that hydrolyzing time was 90min,30℃,enzyme concentration was 0.25mg/ml,the ratio of threeenzymes was 1:1:1. Having this information, ultraviolet mutation wascarried out with the condition such as 15W of irradiating power, 15cmaway from it, 30S 60S 90S 120S 150S as the irradiating time, solidmedia containing osmotic stabilizer as regeneration plate. Finally, acolony was gotten on "A"plate, named as "yuan sheng zhi ti". By de-tecting enzyme activity of the solid fermentation medium, the CMCenzyme activity of the mutant was increased by 3.14IU/g,and thepercentage increases was 255.34%.The crude ferment liquid of mutant 18 and wild strain were filtrated,centrifuged, dialyzed (MWcutoff is 10KDa), condensed by PEG20000and got the enriched sample. With mutant 18 crude cellulase liguiddialyzed equally, chromatography of SephadexG-75 had been carriedout. And there were four peaks by detecting the protein concentration.Among them, the highest peak contained a CMC enzyme activity,which relative activity of enzyme was 0.0011IU/mg. Since cellulasewas a kind of complex protein, the enzyme activity of CMC and FPAcould not be detected when it was purified to a single protein. So itwas almost meaningless to purify to that degree.Through many times of collecting the chromatography liquid withenzyme activity part and the concentration of PEG20000, theSDS-PAGE could be done. The result of electrophoresis showed that itcontains three protein band, which molecular weight were 30, 21,14KD. At the same time mutant 8,18 and wild strain had the sameband in gel like 31,20,17,14KD. The difference between them was... |
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