【作者】 王建设；

【导师】 尹俊； 姚斌；

【作者基本信息】 内蒙古农业大学 ， 遗传学， 2010， 硕士

【摘要】 木聚糖酶能够随机切割木聚糖主链骨架的β-1,4-糖苷键,产生木糖和低聚木糖或带有侧链的寡聚木糖,是木聚糖降解酶系中最关键的酶。由于木聚糖酶在造纸、食品、纺织、饲料等行业上都具有广泛的应用,科学家对不同来源的木聚糖酶进行了深入的研究。特别是来源于嗜热微生物的木聚糖酶具有许多独特的性质。所以,本研究的目的是筛选一些嗜热微生物,从这些菌株克隆木聚糖酶基因,并进行重组酶的高效表达。本研究从云南省保山市的一眼温泉的流淌物中分离到3株嗜热菌A4,A15和E2。通过16S rDNA序列比对鉴定,A4和A15菌株属于脂环酸芽孢杆菌Alicyclobacillus属,并命名为Alicyclobacillus sp. A4和Alicyclobacillus sp. A15;E2菌株属于厌氧芽孢杆菌Anoxybacillus属,并命名为Anoxybacillus sp. E2。研究发现,A4和A15菌株的最适生长温度为60℃,pH值为3.0;E2菌株的最适生长温度为60℃,pH范围在7.0-10.0,而且在pH 12.0也能生长。以筛选到的3株菌为材料,分别克隆到3个木聚糖酶基因。将三个基因分别构建了原核表达载体,在大肠杆菌中进行表达并研究了这些木聚糖酶的性质。来源于A4菌株的木聚糖酶XynA4-2最适温度为55℃、pH值为6.2。其不仅在50℃具有较好的稳定性,而且和商业葡聚糖酶共同作用下,能有效地降低麦芽浆的黏度和过滤速率。因此,在啤酒酿造中具有潜在的应用价值。来源于A15菌株的木聚糖酶XynA15最适温度为50℃,最适pH值为7.0,不仅在较广的温度范围(35-60℃)内具有较高的酶活,而且在50℃具有较好的热稳定性。XynA15在造纸、生物能源、饲料等领域具有潜在的应用价值。来源于E2菌株的木聚糖酶XynE2的最适温度为65℃,最适pH值为7.8,并且在60℃具有很好的热稳定性,处理1 h酶活基本没有变化。XynE2不仅在pH 6.6- 8.6具有90%以上的相对酶活,而且还有较广的pH稳定性(37℃, pH 4.6-12.0处理1 h保存80%以上的酶活)。这些性质使XynE2在造纸及纸浆上具有应用的潜能。本研究从高温环境中筛选到3株嗜热菌,分别克隆了3个木聚糖酶基因。对这些木聚糖酶基因进行异源表达,并进行酶学性质研究。研究表明,这些木聚糖酶具有潜在的应用价值。更多还原

【Abstract】 Xylanase plays an important role in the xylanolytic enzyme system by randomly cleaving theβ-1,4-glycoside linkages in xylan backbone and generating xylose and oligoxylose units with various branched substitutions. Many studies have been conducted to explore xylanases from various sources due to their widespread applications in the paper, food, textile and animal feed industries. Xylanases from the thermophilic microorganisms have favorable properties, thus the researches on these xylanases have been attracting much attention all over the world. The objective of this study is to isolate some thermophilic microorganisms, obtain xylan-degrading enzymes from them, and achieve high-yield expression of the recombinant enzymes.In this study, three thermophilic strains were isolated from the outflow of a hot spring in Baoshan city, Yunnan province, China. These bacteria were named as A4, A15 and E2, respectively. Based on sequence comparison of the 16S ribosomal deoxyribonucleic acid (rDNA), A4 and A15 were classified into the genus Alicyclobacillus, and designated as Alicyclobacillus sp. A4 and Alicyclobacillus sp. A15, respectively. Strain E2 belonged to the genus Anoxybacillus and designated as Anoxybacillus sp. E2. Strain A4 and A15 showed the optimal growth at 60°C and pH 3.0, and the optimal growth of strain E2 occurred at 60°C and pH 7.0–10.0 (still survival at pH 12.0).Three xylanase genes were cloned from the strains described above and expressed in Escherichia coli BL (DE3), respectively. XynA4-2 from Alicyclobacillus sp. A4 exhibited maximum activity at pH 6.2 and 55°C. The enzyme was thermostable at 50°C. When combined with the commercial glucanase from Sunson, XynA4-2 could increase the filtration rate and reduce the viscosity of mash. These favorable properties make XynA4-2 a good candidate in the brewing industry.XynA15 from Alicyclobacillus sp. A15 showed the maximum activity at 50°C and pH 7.0, was highly active over a broad temperature range (35–60°C) and highly thermostable at 50°C. Thus XynA15 had potential for applications in the paper, biofuel, and animal feed industries. XynE2 from Anoxybacillus sp. E2 showed maximal activity at pH 7.8 and 65°C, and was thermostable at 60°C. The enzyme was highly active and stable over a broad pH range, showing more than 90% of maximal activity at pH 6.6–8.6 and retaining more than 80% of activity at pH 4.6–12.0, 37°C for 1 h, respectively. These favorable properties make XynE2 a good candidate in the pulp and paper industries.In summary, three thermophilic bacterial strains were isolated from a high-temperature environment, and xylanase genes were cloned from the genomic DNA of each strain. These genes were heterologously expressed and characterized. All recombinant enzymes showed favorable properties for potential applications.更多还原