發(fā)布時(shí)間：2018-06-24 08:39

  本文選題：中性纖維素內(nèi)切酶 + 生物石洗　； 參考：《華東理工大學(xué)》2013年博士論文

【摘要】：中性纖維素內(nèi)切酶(中性EG)在牛仔布生物石洗中具有很高的應(yīng)用價(jià)值,但國內(nèi)中性EG不具有自主知識產(chǎn)權(quán),因此,發(fā)掘全新中性EG,對于打破國外中性EG的壟斷地位具有重要意義。完整中性EG和截短中性EG在牛仔布生物石洗上有不同表現(xiàn),這與纖維素酶的纖維素結(jié)合區(qū)相關(guān),但深層次原因還尚未發(fā)現(xiàn)。本文主要研究中性EG的生產(chǎn)菌株篩選、發(fā)掘、異源表達(dá)以及酶學(xué)性質(zhì)表征,初步研究中性EG在牛仔布生物石洗上的應(yīng)用,具體研究內(nèi)容和結(jié)論如下。 第一部分,Bacillus subtilis LH中性EG的基因發(fā)掘、異源高效表達(dá)、表征及其應(yīng)用于生物石洗的基礎(chǔ)研究。 1)從魯華生物技術(shù)研究所菌庫中,篩選到產(chǎn)中性EG的菌株B. subtilis LH,獲得B. subtilis LH的中性EG基因。Pichia pastoris GS115和Escherichia.coli Rosetta (DE3)分別表達(dá)了成熟中性EG(分別命名為MBSEG-1和MBSEG-2),結(jié)果表明MBSEG-2表達(dá)水平遠(yuǎn)高于MBSEG-1。采用最優(yōu)產(chǎn)酶條件,未濃縮MBSEG-2的EG酶活可達(dá)25686U/mL,是目前所有報(bào)道的最高水平。 2)MBSEG-1和MBSEG-2的酶學(xué)性質(zhì)相似,最適溫度均為65℃,最適pH值均為6.86,既有內(nèi)切酶活性又有外切酶活性,N-糖基化的有無引起了酶在熱穩(wěn)定性、木瓜蛋白酶有限水解上的差異。 3)基于截短MBSEG-2以及完整MBSEG-1和MBSEG-2水洗牛仔布實(shí)驗(yàn),并結(jié)合蛋白3D模型分析,發(fā)現(xiàn)具有高比例的表面疏水氨基酸和芳香氨基酸的EG有助于提高牛仔布的除毛率和脫色率,而與EG(但必須有催化域)的完整性無關(guān)。 第二部分,真菌Petriella setifera LH的鑒定,中性EG的表征及其應(yīng)用于生物石洗的基礎(chǔ)研究。 從魯華生物技術(shù)研究所的保存土壤中,篩選到一株產(chǎn)中性EG的真菌,基于延伸因子基因(EF-la)部分序列的系統(tǒng)發(fā)育樹的分析,并結(jié)合真菌形態(tài)學(xué)特征,鑒定該真菌為Pe. Setifera,命名為Pe. setifera LH。Pe. setifera LH的中性EG于40℃時(shí)可保持80%以上的相對酶活,最適pH為6-7,水洗牛仔布的效果與商業(yè)化中性EG相當(dāng),揭示了它在牛仔布生物石洗上具有潛在的應(yīng)用價(jià)值。 第三部分,真菌Phaeosphaeria sp. LH21兩個(gè)中性EG的基因發(fā)掘、序列分析及酶學(xué)性質(zhì)表征。 1)從極端環(huán)境的深海淤泥中,篩選到一株產(chǎn)中性EG的真菌,基于EF-1α部分序列和18S rDNA部分序列的比對分析,鑒定該真菌為Phaeosphaeria sp.,命名為Phaeosphaeria sp. LH21。 2)采用基因組走讀技術(shù)和RT-PCR方法,首次克隆了Phaeosphaeria sp. LH的兩個(gè)全新EG(GH45EG-1和GH45EG-2)基因。GH45EG-1和GH45EG-2全長分別為235和233個(gè)氨基酸。通過NCBI BLAST對氨基酸序列進(jìn)行比對分析,表明GH45EG-1和GH45EG-2屬于糖苷水解酶第45家族,僅有催化域無纖維素結(jié)合區(qū),其氨基酸序列和已知EG氨基酸序列分別擁有71%和63%的最大一致性。 3)基于兩個(gè)成熟EG (mGH45EG-1和mGH45EG-2)和Humicola insolens EG V氨基酸序列之間的比對分析,再根據(jù)蛋白3D結(jié)構(gòu)模型的分析,推測nGH45EG-1其中個(gè)活性中心位點(diǎn)為Asp16,推測mGH45EG-2活性中心位點(diǎn)為Asp122和Asp11,推測它們催化機(jī)制為反轉(zhuǎn)機(jī)制。 4)mGH45EG-1和mGH45EG-2分別于P. pastohs GS115中表達(dá)。重組酶mGH45EG-1和mGH45EG-2的底物譜證實(shí)它們屬于內(nèi)切酶類型,對羧甲基纖維素鈉具有最高降解活性,對結(jié)晶纖維素合濾紙降解的相對酶活僅為0.1-0.3%。10mM Ca2+和Mg2+提高了8-27%重組酶mGH45EG-1和nGH45EG-2的相對酶活,多數(shù)金屬離子對它們有抑制作用。重組酶mGH45EG-1和mGH45EG-2最適pH值均為8,在pH5-10時(shí)相對酶活均在75%以上,于50℃及pH3-10保溫1h歷相對殘余酶活均在90%以上,最適溫度分別為65℃和60℃。
[Abstract]:Neutral cellulose endonuclease (neutral EG) has high application value in denim biological stone washing, but neutral EG does not have independent intellectual property right in China. Therefore, it is of great significance to excavate new neutral EG for breaking the monopoly status of foreign neutral EG. Complete neutral EG and short neutral EG have different performance in denim biological stone washing. It is related to cellulose binding area of cellulase, but the deep reason has not yet been found. This paper mainly studies screening, excavating, heterologous expression and characterization of enzymatic properties of neutral EG producing strains, and preliminarily studies the application of neutral EG in denim biological stone washing. The specific contents and conclusions are as follows.
The first part is the gene discovery of Bacillus subtilis LH neutral EG, heterologous high expression, characterization and its application in biological stone washing.
1) from the bacterial Library of the Institute of biotechnology, the strain B. subtilis LH producing neutral EG was screened and the neutral EG gene.Pichia pastoris GS115 of B. subtilis LH was obtained, and the neutral EG gene was expressed respectively. Under the optimal conditions of enzyme production, the EG activity of MBSEG-2 without concentration reached 25686U/mL, which is the highest level reported at all times.
