【作者】 魁红；

【导师】 董守良；

【作者基本信息】 兰州大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 木聚糖是一种结构复杂的多聚五碳糖分子,需要多种酶的协同作用才能完全降解。而木聚糖酶能够水解木聚糖主链的β-1,4-糖苷键,是木聚糖降解酶体系中最关键的水解酶。由于木聚糖酶在工业上的重要应用,因而对木聚糖酶的研究越来越受到关注。而选择从极端环境中筛选产酶菌株,并从中筛选到具备优良性质的酶,是目前重要的研究手段之一。本研究以真菌Bispora betulina及嗜碱细菌Nesterenkonia xinjiangensis为实验材料,分别用加入桦木木聚糖、燕麦木聚糖或麸皮与玉米芯的培养基对实验菌株进行诱导产酶实验。经酶活力测定,这两株菌均能在诱导下产胞外木聚糖酶。通过对木聚糖酶基因的序列分析,分别从真菌B. betulina和嗜碱细菌N.xinjiangensis中克隆得到了两个GH11木聚糖酶基因Xyn11BB与xyn11NX,通过BLAST比对分析：Xyn11BB和xyn11NX与已报道的木聚糖酶的序列的一致性均不超过70%,这表明两个基因均具有一定的新颖性。将Xyn11BB与xyn11NX分别在毕赤酵母和大肠杆菌中进行了表达,在摇床水平上重组后的木聚糖酶表达量分别为121.15和65.5 U/mL,从而验证了基因的功能。对重组木聚糖酶rXyn11BB及rXyn11NX分别进行了纯化及酶学性质研究。其中重组酶rXyn11BB的最适pH是4.5,在pH 4.0-10.0的范围内有良好的pH稳定性,最适温度是50℃；rXyn11BB的比活为470U/mg, Km为113.06 mg/mL, Vmax为303.03μmol·min-1mg-1；它对胶原蛋白酶和胰蛋白酶及α-糜蛋白酶具有良好的抗性。而重组酶rXyn11NX的最适pH为7.0,在中性和碱性条件下有良好的pH稳定性；最适温度为55℃,此酶具有很好的热稳定性,在70℃下保温60 min后能保留近60%的剩余酶活,甚至在90℃下保温15 min后酶活还能保留40%以上。rXyn11NX的比活为2158 U/mg,Km为16.08mg/mL,Vmax为45.66μmol·min-1mg-1。同时重组酶rXyn11NX没有纤维素酶活性,这些特点使rXyn11NX在工业上尤其是造纸业有良好的应用前景。综合酶学性质表明二者可作工业应用及科学研究的良好材料。更多还原

【Abstract】 Because of the complexity of xylan, the complete hydrolysis of xylan requires cooperation of a great variety of enzymes. Among them, xylanase plays an important role in the xylanolytic enzyme system by hydrolyzing theβ-1,4-glycoside linkages in xylan backbone. Due to the important industrial application great attention was focused on xylanase system. Nowadays, screening enzyme-producing strains from special environments and then obtaining enzymes with superior properties have been important research method.In this study, a fungus, Bispora betulina and an alkaliphilic bacterium, Nesterenkonia xinjiangensis were used for the strains. To determine whether they could produce xylanase, mediums containing oat spelt xylan, birchwood xylan, wheat bran or corncob were used to induce them. Results showed that all the carbon sources could induce the two strains to produce xylanase.Based on sequence analysis of xylanase, a GH11 xylanase gene, designated as Xyn11BB, was cloned from B. betulina. Meanwhile, the other GH11 xylanase gene, designated as xyn11NX, was cloned from N. xinjiangensis. By homology searches using BLAST, the sequences of nucleotides and coded amino acids of Xyn11BB or xyn11NX share the highest identities which are less than 70%among the known sequences of GH11 xylanases, respectively. The results demonstrated that both of them are novel.Xyn11BB and xyn11NX were expressed in Pichia pastoris and Escherichia coli, respectively. The corresponding activities were 121.15 and 65.5 U/mL, which verified the function of Xyn11BB and xyn11NX.The recombinant proteins were purified and further characterized. Xyn11BB from Pichia pastoris shows the optimal pH of 4.5, optimal temperature at 50℃, and is stable over pH 4.0-10.0; Km is 113.06 mg/mL; Vmaxis 303.03μmol-min-1mg-1; the specific activity is 470 U/mg when using oat spelt xylan as substrate; rXyn11BB has good resistance to collagenase, trypsin and a-chymotrypsin. Xyn11NX from Escherichia coli shows the optimal pH of 7.0, optimal temperature at 55℃; It is thermostable, retains nearly 60%of the initial activity after incubation at 70℃for 1 h and more than 40%of the activity at 90℃for 15 min; Km is 16.08 mg/mL; Vmaxis 45.66μmol·min-1mg-1; The specific activity is 2158 U/mg when using oat spelt xylan as substrate. In addition, the enzyme has no cellulase activity either. Above all, the characteristics make it promising for various applications, especially in pulp and paper industry. Both of two recombinant xylanases have good prospects in industrial application and scientific research due to the enzymical characterizations.更多还原