堆肥生境微生物區(qū)系動(dòng)態(tài)變化和優(yōu)勢(shì)絲狀真菌胞外酶系功能分析
發(fā)布時(shí)間：2019-06-13 16:09
【摘要】：堆肥是在微生物作用下高效降解木質(zhì)纖維素的系統(tǒng)。本文以三種原料堆肥為研究出發(fā)對(duì)象,利用高通量測序探究了雞舍墊料堆肥、蘑菇渣堆肥、秸稈堆肥三種生物質(zhì)降解系統(tǒng)中微生物區(qū)系隨原料、時(shí)間和空間的動(dòng)態(tài)變化,通過分離純化從玉米秸稈堆肥中分離到一株疏棉狀嗜熱絲孢菌(Thermomyces lanuginosus),并在不同底物培養(yǎng)下,研究了其胞外酶系功能。此外,在畢赤酵母GS115中克隆表達(dá)了該菌重要的內(nèi)切木聚糖酶XYNA,并研究其酶學(xué)性質(zhì)。這些工作將對(duì)微生物菌劑復(fù)配以及利用該菌產(chǎn)工業(yè)用酶具有一定的指導(dǎo)意義。本論文取得的主要研究成果如下：1、三種堆肥生境微生物區(qū)系的動(dòng)態(tài)變化對(duì)蘑菇渣堆肥、雞舍墊料堆肥、秸稈堆肥三種堆肥生境微生物區(qū)系動(dòng)態(tài)變化研究結(jié)果表明,三種堆肥中的細(xì)菌群落存在一致性和差異性。從門層次上來看,三種堆肥系統(tǒng)中優(yōu)勢(shì)菌門均是厚壁菌門、放線菌門、擬桿菌門和變形菌門。隨堆肥進(jìn)程,不同門的比重在不同原料的堆肥中發(fā)生的變化不同。溫度較低的蘑菇渣堆肥中,以擬桿菌門和變形菌門為主。在加入了氮源的雞舍墊料堆肥中,溫度迅速增加,厚壁菌門在中期占據(jù)優(yōu)勢(shì),其次是放線菌門,擬桿菌門和變形菌門明顯減少。在增加碳源并減少氮源的玉米秸稈堆肥中,放線菌門在堆肥中期占據(jù)優(yōu)勢(shì),其次是厚壁菌門,擬桿菌門和變形菌門均明顯減少。對(duì)玉米秸稈堆肥中的真菌多樣性研究結(jié)果表明,子囊菌門中的Thermomyces lanuginosus (疏棉狀嗜熱絲胞菌,以下簡稱T. lanuginosus)以98%以上的比重占據(jù)數(shù)量上的優(yōu)勢(shì)。2、從玉米秸稈堆肥中分離到一株T. lanuginosusT. lanuginosus是產(chǎn)多種耐熱降解酶的絲狀真菌,在堆肥進(jìn)程中對(duì)生物質(zhì)的降解具有十分重要的意義。通過對(duì)來自玉米秸稈堆肥中的菌種篩選和分離,獲得了一株疏棉狀嗜熱絲胞菌,命名為sduw-01。3、探究了不同底物培養(yǎng)條件下T. lanuginosus的胞外降解酶系及其功能分別用木糖、木糖醇、阿拉伯糖等三種組成半纖維素的單糖在液體培養(yǎng)條件下發(fā)酵,發(fā)現(xiàn)誘導(dǎo)產(chǎn)木聚糖酶的能力為,木糖阿拉伯糖木糖醇。分別用玉米秸稈粉、麩皮、玉米秸稈粉和麩皮的混合物等三種底物種固體發(fā)酵培養(yǎng)T.lanuginosus,發(fā)現(xiàn)混合發(fā)酵產(chǎn)木聚糖酶最高。分別在六種底物培養(yǎng)條件下產(chǎn)生的粗酶液中,均檢測到蛋白酶,且以固體發(fā)酵產(chǎn)蛋白酶較高。通過液相色譜技術(shù)和質(zhì)譜技術(shù)聯(lián)用分析用木糖、木聚糖和三種固體底物發(fā)酵產(chǎn)粗酶液的蛋白組,結(jié)果表明,胞外酶系中的降解酶種類少,且T. lanuginosus能夠根據(jù)不同底物中的不同成分作出相應(yīng)的響應(yīng)。以木聚糖為碳源發(fā)酵產(chǎn)1,4-β-內(nèi)切木聚糖酶比例最高為大約為50%,其次是α-葡糖苷酶；玉米秸稈粉中比木聚糖發(fā)酵蛋白質(zhì)組中的幾丁質(zhì)酶和p-1,4-木糖甘酶以及有關(guān)的絲氨酸蛋白酶含量高。此外,還發(fā)現(xiàn),脂肪酸合成酶在代謝酶中的含量比重最大,為該菌迅速生長提供細(xì)胞膜合成需要的脂質(zhì)。4、在畢赤酵母GS115中成功表達(dá)木聚糖酶基因xynA提取了T. lanuginosus 的 RNA并反轉(zhuǎn)錄獲得cDNA后,引物擴(kuò)增木聚糖酶基因xynA,利用pPIC-9K質(zhì)粒載體在畢赤酵母GS115中表達(dá)成功木聚糖酶XYNA。經(jīng)過濃縮純化后,對(duì)其酶學(xué)性質(zhì)研究表明,其最適溫度為70℃,最適pH為6.2,在60℃下處理,2小時(shí)能保持100%的酶活,FACE電泳結(jié)果顯示降解樺木木聚糖的主要產(chǎn)物為木二糖和木三糖,還有少量的木糖和木四糖。
[Abstract]:Composting is a highly effective system for degrading lignocellulose under the action of microorganisms. In this paper, three kinds of raw materials are used as the research object, and the dynamic changes of the microbial flora with the raw materials, time and space in the three kinds of biomass degradation systems, such as the compost of the chicken house, the compost of the mushroom residue and the straw compost, are investigated by high-throughput sequencing. A strain of Thermomyces lanuginosus was isolated from the compost of the corn straw and the function of its extracellular enzyme was studied under different substrate culture. In addition, in Pichia pastoris GS115, the important endoxylanase XYNA was cloned and its enzymatic properties were studied. These work will have a certain guiding significance to the microbial agent formulation and the use of the bacteria-producing industrial enzyme. The main research results in this paper are as follows:1. The dynamic changes of the microbial flora of the three composted habitats have shown that the dynamic changes of the microbial flora of the three types of composted habitats, such as the compost of the mushroom residue, the compost of the chicken house and the composting of the straw, are as follows: There was consistency and difference in the bacterial community in the three composts. From the level of the door, the advantages of the three composting systems are the thick-walled fungus door, the actinomycete door, the Bacteroides and the deformable fungus door. Depending on the composting process, the specific gravity of the different doors is different in the compost of different raw materials. In the compost of the mushroom residue with lower temperature, it is mainly composed of the Bacteroides and the strain. In the compost of the chicken house with the nitrogen source, the temperature was rapidly increased, the thick-walled fungus door occupied the advantage in the medium term, and the second was