平菇漆酶基因生物信息学分析和杂优-2平菇漆酶分离纯化及酶学性质、功能基团研究

发布时间：2018-04-23 08:23

本文选题：漆酶 + 生物信息学　；参考：《西南大学》2017年硕士论文

【摘要】：漆酶(laccase)是一种铜多酚氧化酶,是木质素降解酶系之一,在食用菌栽培过程中,漆酶对菌丝体扭结具有促进作用,能够形成较多的子实体原基,进而提高食用菌的产量,且在工业染料废水处理等方面有重要作用。依据平菇基因组数据,真菌木质素降解酶的编码基因能在基因组水平上得到统计和注释。但是,目前也存在一些亟待解决的问题,如功能基因注释结果的不准确性、漆酶基因转录调控机理不清楚等。本文通过对平菇漆酶的11条基因序列及其推断氨基酸序列进行生物信息学分析,为研究漆酶基因的调控机理、功能基因组及为本文实验提供理论指导。同时,本实验选用尚未报道的杂优-2平菇为实验材料,从中分离纯化出漆酶并对其理化性质进行研究旨在为充分了解杂优-2平菇漆酶的理化性质及对漆酶的进一步研究、食用菌生产和工业应用等方面提供参考。平菇漆酶基因可能受到N源和来自外界压力胁迫调控,但可能不会受到培养基中葡萄糖浓度的影响,除LACC12外,其余漆酶基因可能会共同响应高浓度的铜离子的压力而激活平菇漆酶基因的转录;杂优-2平菇漆酶在中性和碱性条件下稳定性较好,但该酶的热稳定性较低。二硫键在维持杂优-2平菇漆酶的空间结构稳定性有重要作用。研究结果如下:1、启动子区域分析结果显示:LACC1-LACC12(LACC5除外)均含有不同的顺式作用元件,包括热激响应元件(HSE)、压力响应元件(STRE)、金属应答元件(MRE)、异生物质反应元件(XRE)、抗氧化响应元件(ARE)、CreA因子结合位点(CreA-bingding site)、氮因子结合位点(NIT),还包括CAAT框(CAAT Box)、TATA框(TATA Box)等核心转录起始位点。对漆酶基因编码区上游2000 bp和下游2000bp区域的限制性酶切位点分析发现:LACC1-LACC12(LACC5除外)的限制性酶切位点数在21~56范围不等。2、漆酶基因系统进化分析及LACC1-LACC12(LACC5除外)相似性分析结果显示:LACC1与秀珍菇(Pleurotus sajor-caju)的LACC2相似性为99.59%;LACC3与秀珍菇(Pleurotus sajor-caju)的LACC5和凤尾菇(Pleurotus pulmonarius)的LACC7相似性分别为87.90%、87.04%;LACC7与杏鲍菇(Pleurotus eryngii)LACC1相似性为95.96%;LACC10与秀珍菇(Pleurotus sajor-caju)的LACC4相似性为97.93%,11条漆酶基因聚为三支。3、氨基酸序列相似性分析发现:11条推断氨基酸序列聚为4支,在进化关系中,LACC6与LACC8、LACC9与LACC10为一支,亲缘关系最近。氨基酸多序列比对和BLAST及RPS-BLAST结果显示:11条推断氨基酸序列中均有漆酶特征序列L1-L4和3个高度保守的铜氧化酶(Cu-oxidase)结构域;SiganlP4.1及Protcomp9.0结果显示:11推断氨基酸序列中都含信号肽,且都为外分泌蛋白。4、Protparam对11条漆酶推断氨基酸理化性质预测结果显示:AI在83.64~92.81;ExtCof1和ExtCof2几乎没有差异;除LACC1和LACC10外,其余氨基酸的GRAVY均为负值;II在32.81~42.04范围内;MW介于55.68~58.87之间;pI在4.53~7.78范围;除LACC8外,其余氨基酸的TNNCR均高于TNPCR。5、推断氨基酸理化性质和组成分析结果显示:理化性质分为三簇,ClusterⅠ与ClusterⅢ、ClusterⅡ与ClusterⅢ之间存在显著性差异(P0.05),ClusterⅠ与ClusterⅡ之间无显著性差异(P0.05);每个簇内的氨基酸理化性质差异性在三个簇之间无显著性差异(P0.05)。氨基酸组成也分为三簇,ClusterⅠ与ClusterⅡ及ClusterⅢ均有显著性差异(P0.05),ClusterⅡ与ClusterⅢ间无显著性差异(P0.05);ClusterⅠ、ClusterⅡ、ClusterⅢ内的漆酶氨基酸序列组成差异性在三个簇之间无显著差异(P0.05)。6、通过对杂优-2平菇菌丝进行液体培养,发酵液经硫酸铵分级沉淀、DEAE(diethylaminoethyl)-Sepharose fast flow层析和Superdex-200 prep grade层析等方法纯化,获得了电泳纯的杂优-2平菇漆酶。结果显示,培养第6天时漆酶活性最高;杂优-2平菇漆酶比活力为115 U/mg,纯化倍数为111.65,回收率为6.96%。分子质量约为244.0 kD,亚基分子质量约为85.6 kD。最适反应pH值和最适反应温度分别为5.0和55℃,在pH 6.0~8.0及40~55℃范围内稳定性较好;最适条件下,以2,2’-连氮-二(3-乙基苯并噻唑-6-磺酸)(2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate),ABTS)为底物的Km值为2.1mmol/L,最大反应速率(Vmax)为0.117μmol/(min·L)。Fe2+、抗坏血酸对该酶活性具有完全抑制作用,乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)、Ag+、Mg2+、Li+对该酶活性影响较小;草酸、甲醇、正丁醇、K+、Ca2+、Ba2+、Zn2+、Cd2+、Pb2+、Mn2+、Co2+对该酶活性有不同程度的抑制作用;Cu2+激活作用不明显;尿素、乙醇、异丙醇对该酶活性具有激活作用。7、化学修饰剂对杂优-2平菇漆酶功能基团进行修饰,结果表明:二硫键和色氨酸残基是构成杂优-2平菇漆酶活性中心的必需基团;丝氨酸残基、赖氨酸残基和精氨酸残基与杂优-2平菇漆酶活性中心的功能基团构成无直接关系,推测其不是漆酶活性中心的必需基团。
[Abstract]:Laccase (laccase) is a kind of copper polyphenol oxidase, which is one of the lignin degrading enzymes. In the cultivation of edible fungi, laccase can promote the kink of mycelium. It can form a large number of primordial primordium, and then improve the production of edible fungi, and it has important role in the treatment of industrial dye wastewater. The coding genes of the bacterial lignin degrading enzyme can be counted and annotated at the genome level. However, there are still some problems to be solved, such as the inaccuracy of the results of functional gene