【作者】 高慧；

【导师】 孙建义；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2006， 硕士

【摘要】 β-1，4-木聚糖酶(EC 3．2．1．8)是降解木聚糖的关键酶，能大幅度提高半纤维素物质的利用效率，因此广泛应用于饲料、造纸和食品等工业，而植物中木聚糖酶抑制剂的存在影响了酶活性的发挥。本文克隆了小麦木聚糖酶抑制剂TAXI-Ⅰ基因，使其在E．coliBL21中表达，筛选阳性重组子；并对表达产物的抑制特性进行了研究；根据不同木聚糖酶对抑制剂敏感度的差异，从而筛选出一种对抑制剂低敏感的木聚糖酶。主要结果如下：根据GenBank上已登陆的小麦木聚糖酶抑制剂基因TAXI-Ⅰ(AJ438880)设计引物，克隆出长度为1284bp的目的片段。根据已克隆的木聚糖酶抑制剂TAXI-Ⅰ基因的酶切图谱及表达载体pET-30a(+)的多克隆位点(MCS)，设计了一对含限制性酶切位点的表达引物：5TAXI02E(EcoRI)／3TAXI02E(XhoI)。以含木聚糖酶基因的pUCm-TTAXIVector(以下简称T-TAXI)质粒为模板进行PCR扩增，获得了相应的两端带有限制性酶切位点的DNA片段，其大小为1175bp。将以上DNA片段与pUCm-T Vector质粒连接获得亚克隆重组质粒pUCm-T TAXIE Vector(简称T-TAXIE)。T-TAXIE经EcoRI和XhoI双酶切后分别与经相同酶双酶切的表达载体pET-30a(+)连接，获得重组表达质粒pET-30a(+)-TAXIE，然后导入E．coliBL21中表达，筛选出阳性重组子。对E．colipET-30a(+)-TAXIE分泌的木聚糖酶抑制剂抑制特性研究表明，其分子量为46.7KDa，在20℃时抑制剂活性最低；在20℃的条件下，当作用时间不超过30min时，抑制剂活性上升较快，以后随着反应时间的延长，活性没有发生显著性变化；在0℃-70℃处理40min，抑制剂活性不受温度的显著影响；对来自G／11家族的细菌和真菌木聚糖酶进行研究，与细菌的木聚糖酶(TFX、BtXA和MHX)相比，真菌的木聚糖酶(ANX和TrXA)更易被TAXI-Ⅰ抑制，在20℃下处理30min，ANX反应后的残余酶活只剩15.8％，而BtXA和MHX残余酶活还具有53.0％和73.8％，TFX抗抑制能力最强，残余酶活高达88.4％。因此，从对抑制剂的敏感度方面考虑，TFX更适合应用，是一种优良的木聚糖酶。更多还原

【Abstract】 β-1, 4-xylanases （EC 3.2.1.8） can catalyze the hydrolysis of xylan （the major constituent of hemicellulose） and play a significant role in nature by recycling hemicellulose. They are widely applied in animal feed and food industries and in biobleaching. However, substances which inhibit the hydrolytic activity of endoxylanases in cereals affect the functionality and performance of the enzymes. In this study, the Triticum aestivum xylanase inhibitor I （TAXI-I） was cloned and expressed by Escherichia coli BL21. Xylanase inhibitor activities against xylanase were analysed with a purpose to provide a theoretical basis for an accurate screening of xylanase. The main results were as follows:The Triticum aestivum xylanase inhibitor I （TAXI-I, AJ438880） gene was cloned according to secquence information in GenBank. The fragment containing the TAXI-I region was amplified by Polymerase Chain Reaction （PCR） from pUCm-T TAXI Vector by using pyrobes^TM DNA Polymerase and a pair of primes 5TAXI02E/3TAXI02E which were designed by analyzing the restriction site mapping of TAXI and the multiple cloning site （MCS） of vector pET-30a（+）, so introducing EcoRI site at 3-end and XhoI site at 5-end of the gene. The resulting PCR product （1175bp） was subcloned into pUCm-T Vector, the positive clone was double digested by EcoRI and XhoI, the DNA insert was ligated into the EcoRI-XhoI-cut site of pET-30a（+）, constructing recombinant vector pET-30a（+）-TAXIE. The recombinant pET-30a （+）-TAXIE was transformed into Escherichia coli BL21 and the recombinants were obtained.The specificity of TAXI-I indicated that xylanase inhibitor had a molecular weight of 46.7KDa, and the activity was at its lowest at 20℃. The inhibitor activity ascended fast during 30min at 20℃, while its activity had no significant diversification in 30min. The activity increased gently after being treated during 0℃-70℃for 30min. TAXI-I inhibit different endoxylanases to varying extents. High inhibition was found against fungal endoxylanases（ANX and TrXA） as well as low inhibition against bacterial endoxylanases （TFX、BtXA and MHX）. In addition, the results indicated that xylanase activities of ANX and TFX remained 15.8% and 88.4%, respectively. So TFX xylanase is the optimum screen in this experiment.更多还原