the actinomycete door, the Bacteroides and the deformed fungus door were obviously reduced. In the composting of the maize straw which increased the carbon source and the nitrogen source, the actinomycete was dominant in the medium term of the compost, followed by the thick-walled fungus door, the Bacteroides and the deformed bacteria. The results of the study on the diversity of the fungi in the compost of corn straw show that the Thermomyces lanuginosus (the "T. lanuginosus) in the subcapsule is the dominant factor in the number of more than 98%, and a T. lanuginosus T is isolated from the corn straw compost. Lanuginosus is a filamentous fungus producing a variety of heat-resistant and degrading enzymes, which is of great significance to the degradation of the biomass in the composting process. by screening and separating the strains from the compost of the corn straw, a strain of the cotton-like hot-filament cell strain is obtained, named as sduw-01.3, and the extracellular-degrading enzyme system of the T. lanuginosus under different substrate culture conditions and the function thereof are respectively used for xylose and xylitol, The three kinds of monosaccharides, such as arabinose, were fermented under the condition of liquid culture, and the ability of the production of the xylanase was found to be xylose and arabinose. T. lanuginosus was cultured by solid fermentation of three kinds of bottom species, such as corn straw powder, bran, corn straw powder and bran. The protease was detected in the crude enzyme solution, which was produced under six substrate culture conditions, respectively, and the protease produced with solid fermentation was high. The analysis of the proteome of crude enzyme with xylose, xylan and three solid substrates was carried out by liquid chromatography and mass spectrometry. The results showed that the type of degrading enzyme in the extracellular enzyme system was small, and T. lanuginosus was able to respond to different components in different substrates. The proportion of xylanase produced by using xylan as a carbon source was up to about 50%, followed by a yeast-glucanase; the chitinase and the p-1,4-xylose and the related serine protease content in the corn straw powder were higher than the chitinase and the p-1,4-xylose and the related serine protease in the xylanase of the xylanase. in addition, it has been found that that content of the fatty acid synthetase in the metabolizing enzyme is the largest, and the lipid required for the cell membrane synthesis is provided for the rapid growth of the bacterium.4, the xylanase gene xynA is successfully expressed in the Pichia pastoris GS115 to extract the RNA of the T. lanuginosus and reverse transcription to obtain the cDNA, The xylanase gene xynA was amplified by the primer, and the xylanase XYNA was expressed in the Pichia pastoris GS115 by using the pPIC-9K plasmid vector. After the concentration and purification, the optimum temperature was 70 鈩，
本文編號(hào)：2498636
[Abstract]:Composting is a highly effective system for degrading lignocellulose under the action of microorganisms. In this paper, three kinds of raw materials are used as the research object, and the dynamic changes of the microbial flora with the raw materials, time and space in the three kinds of biomass degradation systems, such as the compost of the chicken house, the compost of the mushroom residue and the straw compost, are investigated by high-throughput sequencing. A strain of Thermomyces lanuginosus was isolated from the compost of the corn straw and the function of its extracellular enzyme was studied under different substrate culture. In addition, in Pichia pastoris GS115, the important endoxylanase XYNA was cloned and its enzymatic properties were studied. These work will have a certain guiding significance to the microbial agent formulation and the use of the bacteria-producing industrial enzyme. The main research results in this paper are as follows:1. The dynamic changes of the microbial flora of