annotation, the unclear mechanism of the transcriptional regulation of laccase gene. In this paper, 11 sequences of laccase and the sequence of inferred amino acid in the laccase are carried out in this paper. Bioinformatics analysis, in order to study the regulation mechanism of laccase gene, functional genome, and provide theoretical guidance for the experiment in this paper. At the same time, this experiment selected the unknown -2 Pleurotus ostreatus as experimental material. The laccase was separated and purified from the experiment and its physicochemical properties were studied in order to fully understand the physical and chemical properties of the laccase laccase and the laccase of the -2. The further study of laccase, the production of edible fungi and industrial application. The laccase gene of Pleurotus ostreatus may be regulated by N source and from external pressure stress, but it may not be affected by the glucose concentration in the medium. Except for LACC12, the other laccase genes may be activated by the pressure of high concentration of copper ions. The laccase gene of Pleurotus ostreatus was transcribed, and the stability of the laccase laccase was better under the neutral and alkaline conditions, but the thermal stability of the enzyme was low. The two sulfur bond played an important role in maintaining the spatial structure stability of the lacca lacca laccase of the hybrid -2. The results were as follows: 1, the analysis of the promoter region showed that LACC1-LACC12 (except LACC5) was different. Two Cis acting elements, including heat shock response element (HSE), pressure response element (STRE), metal response element (MRE), heterogeneous reaction element (XRE), antioxidant response element (ARE), CreA factor binding site (CreA-bingding site), nitrogen factor binding site (NIT), and CAAT frame (CAAT Box), and other core transcriptional starting sites. Restriction site analysis on the upstream 2000 BP and downstream 2000bp region of the laccase gene coding region found that the number of restriction sites of LACC1-LACC12 (except LACC5) was.2 in the 21~56 range, the phylogenetic analysis of laccase gene and the similarity analysis of LACC1-LACC12 (LACC5) showed that LACC1 and Pleurotus sajor-caju L The similarity of ACC2 was 99.59%; LACC7 similarity between LACC3 and Pleurotus sajor-caju (Pleurotus pulmonarius) was 87.90%, 87.04%, respectively, and LACC1 similarity between LACC7 and Pleurotus Abalone (Pleurotus eryngii) was 95.96%, 97.93% and 11 laccase genes were three. .3, analysis of amino acid sequence similarity found that 11 deduced amino acid sequences were 4 branches. In the evolutionary relationship, LACC6 and LACC8, LACC9 and LACC10 were one branch. The relationship between amino acid sequence alignment and BLAST and RPS-BLAST showed that 11 deduced amino acid sequences with laccase characteristic sequence L1-L4 and 3 highly conserved copper oxygen. The domain of the enzyme (Cu-oxidase), and the results of SiganlP4.1 and Protcomp9.0 showed that 11 deduced that all the amino acid sequences contained signal peptides and all were exocrine protein.4. Protparam showed that the physicochemical