【作者】 杨越；

【导师】 余泽芬；

【作者基本信息】 云南大学 ， 生物工程， 2012， 硕士

【摘要】 嗜热微生物和其他生活在极端环境的微生物一起增加了生命世界的丰富性,这些微生物具有极其重要的科学价值和应用前景。从嗜热菌分离到的各种嗜热酶具有嗜中温酶不可比拟的许多优点。云南省热泉资源非常丰富,其中蕴涵着丰富的嗜热微生物资源。本研究对云南大理、临沧、德宏、保山和红河地区的共25个温泉的水样和邻近温泉的高温土样进行采集,分离和鉴定了其中的细菌和真菌。为了开发嗜热酶,同时也为了研究嗜热酶的热稳定机制奠定基础,选取一株嗜高温的哈茨木霉,以两株常温哈茨木霉作对照,比较了嗜热和嗜中温的蛋白酶、木聚糖酶、纤维素酶、几丁质酶的最佳发酵条件和部分酶学性质。本研究主要研究结果为：1.嗜热细菌的分离和鉴定。采用纯培养方法,从214份水样和土样中分离纯化获得106株嗜热细菌,包含8个种：Bacillus licheniformis, Bacillus teqilensis, Bacillus subtilis, Bacillus altitudinis, Bacillus axarquiensis, Bacillus flexus, Brevibacillus agri, Brevibacillus breviS。2.嗜热真菌的分离和鉴定。采用纯培养的方法从云南热泉及其周围的土壤中分离得到了79株嗜热真菌,结合形态学特征和ITS序列对嗜热真菌进行了初步鉴定,包括8个种：Myceliophthora thermomyce, Rhizopus microosporus, Rhizopus pusillus, Rhizomucor miehei, Rhizomucor pusillus, Scytalidium thermophilum, Trichoderma harzianum, Thermomyces lanuginosus.其中S. thermophilum是这些地区的优势种,检出率是57%。经生长温度测试后,第一次确定Rhizopus microosporus, Rhizopus pusillus, Trichoderma harzianum是嗜热真菌。3.嗜热酶和嗜中温酶的部分酶学性质研究。对哈茨木霉Trichoderma harzianii嗜热菌T1和中温菌YMF1.403, YMF1.408产生的几丁质酶、蛋白酶、木聚糖酶、纤维素酶进行了初步纯化,并对各个酶的热稳定性等性质进行了研究。(1)嗜热纤维素酶的最适发酵条件为：温度50℃,180r/min,发酵9天；嗜中温酶的最适发酵条件是温度28℃,160r/min,发酵7天。嗜热纤维素酶的最适pH和最适温度分别为4.8和50℃,pH5.5后酶活开始下降,70℃度处理30分种后残留酶活在60%以上。与嗜热酶相比,嗜中温酶的最适pH和最适温度分别为5.5和50℃,pH6.0后酶活开始下降,60℃度处理30分种后残留酶活就下降50%；(2)嗜热木聚糖酶的最适发酵条件为：温度45℃,180r/min,发酵7天,NaCl为最佳无机盐；嗜中温木聚糖酶的最适发酵条件是温度28℃,160r/min,发酵7天。三株菌的最适反应温度均为50℃。嗜热酶在45℃-75℃时酶活较为稳定,80℃酶活降至40%,嗜中温酶在45℃到55℃酶活较为稳定,70℃时酶活降至20%。(3)嗜热蛋白酶的最适发酵条件为：温度50℃,180r/min,发酵6天,嗜中温蛋白酶的最适发酵条件是温度28℃,160r/min,发酵7天,PMSF对三种酶都有较强的抑制作用。嗜热蛋白酶在4℃到60℃都有很高的热稳定性,酶活能保持80%以上,而常温酶在4℃到50℃稳定性较好,50℃以上酶活降至40%以下。嗜热蛋白酶为中性蛋白,在pH7.0时酶活最高,常温酶为碱性蛋白,在pH8.0时酶活最高。(4)嗜热几丁质酶的最适发酵温度：温度45℃,180r/min,发酵7天,嗜中温几丁质酶的最适发酵条件为：温度28℃,160r/min,发酵7天。三种几丁质酶的最适反应温度均为50℃。嗜热菌在4℃-60℃热稳定性较好,酶活能保持在80%以上,而常温菌在50℃以上酶活急剧下降,酶活仅能保存20%左右。更多还原

