【作者】 马鑫；

【导师】 林敏；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 半纤维素作为一类可再生能源物质广泛存在于自然界中。其总量占植物干重的30%左右,是仅次于纤维素的第二大组成部分。因此半纤维素的分解利用对解决环境问题与能源危机都具有重大意义。β-甘露聚糖酶是一类能够降解甘露聚糖的半纤维素水解酶,作为添加剂广泛应用于动物饲料中,同时在食品、药学、医学、造纸和纺织业中也具有重要的应用价值。因此通过各种方法获得高酶活和高耐受能力的β甘露聚糖酶一直是研究的热点。本研究从新疆极端干燥的环境采集土样筛选获得具有较高酶活性的β-甘露聚糖酶的菌株,构建了高产β-甘露聚糖酶的重组大肠杆菌,初步开展了重组大肠杆菌诱导表达的条件优化,并进行了目的蛋白的分离纯化,以及β-甘露聚糖酶的酶学特性的分析。利用水解圈筛选法从新疆极端干燥环境土壤样品中分离到三株具有高酶活β-甘露聚糖酶的菌株。根据16S rDNA序列分析以及菌落形态观察分别鉴定为芽孢杆菌(Bacillus sp.)、柠檬酸杆菌(Citrobacter sp.)、多粘类芽孢杆菌(Paenibacillus sp.)。水解圈相对较大的芽孢杆菌被命名为Bacillus sp. MX,并作为本研究的实验材料。从芽孢杆菌MX基因组DNA中克隆β-甘露聚糖酶基因(man),连接到表达载体pET-28a中,转化大肠杆菌BL21获得转化子BL21(pET-man)。使用IPTG作为诱导物,37℃培养3个小时表达出带有组氨酸标签的β-甘露聚糖酶。SDS-PAGE分析表明,BL21((pET-man)表达出的β-甘露聚糖酶主要以包涵体形式存在于细胞质中。试验证明:β-甘露聚糖酶诱导表达的最佳培养条件为30℃,0.5mM IPTG诱导5小时,在优化条件下获得可溶性β-甘露聚糖酶的含量较高的细胞培养物。SDS-PAGE分析显示该蛋白的相对分子量为43kDa。以Ni树脂为介质,采用不同浓度咪唑进行洗脱,获得纯化的β-甘露聚糖酶。3,5-二硝基水杨酸(DNS)法测定该酶在25-95℃,pH3.0-9.6范围内均具有酶活。最适作用温度和pH值分别为55℃和5.0,酶比活力达4572U/mg。在pH4.0,或80℃和90℃处理10min后,该酶相对酶活力仍保持84%或33%和14%,表明该β-甘露聚糖酶具有较好的耐酸性和热稳定性。为了在真核生物中表达β-甘露聚糖酶,将该酶编码基因插入连接到穿梭质粒pPIC9K中,转化至大肠杆菌JM109,获得携带表达β-甘露聚糖酶基因的多拷贝数的质粒(pPIC9K-man),并将BglII线性化产物电转至巴斯德毕赤酵母宿主菌GS115中。经过甲醇诱导表达出该蛋白,测得比活力为345U/mg,低于在大肠杆菌中表达的比活。其原因可能是由于细菌基因在真菌中表达受到了密码子偏好性的影响,且man基因整合到染色体上,大大降低β-甘露聚糖酶基因的表达量。尚需对β-甘露聚糖酶基因的密码子进行优化以提高酶活。更多还原

【Abstract】 Hemicellulose is a widely used substance which accounts for from 1/4 to 1/3 of plants dry weight. Degradation and utilization of hemicellulose is of great significance in resolving energy crisis, food shortage and environmental pollution. Thus, finding a hemicellulosic hydrolase with high activity and tolerance to adversity seems crucial.β-mannanase is a important kind of hemicellulose hydrolases, which is ubiquitous in animal feed,food, medicine and widely used in paper making and textile industry. In this study, a Bacillus sp. strain with highβ-mannanase activity was isolated from an extreme environment in Xinjiang.Further efforts of expression optimization and enzyme identification were made in the E.coli.Three strains with highβ-mannanase activity were isolated from an extremely dry environment by comparing the size of hydrolyzed circle produced by each strain, which were further identified as belonging to Bacillus sp., Citrobacter sp. and Paenibacillus sp. respectively using 16S rDNA sequence analysis and colonial morphology methods. The Bacillus sp. strain with the biggest hydrolyzed circle, named Bacillus sp. MX, was selected for further research.Theβ-mannanase gene was cloned from Bacillus sp. MX chromosome and inserted into pET-28a, further transduction of this recombinant plasmid into an E.coli BL21 strain resulted in a transformant, named BL21 (pET-man).The target protein with a His-tag was expressed efficiently when BL21 (pET-man) was induced by IPTG at 37℃for 3 hours. SDS-PAGE analysis showed that most of theβ-mannanase expressed in the cytoplasm exists in the form of inclusion bodies.Different temperatures、IPTG concentrations and inducing time were tested for a higher yield of solubleβ-mannanase. The optimum condition found in this study was 0.5mM IPTG at 30℃for 5 hours. Purified fusion protein was obtained using BBST NTA Resin.The molecular mass of the purified protein determined by SDS-PAGE approximates to 43 kDa . The enzyme was identified to be active within the range of 25-95℃and pH3.0-9.6 using the dinitrosalicylic acid (DNS) method, with the optimum temperature at 55℃, pH at 5.0 (specific activity reached 4572 U/mg). Purifiedβ-mannanase remained 84%, or 33% and 14% of its initial activity, respectively, when expose in a pH 4.0 or 80℃or90℃condition for 10min, suggesting good acid resistance and thermo-stability.The gene ofβ-mannanase was cloned and inserted into the shuttle plasmid pPIC9K to develop a recombinant plasmid named pPIC9K-man. The plasmid pPIC9K-man was then digested with endonuclease BglII and integrated into pichia pastoris GS115 chromosome by PEG transformation. The fusionβ-mannanase expressed by the eukaryotic recombinant showed a highest specific activity of 345U/mg, which is lower than that expressed in the E.coli. Presumably, it’s because that the expression of the procaryotic man gene was reduced due to codon bias in the eukaryote system, or due to the reduction in copy number when man was integrated in the GS115 chromosome. Further efforts will be needed on codon optimization and improvement of the enzymic activity.更多还原