properties of amino acids in 11 laccase showed that AI was almost no difference in 83.64~92.81; ExtCof1 and ExtCof2, except LACC1 and LACC10, and the GRAV of the rest of the amino acids. Y is negative; II is in the range of 32.81~42.04; MW is between 55.68~58.87; pI is in the 4.53~7.78 range; except LACC8, the TNNCR of other amino acids is higher than TNPCR.5. It is concluded that the physicochemical properties and composition analysis results show that the physicochemical properties are divided into three clusters, Cluster I and Cluster III. There was no significant difference between Cluster I and Cluster II (P0.05); there was no significant difference between the physical and chemical properties of amino acids in each cluster between three clusters (P0.05). The amino acid composition was also divided into three clusters, Cluster I and Cluster II and Cluster III had significant differences (P0.05), Cluster II and Cluster III had no significant difference (P0.05); The difference in the amino acid sequence composition of laccase in Er I, Cluster II and Cluster III had no significant difference between the three clusters (P0.05).6. Through the liquid culture of the hypha of the hetero -2 mushroom, the fermentation liquid was fractionated by ammonium sulfate, DEAE (diethylaminoethyl) -Sepharose fast flow chromatography and Superdex-200 chromatography and other methods were obtained. The results showed that the laccase activity of laccase -2 was the highest at sixth days. The specific activity of laccase was 115 U/mg, the purification multiple was 111.65, the recovery rate was about 244 kD, the molecular mass of subunit was about 85.6 kD. and the optimum reaction pH value and the optimum reaction temperature were 5 and 55, respectively, in pH 6.0~8.0 and in pH 6.0~8.0. In the range of 40~55 C, the stability is good; under the optimum conditions, the Km value of 2,2 '- two (3- ethyl benzothiazole -6- sulfonic acid) (2,2' -azino-bis (3-ethylbenzothiazoline-6-sulfonate), ABTS) is 2.1mmol/L, the maximum reaction rate (Vmax) is 0.117 micron mol/, and the ascorbic acid has a complete inhibitory effect on the activity of the enzyme, B two Amine four acetic acid (ethylenediaminetetraacetic acid, EDTA), Ag+, Mg2+, Li+ have little effect on the activity of the enzyme, oxalic acid, methanol, n-butanol, K+, Ca2+, Ba2+, Zn2+, Cd2+, Pb2+, etc. have different degrees of inhibition on the activity of the enzyme; the activation effect is not obvious; urea, ethanol and isopropanol have activation effect on the activity of the enzyme, chemical modifier The functional groups of laccase laccase in -2 Pleurotus ostreatus were modified. The results showed that the two sulfur bond and tryptophan residues were essential groups to make the active center of the laccase laccase, and the functional groups of the serine residue, lysine residue and arginine residue and the active center of the laccase laccase were not directly related to the active center of the laccase laccase, and that it was not the active center of laccase in the laccase laccase activity center. The essential group.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q814;Q811.4

【参考文献】