【作者】 孟楠；

【导师】 夏黎明；

【作者基本信息】 浙江大学 ， 生物化工， 2011， 硕士

【摘要】 内切型-β-葡聚糖酶(CMC酶)是纤维素酶的重要组分之一,在纤维素水解糖化、食品、饲料、织物水洗等领域中应用广泛。本文采用基因工程技术,在内切型-p-葡聚糖酶基因重组菌株的构建、筛选及产酶等方面进行了研究。首先,以实验室前期克隆得到的厌氧真菌内切型-p-葡聚糖酶基因afl为研究对象,将目的基因与大肠杆菌表达载体pET-28a(+)连接,转入大肠杆菌表达菌株BL21(DE3),获得重组大肠杆菌BL21 (DE3)/pET-28a(+)-αf1。重组菌的适宜培养条件为：菌体浓度OD600为0.8时加入IPTG至终浓度为0.1 mM,31℃诱导培养。采用SDS-PAGE蛋白电泳对其表达产物进行分析,在40 kDa附近得到与目的基因afl表达蛋白理论值相当的明显条带。酶学性质研究表明,酶蛋白的最适作用条件为pH 6.0,45℃,属于中性纤维素酶。试验结果证明af1基因具有适于表达的完整序列。进一步将af1基因与毕赤酵母表达载体pPIC9K连接,经限制性内切酶BglⅡ线性化后,采用基因导入仪将重组DNA转入毕赤酵母GS115。在选择性培养平板上进行筛选,挑出六个高抗阳性转化子。提取基因组进行PCR鉴定,均获得与目的基因相关的条带,证明af1基因已整合入GS115基因组,有利于稳定遗传。afl基因在甲醇诱导下可在毕赤酵母中表达,取上清液进行SDS-PAGE蛋白电泳分析,在52 kDa附近出现明显条带。分别诱导培养筛选到的六个高抗阳性转化子,发现其产酶性能基本一致。采用重组毕赤酵母在摇瓶条件下分批发酵,结果显示其产酶性能稳定。在pH 6.0,50℃条件下检测CMC酶活力,发酵液中酶活在诱导培养96 h左右可达到600 U/mL以上。该试验结果为进一步利用重组毕赤酵母生产中性纤维素酶奠定了基础。通过试管初筛、摇瓶复筛,从各约300个里氏木霉转化子T/af1(含有厌氧真菌内切型-p-葡聚糖酶基因afl)和T/egll(含有里氏木霉内切型-p-葡聚糖酶基因egⅡ)中获得了T/αf1-3和T/egⅡ-5两个优良转化子。对重组里氏木霉T/af1-3进行摇瓶产酶研究,在pH 6.0,50℃条件下测得发酵液中的中性CMC酶活力可达1280 U/mL,提高到同期原始菌的3.9倍；而对重组里氏木霉T/egⅡ-5进行摇瓶产酶研究,在pH 4.8,50℃条件下测得发酵液中的酸性CMC酶活力可达到3500 U/mL,约是同期原始菌的4.9倍。采用里氏木霉作为基因工程宿主菌具有外源基因表达水平高,产物易于分离提取等优点,本文的研究成果在纤维素酶的定向进化及纤维素酶高产菌株的构建方面具有重要的指导意义。更多还原

【Abstract】 Endo-β-glucanase (CMCase) is one of the most important parts of cellulase, and has great value in cellulose degrading, food, feedstuff production and fabric washing fields. This paper focused on construction, screening and CMCase production of high endo-p-glucanase producing strains based on gene engineering technology.An endo-β-glucanase gene afl from anaerobic fungus was inserted into Escherichia coli expression vector pET-28a(+). Then the recombinant plasmid pET-28a(+)-af1 was transformed into Escherichia coli BL21(DE3) to obtain recombinant strain BL21(DE3)/pET-28a(+)-af1. The appropriate conditions to cultivate recombinant Escherichia coli were:added IPTG to 0.1 mM when the cell concentration reached OD600=0.8, then cultivated at 31℃. SDS-PAGE analysis of its expression product showed a clear protein band near 40 kDa, which accorded with that of AF1 protein theoretic size. The enzyme exhibited maximum CMCase activity at pH 6.0,45℃, which belonged to the neutral cellulase. It had been proved that gene afl had a complete sequence ready to express product.Furthermore, afl was inserted into Pichia pastoris expression vector pPIC9K, digested by enzyme Bglll, and then transformed into Pichia pastoris GS115 using Gene Delivery Instrument. Transformants were screened by selective plates, and 6 positive transformants with high G418 resistance were obtained. PCR analysis of their genome showed clear bands relative to the size of gene afl, which demonstrated that afl was successfully integrated into GS115 genome and contributed to stable inheritance.The expression product was analyzed by SDS-PAGE, and a clear protein band was found near 52 kDa. The 6 positive transformants selected were cultivated respectively and showed similar and stable enzyme producing performance.Moreover, in shaking flask experiments with methonal induction, the CMCase activity of the supernatant could reach 600 U/mL at 96 h. This result laid good foundation for neutral cellulase production using recombinant Pichia pastoris strains.Through secreening processes, T/afl-3 and T/egⅡ-5 with good endo-β-glucanase producing performance were respectively obtained from recombinant Trichoderma reesei T/af1 and T/egⅡ, which included about 300 strains respectively. Shaking flask experiments demonstrated that, T/af1-3 contained endo-β-glucanase gene af1 from anaerobic fungus, and the neutral CMCase activity in the supernatant could reach 1280 U/mL measured at pH 6.0,50℃, which was 3.9-fold compared with that of original strain; T/egⅡ-5 contained endo-β-glucanase gene egⅡfrom Trichoderma reesei, and the acid CMCase activity in the supernatant could reach 3500 U/mL measured at pH 4.8,50℃, which was 4.9 times that of original strain. As a genetic engineering host, Trichoderma reesei system has plentiful advantages, such as high expression level of foreign gene, simple procedures for product separation, etc. This paper is useful for cellulase directed evolution and construction of industrial strains with high cellulase activity.更多还原