發(fā)布時(shí)間：2018-04-23 08:23

  本文選題：漆酶 + 生物信息學(xué)��； 參考：《西南大學(xué)》2017年碩士論文

【摘要】：漆酶(laccase)是一種銅多酚氧化酶,是木質(zhì)素降解酶系之一,在食用菌栽培過程中,漆酶對菌絲體扭結(jié)具有促進(jìn)作用,能夠形成較多的子實(shí)體原基,進(jìn)而提高食用菌的產(chǎn)量,且在工業(yè)染料廢水處理等方面有重要作用。依據(jù)平菇基因組數(shù)據(jù),真菌木質(zhì)素降解酶的編碼基因能在基因組水平上得到統(tǒng)計(jì)和注釋。但是,目前也存在一些亟待解決的問題,如功能基因注釋結(jié)果的不準(zhǔn)確性、漆酶基因轉(zhuǎn)錄調(diào)控機(jī)理不清楚等。本文通過對平菇漆酶的11條基因序列及其推斷氨基酸序列進(jìn)行生物信息學(xué)分析,為研究漆酶基因的調(diào)控機(jī)理、功能基因組及為本文實(shí)驗(yàn)提供理論指導(dǎo)。同時(shí),本實(shí)驗(yàn)選用尚未報(bào)道的雜優(yōu)-2平菇為實(shí)驗(yàn)材料,從中分離純化出漆酶并對其理化性質(zhì)進(jìn)行研究旨在為充分了解雜優(yōu)-2平菇漆酶的理化性質(zhì)及對漆酶的進(jìn)一步研究、食用菌生產(chǎn)和工業(yè)應(yīng)用等方面提供參考。平菇漆酶基因可能受到N源和來自外界壓力脅迫調(diào)控,但可能不會受到培養(yǎng)基中葡萄糖濃度的影響,除LACC12外,其余漆酶基因可能會共同響應(yīng)高濃度的銅離子的壓力而激活平菇漆酶基因的轉(zhuǎn)錄;雜優(yōu)-2平菇漆酶在中性和堿性條件下穩(wěn)定性較好,但該酶的熱穩(wěn)定性較低。二硫鍵在維持雜優(yōu)-2平菇漆酶的空間結(jié)構(gòu)穩(wěn)定性有重要作用。研究結(jié)果如下:1、啟動子區(qū)域分析結(jié)果顯示:LACC1-LACC12(LACC5除外)均含有不同的順式作用元件,包括熱激響應(yīng)元件(HSE)、壓力響應(yīng)元件(STRE)、金屬應(yīng)答元件(MRE)、異生物質(zhì)反應(yīng)元件(XRE)、抗氧化響應(yīng)元件(ARE)、CreA因子結(jié)合位點(diǎn)(CreA-bingding site)、氮因子結(jié)合位點(diǎn)(NIT),還包括CAAT框(CAAT Box)、TATA框(TATA Box)等核心轉(zhuǎn)錄起始位點(diǎn)。對漆酶基因編碼區(qū)上游2000 bp和下游2000bp區(qū)域的限制性酶切位點(diǎn)分析發(fā)現(xiàn):LACC1-LACC12(LACC5除外)的限制性酶切位點(diǎn)數(shù)在21~56范圍不等。2、漆酶基因系統(tǒng)進(jìn)化分析及LACC1-LACC12(LACC5除外)相似性分析結(jié)果顯示:LACC1與秀珍菇(Pleurotus sajor-caju)的LACC2相似性為99.59%;LACC3與秀珍菇(Pleurotus sajor-caju)的LACC5和鳳尾菇(Pleurotus pulmonarius)的LACC7相似性分別為87.90%、87.04%;LACC7與杏鮑菇(Pleurotus eryngii)LACC1相似性為95.96%;LACC10與秀珍菇(Pleurotus sajor-caju)的LACC4相似性為97.93%,11條漆酶基因聚為三支。3、氨基酸序列相似性分析發(fā)現(xiàn):11條推斷氨基酸序列聚為4支,在進(jìn)化關(guān)系中,LACC6與LACC8、LACC9與LACC10為一支,親緣關(guān)系最近。