中性纤维素内切酶的发掘、表征及其应用于生物石洗的基础研究

发布时间：2018-06-24 08:39

本文选题：中性纤维素内切酶 + 生物石洗　；参考：《华东理工大学》2013年博士论文

【摘要】：中性纤维素内切酶(中性EG)在牛仔布生物石洗中具有很高的应用价值,但国内中性EG不具有自主知识产权,因此,发掘全新中性EG,对于打破国外中性EG的垄断地位具有重要意义。完整中性EG和截短中性EG在牛仔布生物石洗上有不同表现,这与纤维素酶的纤维素结合区相关,但深层次原因还尚未发现。本文主要研究中性EG的生产菌株筛选、发掘、异源表达以及酶学性质表征,初步研究中性EG在牛仔布生物石洗上的应用,具体研究内容和结论如下。第一部分,Bacillus subtilis LH中性EG的基因发掘、异源高效表达、表征及其应用于生物石洗的基础研究。 1)从鲁华生物技术研究所菌库中,筛选到产中性EG的菌株B. subtilis LH,获得B. subtilis LH的中性EG基因。Pichia pastoris GS115和Escherichia.coli Rosetta (DE3)分别表达了成熟中性EG(分别命名为MBSEG-1和MBSEG-2),结果表明MBSEG-2表达水平远高于MBSEG-1。采用最优产酶条件,未浓缩MBSEG-2的EG酶活可达25686U/mL,是目前所有报道的最高水平。 2)MBSEG-1和MBSEG-2的酶学性质相似,最适温度均为65℃,最适pH值均为6.86,既有内切酶活性又有外切酶活性,N-糖基化的有无引起了酶在热稳定性、木瓜蛋白酶有限水解上的差异。 3)基于截短MBSEG-2以及完整MBSEG-1和MBSEG-2水洗牛仔布实验,并结合蛋白3D模型分析,发现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脱色率,而与EG(但必须有催化域)的完整性无关。第二部分,真菌Petriella setifera LH的鉴定,中性EG的表征及其应用于生物石洗的基础研究。从鲁华生物技术研究所的保存土壤中,筛选到一株产中性EG的真菌,基于延伸因子基因(EF-la)部分序列的系统发育树的分析,并结合真菌形态学特征,鉴定该真菌为Pe. Setifera,命名为Pe. setifera LH。Pe. setifera LH的中性EG于40℃时可保持80%以上的相对酶活,最适pH为6-7,水洗牛仔布的效果与商业化中性EG相当,揭示了它在牛仔布生物石洗上具有潜在的应用价值。第三部分,真菌Phaeosphaeria sp. LH21两个中性EG的基因发掘、序列分析及酶学性质表征。 1)从极端环境的深海淤泥中,筛选到一株产中性EG的真菌,基于EF-1α部分序列和18S rDNA部分序列的比对分析,鉴定该真菌为Phaeosphaeria sp.,命名为Phaeosphaeria sp. LH21。 2)采用基因组走读技术和RT-PCR方法,首次克隆了Phaeosphaeria sp. LH的两个全新EG(GH45EG-1和GH45EG-2)基因。GH45EG-1和GH45EG-2全长分别为235和233个氨基酸。通过NCBI BLAST对氨基酸序列进行比对分析,表明GH45EG-1和GH45EG-2属于糖苷水解酶第45家族,仅有催化域无纤维素结合区,其氨基酸序列和已知EG氨基酸序列分别拥有71%和63%的最大一致性。 3)基于两个成熟EG (mGH45EG-1和mGH45EG-2)和Humicola insolens EG V氨基酸序列之间的比对分析,再根据蛋白3D结构模型的分析,推测nGH45EG-1其中个活性中心位点为Asp16,推测mGH45EG-2活性中心位点为Asp122和Asp11,推测它们催化机制为反转机制。 4)mGH45EG-1和mGH45EG-2分别于P. pastohs GS115中表达。重组酶mGH45EG-1和mGH45EG-2的底物谱证实它们属于内切酶类型,对羧甲基纤维素钠具有最高降解活性,对结晶纤维素合滤纸降解的相对酶活仅为0.1-0.3%。10mM Ca2+和Mg2+提高了8-27%重组酶mGH45EG-1和nGH45EG-2的相对酶活,多数金属离子对它们有抑制作用。重组酶mGH45EG-1和mGH45EG-2最适pH值均为8,在pH5-10时相对酶活均在75%以上,于50℃及pH3-10保温1h历相对残余酶活均在90%以上,最适温度分别为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.
【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：Q814

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