【作者】 徐荣燕；

【导师】 李多川；

【作者基本信息】 山东农业大学 ， 植物病理学， 2010， 博士

【摘要】 纤维素是地球上最丰富的碳水化合物,可被纤维素酶降解转化成为葡萄糖,对于人类社会解决能源危机、食物短缺和环境污染具有重大的现实意义。传统上采用化学方法控制纤维素降解难度大、成本高,对环境污染严重,而纤维素的生物降解有效克服了这些问题。纤维素酶是一类能够水解纤维素β-D-糖苷键生成葡萄糖的多组分酶的总称,纤维素的彻底降解需要内切葡聚糖酶、外切葡聚糖酶和β-葡萄糖苷酶三种组分的协同作用,β-葡萄糖苷酶是纤维素酶解的瓶颈。在纤维素水解时,增加β-葡萄糖苷酶活性,会有效提高纤维素酶解效率。近些年来,许多专家学者通过分子生物学等手段不断提高酶活力和获得新性能的重组纤维素酶。糖化酶是一种具有外切活性的酶,它能从非还原性末端水解淀粉、糊精、糖原等的α-1, 4-葡萄糖苷键,得到终产物β-D-葡萄糖,是淀粉转化为葡萄糖过程中的主要酶类之一。糖化酶已在酿造、食品、医学等工业和农业领域中得到了广泛的应用,具有重要商业价值。但市场应用的糖化酶主要来源于常温菌,因其酶活力和产率低,热稳定性差导致产品成本高,储存期短,而制约糖化酶在农业、工业上的应用。由此可见,热稳定性糖化酶的研究和开发具有重要意义。疏绵状嗜热丝孢菌(Thermomyces lanuginosus)和嗜热毛壳菌(Chaetomium thermophilum CT2)是广泛分布的,生长上限温度较高的嗜热真菌,从这两种真菌中已分离了多种嗜热酶,具有极大的研究和应用价值。本研究通过RT-PCR和RACE-PCR从嗜热毛壳菌中首次分离到了一个新的β-葡萄糖苷酶基因,GenBank登录号为EF648280,序列分析表明该基因全长核酸序列为3213 bp,包含一个2604 bp的开放阅读框ORF,编码867个氨基酸的蛋白质,酵母表达后酶蛋白表达量为1.644 U/mg,经硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换等步骤纯化了该重组蛋白,SDS-PAGE显示该重组蛋白大小约为119 kDa,比推测的蛋白分子量稍大,可能与该蛋白的糖基化有关。重组酶的最适反应温度和最适pH值分别为60℃和5.0,该酶具有较高的热稳定性,70℃保温10 min后剩余酶活为29.7 %。本研究还从疏绵状嗜热丝孢菌中克隆到一种糖化酶基因(GenBank登录号为EF545003)并将其在毕赤酵母中进行了高效表达,经甲醇诱导表达后酶活最高可达11.6 U/mL,经硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换等步骤纯化了该重组蛋白,SDS-PAGE显示该重组蛋白大小约为67 kDa,该重组酶的最适反应温度和最适pH值分别为60℃和5.0,在pH 4.0-9.0的范围内是很稳定的。对来自嗜热毛壳菌的β-葡萄糖苷酶基因的非保守氨基酸和保守氨基酸进行定点突变。三个非保守氨基酸的突变为K275R、N276S和279D,表达筛选后发现与原始菌GS-BGL相比,三个突变子的酶活都有不同程度的降低,突变子的酶活分别是原始菌酶活的81.3 %、63 %和65 %,但最适反应温度均提高了5℃;突变子N276S和G279D的最适pH由5变为4,而突变子K275R最适pH值仍保持在5;在热稳定性方面,三个突变子表现不同,60℃处理60 min,突变子G279D稳定性提高最多,仍有80.6 %的酶活,突变子N276S稳定性次之,酶活剩余72.1 %,原始酶仅剩69.55 %的活性,而突变子K275R酶活剩余59.5 %,与原始酶相比稳定性下降。而当保守氨基酸第287位的天冬氨酸突变为非亲核的甘氨酸后,突变子失去了糖苷水解酶的活性,证明了该氨基酸为维持水解酶活性的必需氨基酸,但目前还没有检测到糖苷合成酶的活性。由于定点突变主要是针对天然酶蛋白中少数的氨基酸残基进行替换,蛋白的高级结构基本保持不变,而且本研究中的β-葡萄糖苷酶的结构迄今为止还没有报道,因而对酶蛋白的改造有限。为了提高β-葡萄糖苷酶的酶活力和稳定性,采用定向进化的方法对β-葡萄糖苷酶的基因进行改造。本研究以β-葡萄糖苷酶bgl基因为出发基因,采用易错PCR的方法建立突变体文库,以高活力为筛选压力,筛选方法采用平板荧光初筛和小量发酵法相结合。经过突变和筛选,获得了一个酶活力提高0.74倍的突变体,序列测定表明:突变基因与出发基因比较,突变子BE857有6个氨基酸发生了变化。通过硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析等步骤纯化了该蛋白,酶学性质与出发酶进行了比较,该突变酶的最适温度为65℃,比出发酶提高了5℃,最适pH与出发酶一致均为5.0,在稳定性方面,突变子株BE857有所提高,60℃处理1 h后酶活仍剩余75.58 %,而出发菌株剩余酶活为69.55 %。更多还原

