| Cellulases are a multi-component enzyme system in which cellulose is degraded into glucose. Generally, cellulases are classified into three groups, such as endoglucanase (endo-1,4-(3-D-glucanase, EG, EC.2.1.4), cellobiohydrolase (exo-1,4-β-D-glucanase, CBH, EC3.2.1.91), and β-glucosidase (1,4-β-D-glucosidase, BG, EC3.2.1.21). In this thesis, Trichodeerma koningii AS3.2774was used for cellulase production, including determination of fermentation conditions, control of metabolism, purification and identification. The results were as follows:1. The optimum fermentation conditions of T. koningii AS3.2774were determined by methods of single factor test and orthogonal design in this thesis. The optimum temperature was28℃. Basic medium consisted of40-mesh straw powder (inductor)8.0g, wheat bran4.0g, and tap water23mL with natural pH. Fermentation peroid was4.5days during which the mould koji of medium was turned over axenic once a day for ventilation. The filter paper activity (FPA) of cellulase reached0.38IU/mL.2. The effect of molasses of alcohol stillage (MAS) on the production of cellulase from T. koningii AS3.2774was investigated. The result showed that in the medium containing15"BX of waste of molasses of alcohol stillage23mL, the FPA of cellulase increased8.6-fold by comparison with control, whose activity reaching3.27IU/mL3. The active component named Ajo was purified from waste of molasses of alcohol stillage. And A10could enhance the FPA of cellulase from T. koningii AS3.2774significantly. It’s a novel polysaccharide which was polymerized with6molecular of rhamnose,17molecular of xylose,4molecular of fructose and2molecular of glucose with molecular weight of4,200Da in one unit.4. The cellulolytic systems from T. koningii AS3.2774were inductive cellulase by metabolism control. It’s revealed that cellulase of T. koningii AS3.2774was induced moderately on basic fermentation medium III which contained40-mesh straw powder (inductor)8.0g, wheat bran4.0g, and tap water23mL, and FPA could reach0.38IU/mL. It was strongly repressed on suppressive medium Ⅴ which contained40-mesh straw powder8.0g, wheat bran4.0g, glcuose2.0g (impressor), and tap water23mL with FPA0.11IU/mL. However, it was highly induced on inductive medium Ⅳ which contained40-mesh straw powder8.0g, wheat bran4.0g, and15"BX of waste of molasses of alcohol stillage (inductor)23mL with FPA3.3IU/mL.5. Fermentation dynamics of T. koningii AS3.2774was investigated systematically. The result indicated that it belonged to the Ⅲ type of fermentation in which the growth of the fungi do not match with production of cellulase. It is very complicated that quantitative relationship between production rate and consumption of carbon resource can not exist. The biomass, ratio of production of cellulase to biomass and morphogenesis of T. koningii AS3.2774were various on different medium. When T. koningii AS3.2774was cultured on inductive medium Ⅳ, the biomass were few, ratio of spore was low, ratio of mycelium was high and ratio of velocity of cellulase to biomass was also high. When T. koningii AS3.2774was cultured on basic medium Ⅲ, the biomass was much more, ratio of mycelial was low while ratio of spore was high, and ratio of velocity of cellulase to biomass was yet low. When T. koningii AS3.2774was cultured on suppressive medium Ⅴ, the ratio of velocity of cellulase to biomass increased by2times but lower than that on inductive medium Ⅳ. It indicated that suppression was released when glucose was used up during anaphase.6. Cellulolytic metabolism of T. koningii AS3.2774was controlled in gene level. The actual control model of cellulase was to promote cellulolytic transcription on large scale. The amounts of secreted proteins between inductive medium Ⅳ and suppresive medium Ⅴ were not significant. The amount of cellulases from inductive medium Ⅳ by T. koningii AS3.2774was37times higher than that from suppressive medium. It showed that microbes preferred existing resource in the evolution level.7. The optimum isolation conditions of cellulases by native electrophoresis and electric elution were investigated, including electrophoresis time10-12h on the separation gel (4%-8%) of180mm in length, voltage180-200V, current<30mA, pH8.8and12.5mM Tris-glycin96mM. In particular, the best concentration of the electrophoresis buffer was only a half of that described by Schagger and Offord.8. In this study, a novel method named "two cycle native gradient-polyacrylamide gel electrophoresis and electro-elution (NGGEE)" was put forward to purify enzymatic proteins from T. koningii AS3.2774. The purification step including:(1) First cycle of native electrophoresis with4%-8%native gradient-polyacrylamide gel;(2) Partition of gel regions for coloration;(3) Location of cellulase in gels;(4) Recovering native cellulase bands from gels;(5) Electro-elution and recovery of native proteins;(6) Dialysis and lyophilization for the recovery of cellulase;(7) Second cycle of electrophoresis on the uniform gel which depended on the protein’s migration in the first electrophoresis. When the procedures went until the step "Dialysis and lyophilization for the recovery of cellulase" at the second time, the isolation was finished. Finally,9cellulolytic proteins of T. koningii AS3.2774were purified. All of them had same efficiency of purification by NGGEE. For example, the first cycle of electrophoresis and electro-elution resulted in a17-fold purification of P3(BG) with a recovery rate of10.6%. And the second cycle of electrophoresis and electro-elution resulted in a24-fold P3(BG) purification with a recovery rate of5.5%. And a specific activity of994.6IU/(mg protein). The final yield of BG reached8μg under purifying procedure of NGGEE.9. The biological function of cellulolytic systems (P1-P10) of T.koningii AS3.2774were identified. The results showed P1had no activities of BG, EG and CBH, but it can be induced and suppressed synchronously with cellulolytic systems. So P1should be one of cellulolytic systems, but its function was undiscovered. P3was identified as BG. P4and P10were identified as CBH. P5, P6and P7were identified as EG. P2and P8were identified as cellulolytic systems but had lower activities of BG, EG and CBH. P9had no activities of BG, EG and CBH, it could not yet be induced and suppressed synchronously with cellulolytic systems. So P9did not belong to cellulolytic systems of T. koningii AS3.2774. Moreover, P3and P4were also were determined as BG, CBH respectively by MALDI-TOFMS and peptide mass fingerprinting. Especially, the enzyme characteristic of P3was found that it’s had one subunit with a molecular mass of69.1kDa. This purified BG had an optimum pH of5.0, an optimum temperature of50℃, an isoelectric point of5.68and a Km for p-nitrophenyl-β-D-glucopyranoside of2.67mmol/L... |
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