2) the enzymatic properties of MBSEG-1 and MBSEG-2 are similar, the optimum temperature is 65, the optimum pH value is 6.86, both endonuclease activity and exonuclease activity. N- glycosylation has caused the difference in the enzyme's thermal stability and the limited hydrolysis of papain.
3) based on the truncated MBSEG-2 and the complete MBSEG-1 and MBSEG-2 washing denim experiment, and combined with the protein 3D model analysis, it is found that the high proportion of the hydrophobic amino acid and the aromatic amino acid EG are helpful to improve the denim's hair removal rate and decolorization rate, but have nothing to do with the integrity of the EG (but must have a catalytic domain).
The second part is the identification of fungal Petriella SETIFERA LH, the characterization of neutral EG and its application in biological stone washing.
From the preserved soil of the Shandong Institute of biotechnology, we screened a fungus producing neutral EG, based on the phylogenetic tree of the extension factor gene (EF-la) sequence, and combined with the morphological characteristics of the fungi to identify the fungus as Pe. Setifera, and the neutral EG named Pe. SETIFERA LH.Pe. SETIFERA LH can maintain more than 80% at 40. The relative enzyme activity, the optimum pH is 6-7, and the washing denim effect is equivalent to commercial neutral EG. It reveals its potential application in denim biological stone washing.
The third part is the gene discovery, sequence analysis and characterization of two neutral EG of fungal Phaeosphaeria sp. LH21.
1) a fungus producing neutral EG was screened from the deep-sea silt of extreme environment. Based on the comparison and analysis of the partial sequence of EF-1 A and the partial sequence of 18S rDNA, the fungus was identified as Phaeosphaeria sp., named Phaeosphaeria sp. LH21..
2) two new EG (GH45EG-1 and GH45EG-2) genes of EG (GH45EG-1 and GH45EG-2) gene were cloned for the first time by genomic access technique and RT-PCR method. The total length of.GH45EG-1 and GH45EG-2 were 235 and 233 respectively. The sequence of amino acids was compared by NCBI BLAST, indicating that GH45EG-1 and glycosides belong to the forty-fifth family of glucoside hydrolase. There is no cellulose binding region in the catalytic domain. The amino acid sequence and the known EG amino acid sequence have the largest consistency of 71% and 63% respectively.
3) based on the comparison and analysis of two mature EG (mGH45EG-1 and mGH45EG-2) and Humicola insolens EG V amino acid sequences, according to the analysis of the structure model of protein 3D, we speculated that the active center site of nGH45EG-1 was Asp16.
4) mGH45EG-1 and mGH45EG-2 were expressed in P. pastohs GS115, respectively. The substrate spectra of the recombinant enzyme mGH45EG-1 and mGH45EG-2 confirmed that they belonged to the type of endonuclease, and had the highest degradation activity to sodium carboxymethyl cellulose. The relative enzyme activity for the degradation of the crystalline cellulose filter paper was only 0.1-0.3%.10mM Ca2+ and Mg2+ increased the 8-27% recombinant enzyme. The relative enzyme activity of H45EG-2 was inhibited by most metal ions. The optimum pH value of the recombinant enzyme mGH45EG-1 and mGH45EG-2 was 8, and the relative enzyme activity was above 75% at pH5-10, and the relative residual enzyme activity at 50 and pH3-10 was above 90%, and the optimum temperature was 65 and 60, respectively.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：Q814

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