本文選題：低溫酶 + 基因克隆��； 參考：《天津科技大學(xué)》2017年碩士論文

【摘要】：木聚糖是植物細(xì)胞壁的主要組成成分,它的完全降解需要多種降解酶的共同作用,其中木聚糖酶和木糖苷酶是木聚糖降解過程中的關(guān)鍵酶。木聚糖降解酶已經(jīng)廣泛的應(yīng)用飼料、食品、造紙和生物能源等多個工業(yè)領(lǐng)域。近年來,人們發(fā)現(xiàn)在食品加工等行業(yè)中應(yīng)用嗜冷木聚糖降解酶可以有效減少產(chǎn)品活性成分、風(fēng)味物質(zhì)和營養(yǎng)成分的損失,降低生產(chǎn)成本。然而,目前發(fā)現(xiàn)的木聚糖酶和木糖苷酶絕大多數(shù)都屬于中高溫酶(http://www.brenda-enzymes.org),已報道有基因序列和酶學(xué)性質(zhì)研究的低溫木糖苷酶仍然匱乏,亟待發(fā)掘性質(zhì)優(yōu)良的嗜冷木聚糖酶和木糖苷酶基因資源,并對其嗜冷機(jī)制進(jìn)行研究,本研究獲得了一個序列和性質(zhì)新穎的嗜冷木聚糖酶Xyn27,并且對嗜冷木糖苷酶AX543的嗜冷機(jī)制進(jìn)行了初步分析和突變研究。具體如下:通過Touch-down PCR和熱不對稱交錯(TAIL) PCR方法,從枝頂孢屬菌的基因組DNA中獲得了 GH10家族(Xyn27)的新型木聚糖酶,Xyn27包含一個信號肽(20個殘基),編碼346個氨基酸,與報導(dǎo)的GH10木聚糖酶(XM_003662144)最高一致性為83 %。重組Xyn27在畢赤酵母GS115中成功表達(dá),最適誘導(dǎo)條件為35℃、48h。Xyn27是一種嗜冷木聚糖酶,其中在35℃時的活性最高,在20℃,10℃和0℃下的相對活性仍為60.25%, 38.70%和10.8%。進(jìn)一步分析表明,與嗜溫或嗜熱的對應(yīng)物相比,Xyn27具有較少的精氨酸而有更多的丙氨酸殘基。Xyn27的最適pH為7.0,在3.0～9.0的pH范圍內(nèi)反應(yīng)lh后仍較穩(wěn)定。此外,Xyn27對于大多數(shù)金屬離子和有機(jī)溶劑有耐受性,優(yōu)于其他GH10嗜冷木聚糖酶,其中對于重金屬離子Ca2+,. Mn2+和Zn2+活性顯著增強(qiáng)。性質(zhì)研究表明,以1%櫸木木聚糖為底物時的Km,Vmax,kcat和 kcat . Km-1 分別為 13.42 mg . mL-1, 9.07 umol · min-1 . mg-1, 192.98 min-1和 14.38 mL.min-1.mg,Xyn27可將木聚糖完全降解為木二糖�；谀揪厶敲竂yn27的嗜冷活性和金屬離子耐受性,表明其在基礎(chǔ)研究和食品等行業(yè)有潛在應(yīng)用。木糖苷酶AX543最適溫度為25℃,為目前報道的最適溫度較低的酶,通過一系列的序列分析和結(jié)構(gòu)比對,推斷l(xiāng)oop區(qū)結(jié)構(gòu)的差異可能是低溫酶嗜冷的主要原因之一,其次還包括氨基酸殘基的不同,經(jīng)分析發(fā)現(xiàn)AX543在5個位置的loop區(qū)存在顯著差異,分別是 loopl (31-46)、loop2 (54-59)、loop3 (164-183)、loop4 (209-224)和 loop5(296-298),于是針對這些位置,設(shè)計引物進(jìn)行突變,成功獲得六個質(zhì)粒,其中前五個突變體成功在畢赤酵母中表達(dá),但由于活性均比較低,未進(jìn)行下一步研究。
[Abstract]:Xylan is the main component of plant cell wall, and its complete degradation requires the joint action of many kinds of degradation enzymes, among which xylanase and xylosidase are the key enzymes in the process of xylan degradation. Xylanase has been widely used in feed, food, paper making and bioenergy industries. In recent years, it has been found that the application of cold-eosinophilic xylanase in food processing and other industries can effectively reduce the loss of active ingredients, flavor substances and nutrients, and reduce the production cost. However, most of the xylanase and xylosidase found at present belong to medium high temperature enzyme http: / www.brenda-enzymes.org.There is still a lack of low-temperature xylosidase, which has been reported to have been studied in gene sequence and enzymology. It is urgent to explore the chilled xylanase and xylanase gene resources, and to study the mechanism of psychrophilic xylanase. In this study, a novel chilled xylanase Xyn27 was obtained, and the mechanism of the chilled xylanase AX543 was preliminarily analyzed and mutated. The results are as follows: a novel xylanase Xyn27 containing a signal peptide (20 residues, encoding 346 amino acids) was obtained from the genomic DNA of the genus Cladosporium by Touch-down PCR and thermal asymmetric interlacing (TAILL) PCR, and a novel xylanase, Xyn27, was obtained from the genomic DNA of the genus Cladosporium. The highest consistency with the reported GH10 xylanase X _ M _ S _ 003662144) was 83. The recombinant Xyn27 was successfully expressed in Pichia pastoris GS115. The optimal inducing condition was that Xyn27 was a chilled xylanase at 35 鈩，

本文編號：1883992