堆肥生境微生物区系动态变化和优势丝状真菌胞外酶系功能分析
发布时间：2019-06-13 16:09
【摘要】：堆肥是在微生物作用下高效降解木质纤维素的系统。本文以三种原料堆肥为研究出发对象,利用高通量测序探究了鸡舍垫料堆肥、蘑菇渣堆肥、秸秆堆肥三种生物质降解系统中微生物区系随原料、时间和空间的动态变化,通过分离纯化从玉米秸秆堆肥中分离到一株疏棉状嗜热丝孢菌(Thermomyces lanuginosus),并在不同底物培养下,研究了其胞外酶系功能。此外,在毕赤酵母GS115中克隆表达了该菌重要的内切木聚糖酶XYNA,并研究其酶学性质。这些工作将对微生物菌剂复配以及利用该菌产工业用酶具有一定的指导意义。本论文取得的主要研究成果如下：1、三种堆肥生境微生物区系的动态变化对蘑菇渣堆肥、鸡舍垫料堆肥、秸秆堆肥三种堆肥生境微生物区系动态变化研究结果表明,三种堆肥中的细菌群落存在一致性和差异性。从门层次上来看,三种堆肥系统中优势菌门均是厚壁菌门、放线菌门、拟杆菌门和变形菌门。随堆肥进程,不同门的比重在不同原料的堆肥中发生的变化不同。温度较低的蘑菇渣堆肥中,以拟杆菌门和变形菌门为主。在加入了氮源的鸡舍垫料堆肥中,温度迅速增加,厚壁菌门在中期占据优势,其次是放线菌门,拟杆菌门和变形菌门明显减少。在增加碳源并减少氮源的玉米秸秆堆肥中,放线菌门在堆肥中期占据优势,其次是厚壁菌门,拟杆菌门和变形菌门均明显减少。对玉米秸秆堆肥中的真菌多样性研究结果表明,子囊菌门中的Thermomyces lanuginosus (疏棉状嗜热丝胞菌,以下简称T. lanuginosus)以98%以上的比重占据数量上的优势。2、从玉米秸秆堆肥中分离到一株T. lanuginosusT. lanuginosus是产多种耐热降解酶的丝状真菌,在堆肥进程中对生物质的降解具有十分重要的意义。通过对来自玉米秸秆堆肥中的菌种筛选和分离,获得了一株疏棉状嗜热丝胞菌,命名为sduw-01。3、探究了不同底物培养条件下T. lanuginosus的胞外降解酶系及其功能分别用木糖、木糖醇、阿拉伯糖等三种组成半纤维素的单糖在液体培养条件下发酵,发现诱导产木聚糖酶的能力为,木糖阿拉伯糖木糖醇。分别用玉米秸秆粉、麸皮、玉米秸秆粉和麸皮的混合物等三种底物种固体发酵培养T.lanuginosus,发现混合发酵产木聚糖酶最高。分别在六种底物培养条件下产生的粗酶液中,均检测到蛋白酶,且以固体发酵产蛋白酶较高。通过液相色谱技术和质谱技术联用分析用木糖、木聚糖和三种固体底物发酵产粗酶液的蛋白组,结果表明,胞外酶系中的降解酶种类少,且T. lanuginosus能够根据不同底物中的不同成分作出相应的响应。以木聚糖为碳源发酵产1,4-β-内切木聚糖酶比例最高为大约为50%,其次是α-葡糖苷酶；玉米秸秆粉中比木聚糖发酵蛋白质组中的几丁质酶和p-1,4-木糖甘酶以及有关的丝氨酸蛋白酶含量高。此外,还发现,脂肪酸合成酶在代谢酶中的含量比重最大,为该菌迅速生长提供细胞膜合成需要的脂质。4、在毕赤酵母GS115中成功表达木聚糖酶基因xynA提取了T. lanuginosus 的 RNA并反转录获得cDNA后,引物扩增木聚糖酶基因xynA,利用pPIC-9K质粒载体在毕赤酵母GS115中表达成功木聚糖酶XYNA。经过浓缩纯化后,对其酶学性质研究表明,其最适温度为70℃,最适pH为6.2,在60℃下处理,2小时能保持100%的酶活,FACE电泳结果显示降解桦木木聚糖的主要产物为木二糖和木三糖,还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，
本文编号：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，
本文编号：2498636
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