【摘要】：嗜熱毛殼菌(Chaetomium thermophile CT2)是一種最適生長溫度較高的極端微生物,其產(chǎn)生的纖維素酶在高溫下可以保持優(yōu)良的催化活性,此特性利于纖維素酶的工業(yè)應(yīng)用。本研究以嗜熱毛殼菌為研究對象,通過RT-PCR擴(kuò)增出編碼嗜熱毛殼菌外切纖維二糖水解酶CBH3及內(nèi)切葡聚糖酶EG2成熟肽的基因,利用畢赤酵母誘導(dǎo)表達(dá)體系分別對這兩個(gè)蛋白進(jìn)行了高效表達(dá)、純化及性質(zhì)研究;對外切纖維二糖水解酶CBH3進(jìn)行同源建模,選定了5個(gè)氨基酸位點(diǎn)并且分別進(jìn)行了定點(diǎn)突變,利用畢赤酵母表達(dá)體系同樣對5個(gè)突變酶分別進(jìn)行了高效表達(dá)、純化及性質(zhì)研究。酵母工程菌WTCBH3表達(dá)的CBH3酶,其蛋白分子量大小約為47kDa,最適反應(yīng)溫度為60℃,最適反應(yīng)pH為5。該酶在70℃條件下保溫1h后,剩余酶活力仍有最高酶活的80%,具有良好的熱穩(wěn)定性。CBH3在50℃~60℃時(shí),酶活力保持在最高酶活的90%以上,但當(dāng)反應(yīng)溫度高于65℃后,CBH3酶活力會隨著溫度的升高而迅速降低,當(dāng)溫度達(dá)到70℃時(shí),剩余酶活力不足20%;酵母工程菌WTEG2表達(dá)的EG2酶,其蛋白分子量大小約為45kDa,最適反應(yīng)溫度為55℃,最適反應(yīng)pH為5,該酶在70℃條件下保溫1h后,剩余酶活力有70%。外切纖維二糖水解酶Cel7B與CBH3的氨基酸序列相似度達(dá)到80.65%,且Cel7B的空間三維結(jié)構(gòu)已通過X射線衍射技術(shù)了解清楚,Cel7B各項(xiàng)參數(shù)符合同源建模的模板標(biāo)準(zhǔn)。將CBH3與Cel7B進(jìn)行同源建模后,選定了CBH3上5個(gè)與底物結(jié)合相關(guān)的氨基酸位點(diǎn)為突變位點(diǎn)。通過定點(diǎn)突變的方法,將5個(gè)選定的氨基酸分別突變?yōu)楸彼岷?進(jìn)行酵母誘導(dǎo)表達(dá)、純化、收集蛋白,測定純化蛋白的酶學(xué)性質(zhì),期待篩選出CBH3結(jié)構(gòu)域中底物結(jié)合區(qū)的關(guān)鍵氨基酸。對純化的5個(gè)突變酶CBH3-246Y、CBH3-250R、CBH3-261D、CBH3-337D、CBH3-391R進(jìn)行活性測定,5個(gè)突變酶的最適溫度均為60℃,最適pH均為5。5個(gè)突變酶的酶活力與原酶CBH3相比都產(chǎn)生了不同程度的降低,其中突變體CBH3-391R的酶活力降低了約60%;突變體CBH3-246Y、CBH3-261D、CBH3-337D的酶活力均降低了25%以上。在反應(yīng)溫度為40℃時(shí),除CBH3-250R外,其它四個(gè)突變酶活力均降低到最高酶活的30%以下。根據(jù)實(shí)驗(yàn)結(jié)果可以推斷出,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催化反應(yīng)過程中與底物結(jié)合相關(guān)的重要氨基酸,其中第391位的精氨酸的突變對酶分子的活性影響最大。
[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，

本文編號：2488334