【Abstract】 Thermophilie microbe and other microbe living in extreme environments increase the diversity of life. These extreme microorganisms are important in the study of scientific theory and application. Thermophilic enzymes are superior to mesophilic enzymes in many aspects. There are many hot springs in Yunnan Province, in which thermophilie microorganisms are abundant. In this study, soil and water samples were collected from25hot springs and neighboring soil distributed in Dali, Lincang, Dehong, Baoshan and Honghe region of Yunnan Province. Bacteria and fungi were isolated from these samples, and then were identified. In order to exploit thermophilic enzyme and further study their mechanism of stability, a thermophilic strain and two mesophilic strains of Trichoderma harzianum were selected. The characters of thermophilic and mesophilic proteae, cellulase, xylanase, chitinase were compared respectively. Results as following:1. Isolation and identification of thermophilic bacteria.106strains were acquired by the pure culture, including8species:Bacillus licheniformis, Bacillus teqilensis, Bacillus subtilis, Bacillus altitudinis, Bacillus axarquiensis, Bacillus flexus, Brevibacillus agri, Brevibacillus brevis.2. Isolation and identification of thermophilic fungi.79strains fungi were acquired by the pure culture, which were identified by combing morphological characters and ITS sequence, including8species:Myceliophthora thermomyce, Rhizopus microosporus, Rhizopus pusillus, Rhizomucor miehei, Rhizomucor pusillus, Scytalidium thermophilum, Trichoderma harzianum, Thermomyces lanuginosus. Among them, S. thermophilum is an dominant species, representing57%of the sample. Rhizopus microosporus, Rhizopus pusillus were first recorded as thermophilic fungi after these strains were tested their growth temperater.3. Part characters of Thermophilic and mesophilic enzymes. Thermophilic and mesophilic proteae, cellulase, xylanase, chitinase produced by T. harzianum thermophilic strain T1, mesophilic strains YMF1.403, YMF1.408were compared from including thermostability et al. aspect(1) The optimal fermentation parameter of thermophilic cellulase are50℃,180r/min,9days, while those of normal temperature cellulase are28℃,160r/min,7days. The optimal temperature and pH for the thermophilic cellulase activity was50℃and4.8, and activity began decline when pH was more than5.5. The residual activity was up to60%after the enzyme was incubated at70℃for30min. The optimal temperature and pH for the mesophilic cellulase activity was5.5and50℃, and activity began decline when pH was more than6.0. The residual activity was less than50%after the enzyme was incubated at60℃for30min.(2) The optimal fermentation parameter of thermophilic xylanase are45℃,180r/min,7days, while those of normal temperature xylanase are28℃,160r/min,7days. The optimal temperature for activities of the xylanase from all three strains was50℃. The activity of thermophilic xylanase was stable when temperature was between45℃and75℃, and the residual activity was40%when the temperature is up to80℃. The activity of mesophilic xylanase was stable when temperature was between45℃and55℃, and the residual activity was20%when the temperature is up to70℃.(3) The optimal fermentation parameter of thermophilic protease are50℃,180r/min,6days, while those of normal temperature protease are28℃,160r/min,7days. PMSF shows a strong inhibition to all three kinds of proteases. The activity of thermophilic protease remained above80%when temperature was between4℃and60℃. The activity of normal temperature protease was stable when temperature was between4℃and50℃, but the residual activity was less than40%when the temperature was up to50℃. The optimal pH for thermophilic protease and mesophilic protease was7.0and8.0respectively.(4)The optimal fermentation parameter of thermophilic chitinase are45℃,180r/min,7days, while those of normal temperature chitinase are28℃,160r/min,7days. The optimal temperature for activities of the chitinase from all three strains was 50℃. The activity of thermophilic chitinase remained above80%when temperature was between4℃and60℃. And the residual activity of mesophilic chitinase remained only20%when the temperature was up to50℃.更多还原