發(fā)布時(shí)間：2018-03-23 17:22

  本文選題：角蛋白酶　切入點(diǎn)：微白黃鏈霉菌Fea-10　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：角蛋白酶(keratinase)是一類可以降解頑固的角蛋白(如羽毛、羊毛、頭發(fā)等)的蛋白酶,廣泛存在于自然界中。角蛋白酶一般可以分類為絲氨酸蛋白酶和金屬蛋白酶。由于角蛋白不能被普通的蛋白酶降解,且數(shù)量巨大,每年都會(huì)有數(shù)百萬(wàn)噸的含角蛋白廢棄物產(chǎn)生,但是它們并不會(huì)在自然界中堆積,這種要是角蛋白降解微生物產(chǎn)生的角蛋白酶將這些廢棄物分解,從而減輕了環(huán)境的負(fù)擔(dān)。目前,已知細(xì)菌、真菌和放線菌中均有可以產(chǎn)生角蛋白酶的菌株,古細(xì)菌中也有可以產(chǎn)生角蛋白酶的報(bào)道。角蛋白降解微生物可以利用廉價(jià)的角質(zhì)廢棄物作為培養(yǎng)基質(zhì)來(lái)產(chǎn)生角蛋白酶,使得角蛋白酶具有較低的生產(chǎn)成本,且角蛋白酶在各種工業(yè)領(lǐng)域具有很好的應(yīng)用,具有很高的利用價(jià)值。本文在養(yǎng)雞場(chǎng)堆積羽毛的土壤中,分離得到了一株角蛋白降解菌Fea-10。經(jīng)過(guò)初步鑒定,Fea-10為微白黃鏈霉菌Streptomyces albidoflavus。Fea-10具有很高的角蛋白酶活性,可以完整的降解羽毛。通過(guò)已知的角蛋白酶的N端序列,在Fea-10基因組中找到兩個(gè)可能的角蛋白酶基因gm2886和gm2888。這兩個(gè)基因均由信號(hào)肽、前肽和成熟酶三個(gè)部分構(gòu)成。將這兩個(gè)基因的完整基因分別構(gòu)建在pET15b-SUMO載體上,轉(zhuǎn)入BL21(DE3)均不能表達(dá);更換為pET28a載體和Rossta(DE3)宿主后,只有g(shù)m2888可以獲得無(wú)活性的包涵體表達(dá);將這兩個(gè)基因去掉信號(hào)肽的酶原基因分別構(gòu)建在pET22b載體上,轉(zhuǎn)入Rossta(DE3),只有2886可以獲得可溶性表達(dá),但并沒(méi)有角蛋白酶活性�？紤]到可能鏈霉菌表達(dá)系統(tǒng)更適于鏈霉菌來(lái)源的角蛋白酶表達(dá)并折疊為有活性的成熟酶,使用鏈霉菌-大腸桿菌穿梭整合型質(zhì)粒pSET152進(jìn)行表達(dá)。表達(dá)宿主菌選擇微弱角蛋白酶活的密旋鏈霉菌ACT12。構(gòu)建gm2886帶有原始啟動(dòng)子和紅霉素啟動(dòng)子的表達(dá)載體和gm2888帶有紅霉素啟動(dòng)子的表達(dá)載體,分別接合轉(zhuǎn)移導(dǎo)入ACT12。使用牛奶平板初篩接合子,并使用TSBY培養(yǎng)基發(fā)酵降解圈大的接合子,使用硫酸銨沉淀后,檢測(cè)只有g(shù)m2886帶紅霉素的載體有角蛋白酶表達(dá)。使用Ni柱純化重組蛋白,并測(cè)定其酶學(xué)性質(zhì)。重組蛋白的最適溫度為50℃,最適pH10.0,在40℃保溫30 min后酶活仍有80%以上。金屬離子Ca~(2+)、Mg~(2+)、K~+均抑制重組蛋白的活性;PMSF顯著抑制重組蛋白的活性,重組蛋白為絲氨酸蛋白酶。重組蛋白對(duì)可溶的casein、Azo-casein、BSA和hemoglobin以及不可溶的羽毛和天青角蛋白均有活性。
[Abstract]:Keratinase is a kind of protease that degrades stubborn keratin (such as feathers, wool, hair, etc.). Keratin is widely distributed in nature. Keratin can be classified as serine protease and metalloproteinase. Because keratin can not be degraded by ordinary protease, and in large quantities, millions of tons of keratin waste are produced every year. But they don't pile up in nature, if keratinase, which is produced by keratin degradation microbes, breaks down these wastes, reducing the burden on the environment. Both fungi and actinomycetes have strains that produce keratinases, and there are reports of keratinases in ancient bacteria. Keratin degrading microbes can use cheap keratinized waste as a culture substrate to produce keratinases. Keratinase has a low production cost, and keratinase has a good application in various industrial fields, and has a high value of utilization. A keratin degrading strain Fea-10 was isolated. It was preliminarily identified that Streptomyces albidoflavus.Fea-10 had high keratinase activity and could completely degrade feathers. Two possible keratinase genes gm2886 and gm2888were found in the Fea-10 genome. The two genes were composed of signal peptide, prepeptide and mature enzyme. The complete genes of these two genes were constructed on pET15b-SUMO vector and transformed into BL21DE3). Only gm2888 could obtain inactive inclusion body expression after being replaced by pET28a vector and Rosstafen DE3) host, and the proenzyme genes which removed the signal peptide from the two genes were constructed on the pET22b vector respectively and transferred into pET22b vector, only 2886 of them could be expressed in a soluble way. Considering that Streptomyces expression systems may be more suitable for streptomyces to express and fold into active mature enzymes, Streptomycin-Escherichia coli shuttle integrated plasmid pSET152 was used to express the recombinant plasmid ACT12. Streptomyces sp. ACT12, which expressed the weak keratinase activity of the host strain, was used to construct the expression vector of gm2886 with the original promoter and erythromycin promoter and gm2888 with the expression of ACT12. The expression vector of erythromycin promoter, The conjugate transfer was introduced into ACT12. the conjugate was first screened with milk plate and fermented with TSBY medium to degrade the conjugate with large circle. After precipitation with ammonium sulfate, the conjugate was incubated with ammonium sulfate. The recombinant protein was purified by Ni column and its enzymatic properties were determined. The optimum temperature of recombinant protein was 50 鈩，

本文編號(hào)：1654438