嗜热毛壳菌第七家族纤维素酶基因的克
发布时间：2019-05-29 23:58
【摘要】：嗜热毛壳菌(Chaetomium thermophile CT2)是一种最适生长温度较高的极端微生物,其产生的纤维素酶在高温下可以保持优良的催化活性,此特性利于纤维素酶的工业应用。本研究以嗜热毛壳菌为研究对象,通过RT-PCR扩增出编码嗜热毛壳菌外切纤维二糖水解酶CBH3及内切葡聚糖酶EG2成熟肽的基因,利用毕赤酵母诱导表达体系分别对这两个蛋白进行了高效表达、纯化及性质研究;对外切纤维二糖水解酶CBH3进行同源建模,选定了5个氨基酸位点并且分别进行了定点突变,利用毕赤酵母表达体系同样对5个突变酶分别进行了高效表达、纯化及性质研究。酵母工程菌WTCBH3表达的CBH3酶,其蛋白分子量大小约为47kDa,最适反应温度为60℃,最适反应pH为5。该酶在70℃条件下保温1h后,剩余酶活力仍有最高酶活的80%,具有良好的热稳定性。CBH3在50℃~60℃时,酶活力保持在最高酶活的90%以上,但当反应温度高于65℃后,CBH3酶活力会随着温度的升高而迅速降低,当温度达到70℃时,剩余酶活力不足20%;酵母工程菌WTEG2表达的EG2酶,其蛋白分子量大小约为45kDa,最适反应温度为55℃,最适反应pH为5,该酶在70℃条件下保温1h后,剩余酶活力有70%。外切纤维二糖水解酶Cel7B与CBH3的氨基酸序列相似度达到80.65%,且Cel7B的空间三维结构已通过X射线衍射技术了解清楚,Cel7B各项参数符合同源建模的模板标准。将CBH3与Cel7B进行同源建模后,选定了CBH3上5个与底物结合相关的氨基酸位点为突变位点。通过定点突变的方法,将5个选定的氨基酸分别突变为丙氨酸后,进行酵母诱导表达、纯化、收集蛋白,测定纯化蛋白的酶学性质,期待筛选出CBH3结构域中底物结合区的关键氨基酸。对纯化的5个突变酶CBH3-246Y、CBH3-250R、CBH3-261D、CBH3-337D、CBH3-391R进行活性测定,5个突变酶的最适温度均为60℃,最适pH均为5。5个突变酶的酶活力与原酶CBH3相比都产生了不同程度的降低,其中突变体CBH3-391R的酶活力降低了约60%;突变体CBH3-246Y、CBH3-261D、CBH3-337D的酶活力均降低了25%以上。在反应温度为40℃时,除CBH3-250R外,其它四个突变酶活力均降低到最高酶活的30%以下。根据实验结果可以推断出,CBH3氨基酸序列中第246位的酪氨酸、第261位的天冬氨酸、第337位的天冬氨酸、第391位的精氨酸都是CBH3催化反应过程中与底物结合相关的重要氨基酸,其中第391位的精氨酸的突变对酶分子的活性影响最大。
[Abstract]:(Chaetomium thermophile CT2) is an extreme microorganism with the optimum growth temperature. The cellulase produced by it can maintain excellent catalytic activity at high temperature, which is beneficial to the industrial application of cellulase. In this study, the genes encoding external fiber disaccharide hydrolase CBH3 and endoglucanase EG2 mature peptide of Chlamydomyces thermophilus were amplified by RT-PCR. The two proteins were highly expressed, purified and characterized by Pichia pastoris induced expression system. The homologous modeling of CBH3 was carried out. Five amino acid sites were selected and site-directed mutations were carried out. The high expression, purification and properties of the five mutases were also studied by using Pichia pastoris expression system. The protein molecular weight of CBH3 enzyme expressed by yeast engineering strain WTCBH3 was about 47kDa. the optimum reaction temperature was 60 鈩，
本文编号：2488334
[Abstract]:(Chaetomium thermophile CT2) is an extreme microorganism with the optimum growth temperature. The cellulase produced by it can maintain excellent catalytic activity at high temperature, which is beneficial to the industrial application of cellulase. In this study, the genes encoding external fiber disaccharide hydrolase CBH3 and endoglucanase EG2 mature peptide of Chlamydomyces thermophilus were amplified by RT-PCR. The two proteins were highly expressed, purified and characterized by Pichia pastoris induced expression system. The homologous modeling of CBH3 was carried out. Five amino acid sites were selected and site-directed mutations were carried out. The high expression, purification and properties of the five mutases were also studied by using Pichia pastoris expression system. The protein molecular weight of CBH3 enzyme expressed by yeast engineering strain WTCBH3 was about 47kDa. the optimum reaction temperature was 60 鈩，
本文编号：2488334
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