【Abstract】 Cellulose is the most abundant carbohydrate available on earth, it can be converted into glucose by cellulases. It has great practical significance to solve the energy crisis, food shortage and environmental pollution. Traditionally, the chemical method to break down cellulose uncontrollable, costly and environmental pollution, but the biological degradation of cellulose effectively overcome these problems. Cellulase is a general term for multi-component enzyme that can hydrolyze cellulose byβ-D-glucoside bond. The completely degradation of cellulose requires synergy of three components: endoglucanase, exo-glucanase andβ-glucosidase, theβ-glucosidase is the bottleneck of cellulose hydrolysis. Increased mount or enhencedβ-glucosidase activity, will effectively improve the efficiency of cellulose hydrolysis. In recent years, many experts and scholars continue to improve the performance of activity and access to new recombinant cellulase by means of molecular biology.Glucoamylase is an exo-acting enzyme that removes the glucose unites from the non-reducing ends of amylose, amylopectin and glycogen by hydrolyzingα-1, 4 linkages in a consecutive manner, producingβ-D-glucose as the sole product. Glucoamylase was widely used in dextrose production, baking industry, brewing of low calorie beer and whole grain hydrolysis for the alcohol industry. In recent years glucoamylases from thermophilic fungi, which are expected to be thermostable, have aroused increasing attention among researchers. The glucoamylase used in the market mainly from room temperature microorganisms. Due to high production costs, poor thermal stability, low activity and yield, the application of glucoamylase is restricted in agriculture and industrial applications. Thus, the research and development of thermorphilic glucoamylase have great importance.T.lanuginosus and C.thermophilum are widely distributed soil-inhibiting fungi of considerable interest producers of thermostable enzymes, which have great value in research and application. In this study, aβ-glucosidase gene was isolated firstly from C.thermophilum CT2 by RT-PCR and RACE-PCR. The GenBank accession number was EF648280, sequence analysis showed that the full nucleotide sequence of the gene was 3213 bp, contained a 2604bp open reading frame, encoding 867 amino acids. The amount of enzyme protein was 1.644 U/mg after yeast expression. The recombinant protein was purified by ammonium sulfate precipitation, DEAE-Sepharose Fast Flow anion exchange. The SDS-PAGE showed that the recombinant protein was about 119 kDa, slightly larger than the deduced molecular weight, which may be related to the glycosylation. The optimal temperature and optimal pH were 60℃and 5.0 separately, the enzyme was relatively stable, after incubated at 70℃for 10 min still remained 29.7 % activity.In this study, a glucoamylase gene from T.lanuginosus was also cloned (GenBank accession number is EF545003) and highly expressed in P.pastoris, the enzyme activity was 11.6 U/mL. The recombinant protein was purified by ammonium sulfate precipitation, DEAE-Sepharose Fast Flow anion exchange. SDS-PAGE showed that the recombinant protein was about 67 kDa, the optimal temperature and the optimum pH were 60℃and 5.0, separately. The enzyme was stable in the range pH 4.0-9.0.The site-directed mutagenesis was acted toβ-glucosidase from C.thermophilum CT2. Three non-conservative amino acid mutation K275R, N276S, and 279D were expressed in p.pastoris, three mutants activities were 81.3 %, 63 % and 65 % respectively compared to GS-BGL, but the optimum temperature of three mutants were increased 5℃; mutants N276S and G279D optimum pH changed from 5 into 4 and mutant K275R optimum pH remained at 5. The performance of the three mutants were different in thermal stability, after incubated at 60℃for 60 min, G279D still have 80.6 % of the activity, N276S have 72.1 % of the activity, the activity of K275R was 59.5 %, while the original enzyme activity remaining 69.55 %. The conservative amino acid Asp287(nucleophile) mutated to Gly(non-nucleophile), After expressed in p.pastoris, found the mutant lost the glycoside hydrolase activity, but not yet detected glycoside synthase activity.Since site-directed mutagenesis is mainly directed against a small number of amino acid residues, the high-level protein structure remained unchanged. The structure ofβ-glucosidase in this study enzyme was not reported so far, in order to improve theβ-glucosidase activity and stability, the directed evolution was used to alert theβ-glucosidase gene. In this study, the mutant library was constructed by error-prone PCR, fluorescence screening and small amount ferment were used to screen mutants with higher activity. A mutant BE857 with 0.74-fold increased enzyme activity was acquired. The sequencing showed the mutant have 6 amino acids changed. The optimum temperature of BE857 was 65℃, 5℃higher than the starting enzyme, the optimum pH kept invariant; the stability also increased, after treatment at 60℃for 1 h, the mutant still remaining 75.58 % activity, while the remaining activity of wild enzyme was 69.55 %.更多还原