the three composted habitats have shown that the dynamic changes of the microbial flora of the three types of composted habitats, such as the compost of the mushroom residue, the compost of the chicken house and the composting of the straw, are as follows: There was consistency and difference in the bacterial community in the three composts. From the level of the door, the advantages of the three composting systems are the thick-walled fungus door, the actinomycete door, the Bacteroides and the deformable fungus door. Depending on the composting process, the specific gravity of the different doors is different in the compost of different raw materials. In the compost of the mushroom residue with lower temperature, it is mainly composed of the Bacteroides and the strain. In the compost of the chicken house with the nitrogen source, the temperature was rapidly increased, the thick-walled fungus door occupied the advantage in the medium term, and the second was the actinomycete door, the Bacteroides and the deformed fungus door were obviously reduced. In the composting of the maize straw which increased the carbon source and the nitrogen source, the actinomycete was dominant in the medium term of the compost, followed by the thick-walled fungus door, the Bacteroides and the deformed bacteria. The results of the study on the diversity of the fungi in the compost of corn straw show that the Thermomyces lanuginosus (the "T. lanuginosus) in the subcapsule is the dominant factor in the number of more than 98%, and a T. lanuginosus T is isolated from the corn straw compost. Lanuginosus is a filamentous fungus producing a variety of heat-resistant and degrading enzymes, which is of great significance to the degradation of the biomass in the composting process. by screening and separating the strains from the compost of the corn straw, a strain of the cotton-like hot-filament cell strain is obtained, named as sduw-01.3, and the extracellular-degrading enzyme system of the T. lanuginosus under different substrate culture conditions and the function thereof are respectively used for xylose and xylitol, The three kinds of monosaccharides, such as arabinose, were fermented under the condition of liquid culture, and the ability of the production of the xylanase was found to be xylose and arabinose. T. lanuginosus was cultured by solid fermentation of three kinds of bottom species, such as corn straw powder, bran, corn straw powder and bran. The protease was detected in the crude enzyme solution, which was produced under six substrate culture conditions, respectively, and the protease produced with solid fermentation was high. The analysis of the proteome of crude enzyme with xylose, xylan and three solid substrates was carried out by liquid chromatography and mass spectrometry. The results showed that the type of degrading enzyme in the extracellular enzyme system was small, and T. lanuginosus was able to respond to different components in different substrates. The proportion of xylanase produced by using xylan as a carbon source was up to about 50%, followed by a yeast-glucanase; the chitinase and the p-1,4-xylose and the related serine protease content in the corn straw powder were higher than the chitinase and the p-1,4-xylose and the related serine protease in the xylanase of the xylanase. in addition, it has been found that that content of the fatty acid synthetase in the metabolizing enzyme is the largest, and the lipid required for the cell membrane synthesis is provided for the rapid growth of the bacterium.4, the xylanase gene xynA is successfully expressed in the Pichia pastoris GS115 to extract the RNA of the T. lanuginosus and reverse transcription to obtain the cDNA, The xylanase gene xynA was amplified by the primer, and the xylanase XYNA was expressed in the Pichia pastoris GS115 by using the pPIC-9K plasmid vector. After the concentration and purification, the optimum temperature was 70 鈩，
本文編號(hào)：2498636
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