氨基酸多序列比對和BLAST及RPS-BLAST結(jié)果顯示:11條推斷氨基酸序列中均有漆酶特征序列L1-L4和3個(gè)高度保守的銅氧化酶(Cu-oxidase)結(jié)構(gòu)域;SiganlP4.1及Protcomp9.0結(jié)果顯示:11推斷氨基酸序列中都含信號肽,且都為外分泌蛋白。4、Protparam對11條漆酶推斷氨基酸理化性質(zhì)預(yù)測結(jié)果顯示:AI在83.64~92.81;ExtCof1和ExtCof2幾乎沒有差異;除LACC1和LACC10外,其余氨基酸的GRAVY均為負(fù)值;II在32.81~42.04范圍內(nèi);MW介于55.68~58.87之間;pI在4.53~7.78范圍;除LACC8外,其余氨基酸的TNNCR均高于TNPCR。5、推斷氨基酸理化性質(zhì)和組成分析結(jié)果顯示:理化性質(zhì)分為三簇,ClusterⅠ與ClusterⅢ、ClusterⅡ與ClusterⅢ之間存在顯著性差異(P0.05),ClusterⅠ與ClusterⅡ之間無顯著性差異(P0.05);每個(gè)簇內(nèi)的氨基酸理化性質(zhì)差異性在三個(gè)簇之間無顯著性差異(P0.05)。氨基酸組成也分為三簇,ClusterⅠ與ClusterⅡ及ClusterⅢ均有顯著性差異(P0.05),ClusterⅡ與ClusterⅢ間無顯著性差異(P0.05);ClusterⅠ、ClusterⅡ、ClusterⅢ內(nèi)的漆酶氨基酸序列組成差異性在三個(gè)簇之間無顯著差異(P0.05)。6、通過對雜優(yōu)-2平菇菌絲進(jìn)行液體培養(yǎng),發(fā)酵液經(jīng)硫酸銨分級沉淀、DEAE(diethylaminoethyl)-Sepharose fast flow層析和Superdex-200 prep grade層析等方法純化,獲得了電泳純的雜優(yōu)-2平菇漆酶。結(jié)果顯示,培養(yǎng)第6天時(shí)漆酶活性最高;雜優(yōu)-2平菇漆酶比活力為115 U/mg,純化倍數(shù)為111.65,回收率為6.96%。分子質(zhì)量約為244.0 kD,亞基分子質(zhì)量約為85.6 kD。最適反應(yīng)pH值和最適反應(yīng)溫度分別為5.0和55℃,在pH 6.0~8.0及40~55℃范圍內(nèi)穩(wěn)定性較好;最適條件下,以2,2’-連氮-二(3-乙基苯并噻唑-6-磺酸)(2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate),ABTS)為底物的Km值為2.1mmol/L,最大反應(yīng)速率(Vmax)為0.117μmol/(min·L)。Fe2+、抗壞血酸對該酶活性具有完全抑制作用,乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)、Ag+、Mg2+、Li+對該酶活性影響較小;草酸、甲醇、正丁醇、K+、Ca2+、Ba2+、Zn2+、Cd2+、Pb2+、Mn2+、Co2+對該酶活性有不同程度的抑制作用;Cu2+激活作用不明顯;尿素、乙醇、異丙醇對該酶活性具有激活作用。7、化學(xué)修飾劑對雜優(yōu)-2平菇漆酶功能基團(tuán)進(jìn)行修飾,結(jié)果表明:二硫鍵和色氨酸殘基是構(gòu)成雜優(yōu)-2平菇漆酶活性中心的必需基團(tuán);絲氨酸殘基、賴氨酸殘基和精氨酸殘基與雜優(yōu)-2平菇漆酶活性中心的功能基團(tuán)構(gòu)成無直接關(guān)系,推測其不是漆酶活性中心的必需基團(tuán)。
[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.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q814;Q811.4

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