本文關(guān)鍵詞：Aspergillus oryzae溶解多糖單加氧酶基因的克隆，異源表達(dá)及酶學(xué)特性分析　出處：《云南大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 溶解多糖單加氧酶 米曲霉 畢赤酵母 異源表達(dá) 酶活

【摘要】：溶解多糖單加氧酶(Lytic polysaccharide monooxygenases, LPMOs)是一類能夠促進(jìn)生物質(zhì)如幾丁質(zhì)和纖維素等降解獲得可溶性寡糖的氧化酶。本研究根據(jù)己報(bào)道的米曲霉LPMOs基因序列(GenBank:BAE61530.1)設(shè)計(jì)特異性引物,PCR擴(kuò)增得到長度為1266 bp的溶解多糖單加氧酶基因lpmo,將該基因克隆到T載體上進(jìn)行測序驗(yàn)證,測序結(jié)果和己知序列相似度100%。基因lpmo和載體pET32a、pPIC9K用相同的限制性內(nèi)切酶進(jìn)行雙酶切、轉(zhuǎn)化到大腸桿菌DH5a中,挑取轉(zhuǎn)化子進(jìn)行酶切和測序驗(yàn)證,獲得了重組表達(dá)載體pET32a-l-pmo(ZH-1)和pPIC9K-lpmo(ZH-2).重組載體pET32a-lpmo和pPIC9K-lpmo分別通過化學(xué)轉(zhuǎn)化法和電轉(zhuǎn)法導(dǎo)入大腸桿菌BL21和畢赤酵母GS115中。重組畢赤酵母GS115/pPIC9K-lpmo經(jīng)甲醇誘導(dǎo)5天后,其酶活較高。重組大腸桿菌BL21/pET32a-lpmo I在IPTG濃度0.2-1mg/mL時(shí)誘導(dǎo)表達(dá)16h后表達(dá)量均為600 mg/L,其最適表達(dá)溫度為16-28℃。表達(dá)的蛋白用Ni親和層析柱純化出相對(duì)較單一重組蛋白,經(jīng)過SDS-PAGE電泳檢測結(jié)果表明,純化后的重組蛋白的分子量大小約為68 kDa。根據(jù)酶學(xué)性質(zhì)研究表明,純化后的LPMOs對(duì)底物殼聚糖的最適反應(yīng)溫度為50℃；超過50℃,顯色反應(yīng)表明,酶活隨溫度升高逐漸降低；低于50℃,酶活隨溫度降低而隨之變化。其最適反應(yīng)的pH值約為6.0,當(dāng)pH大于6.0時(shí)酶活逐漸降低,說明LPMOs在弱酸條件下活性較高。金屬離子對(duì)重組LPMOs酶活也有一定程度的影響,低濃度Cu2+、Al3+、Fe3+金屬離子對(duì)重組LPMOs酶活具有促進(jìn)作用,高濃度Cu2+、Al3+、Fe3+對(duì)酶活具有抑制作用；Zn2+對(duì)酶活無影響。本實(shí)驗(yàn)研究表明,通過構(gòu)建異源表達(dá)載體,提高LPMOs表達(dá)量。LPMOs在50℃和pH 6.0條件下酶活相對(duì)較高。為今后纖維素降解工業(yè)生產(chǎn)乙醇提供一定技術(shù)支持。
[Abstract]:Lytic polysaccharide monooxygenases. LPMOs is a kind of oxidase that can promote the degradation of biomass such as chitin and cellulose to obtain soluble oligosaccharides. The LPMOs gene sequence of Aspergillus oryzae has been reported in this study. GenBank: BAE61530.1). A 1266bp LPM gene was amplified by PCR and cloned into T vector for sequencing. Lpmo and pET32a pPIC9K were digested with the same restriction endonuclease and transformed into Escherichia coli DH5a. The transformants were digested and sequenced. Recombinant expression vectors pET32a-l-pmoguanZH-1) and pPIC9K-lpmo-ZH-2) were obtained. The recombinant vectors pET32a-lpmo and pPIC9K-lpmo were introduced into Escherichia coli BL21 and Pichia pastoris GS115 by chemical transformation and electrotransposition respectively. The recombinant Pichia pastoris G. S115 / pPIC9K-lpmo was induced by methanol for 5 days. The recombinant Escherichia coli BL21/pET32a-lpmo I was induced to express at 0.2-1 mg / mL IPTG for 16 h and the expression level was 600 mg/L. The optimal expression temperature was 16-28 鈩，

本文編號(hào)：1404963