【作者】 林元山；

【导师】 梁智群；

【作者基本信息】 广西大学 ， 微生物学， 2011， 博士

【摘要】 纤维素酶(cellulase)是降解纤维素生成葡萄糖的一组酶的总称,一般将纤维素酶分为三类：葡聚糖内切酶(1,4-β-D-glucan glucanohydrolase E.C3.2.1.4),葡聚糖外切酶(1,4-β-D-glucan cellobiohydrolase E. C3.2.1.91),p-葡聚糖苷酶(β-glucosidase, E. C3.2.1.21)。本论文以康氏木霉(Trichodeerma koningii) AS3.2774为实验菌株,对康氏木霉纤维素酶系的发酵条件,纤维素酶系的代谢调控,纤维素酶系的分离纯化方法,酶系组分的生物学功能鉴定进行了系统研究,结果如下：1、通过康氏木霉AS3.2774固体发酵纤维素酶的单因素及正交试验研究,得出最优发酵条件为：发酵温度28℃,稻草粉(40目)8g,麸皮为4g,培养基含水量23mL,每天翻曲1次,发酵周期4.5天,在此在条件下,康氏木霉产纤维素酶活力达0.38IU/mL.2、通过研究糖蜜酒精废液对康氏木霉发酵纤维素酶的影响,发现用15"BX的糖蜜酒精废液配制的培养基,康氏木霉发酵纤维素酶的滤纸酶活力是对照的8.6倍,达3.3IU/mL。3、经分离纯化,从糖蜜酒精废液中得到对康氏木霉发酵纤维素酶具有促进作用的活性物质A10；A10是一种新型多糖,一分子A10含有6个鼠李糖分子、17个木糖分子、4个果糖分子以及2个葡萄糖分子,分子量为4,200Da左右。4、通过对康氏木霉AS3.2774的碳源代谢调控研究,发现其纤维素酶系是一组诱导酶,可以通过诱导物(纤维素或A10)与阻遏物(纤维素酶酶解纤维素末端产物葡萄糖)进行代谢调控。康氏木霉在基本发酵培养基Ⅲ(配方为：稻草粉8g,麸皮4g,自来水23mL,自然pH)产纤维素酶属于一般性诱导,纤维素酶活力达0.38IU/mL。若将基本发酵培养基Ⅲ中添加2.0g葡萄糖,则成为高度阻遏培养基V,纤维素酶活力仅为0.11IU/mL。若将基本发酵培养基Ⅲ中自来水替换为15"BX废糖蜜23mL,则成为高度诱导培养基Ⅳ,纤维素酶活力高达3.3IU/mL。5、经发酵动力学研究,发现康氏木霉AS3.2774的发酵纤维素酶属于菌体生长与产物合成非偶联类型(Ⅲ型),纤维素酶合成速度与碳源利用也不存在定量关系,而且情况很复杂。在不同的培养基上呈现不同的特点,引起生物量、纤维素酶产物比生产率和菌体形态分化差异。其中,在诱导培养基Ⅳ中,菌丝体比重较大且饱满壮实,孢子比重较小,生物量较少,纤维素酶产物比生产率较快；在一般性诱导培养基Ⅲ中,菌丝体比重较小且易衰竭,孢子比重较大,生物量较多,纤维素酶产物比生产率较慢；康氏木霉在阻遏培养基V发酵过程中,出现两次产物比生产率的增速过程,但幅度比诱导培养基低,体现阻遏解除的机制。6、康氏木霉AS3.2774的纤维素酶的代谢调控实质基因水平的诱导与阻遏,是纤维素酶蛋白在合成“量”上的调节,二者胞外蛋白差异十分显著,其中被诱导的纤维素酶胞外蛋白是被阻遏的纤维素酶胞外蛋白的38倍以上,体现康氏木霉优先利用现有资源的进化机制。7、论文研究了“聚丙烯酰胺凝胶活性电泳电洗脱二步循环法”分离康氏木霉纤维素酶系的条件,获得有别于Schagger和Offord的电泳方案的最优条件为：分离胶梯度4%-8%；胶长180mm；浓缩胶电压,80-100V,电流20-30mA；分离胶电压180-200V,电流<30mA；电泳缓冲液离子浓度,12.5mmol/L Tris-glycin96mmol/L,电泳时间为10-12h,4℃环境条件操作。8、通过对纤维素酶系的分离纯化研究,提出了“聚丙烯酰胺凝胶活性电泳电洗脱二步循环法”分离纯化纤维素酶的新方案,包括：(1)第一次电泳,即4%-8%梯度胶电泳分离；(2)分区染色；(3)胶内蛋白定位；(4)切胶分类蛋白；(5)电洗脱回收蛋白；(6)透析脱盐、冻干；(7)第二次电泳,即均一胶活性电泳,均一胶浓度由目的蛋白在第一次电泳胶的迁移率决定,分离程序进行至第二次透析脱盐、冻干时结束。经分离纯化获得9个单一组分,各组分的纯化效果基本一致。其中,活性蛋白P3(β-葡萄糖苷酶)在第一次活性电泳分离时,纯化倍数为17倍,回收率为10.6%；在第二次活性电泳分离时,纯化倍数为24倍,回收率为5.5%,比活力达994.6IU/(mg protein),在整个纯化完成后,最后获得8μg电泳纯的β-葡萄糖苷酶。9、康氏木霉纤维素酶系组分经生物学功能测定,组分P1无β葡萄糖苷酶活性、外切葡聚糖酶活性、内切葡聚糖酶活性。与纤维素酶系其它组分同时被诱导与阻遏调控,属于纤维素酶系,功能未知；P3为β葡萄糖酶；P4、P10为外切葡聚糖酶；P5、P6P7为内切葡聚糖酶；P2、P8纤维素酶活性较弱,属于纤维素酶系；P9基本无纤维素酶活性,不能与纤维素酶系其它组分同时被诱导、被阻遏调控,不属于康氏木霉纤维素酶系。经胰蛋白酶酶切、基质辅助激光解吸/电离飞行时间质谱、肽指纹图谱鉴定P3为β葡萄糖酶,P4外切葡聚糖酶,进一步证明“聚丙烯酰胺梯度凝胶活性电泳电洗脱二步循环法”分离纯化纤维素酶的方案是可靠的。经酶学特性研究,P3的分子量为69.1KDa,等电点pI5.63,最适反应温度为50℃,最适反应pH5.0, Km=2.67mmol/L p-NPG。更多还原

【Abstract】 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更多还原