【作者】 柏映国；

【导师】 张志芳；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2010， 博士

【摘要】 嗜酸菌是一类广泛分布在酸性环境中极其重要的微生物,然而目前对嗜酸菌的了解还十分有限。脂环酸芽孢杆菌(Alicyclobacillus)作为典型的一个嗜酸耐热菌属,近些年来受到了极大的关注。本文从云南保山高温温泉出水处的土样中分离得到一株嗜酸嗜热的菌株,经16S rDNA比较鉴定,初步确定为脂环酸芽孢杆菌,命名为Alicyclobacillus sp. A4。生长条件实验表明:该株菌的最适生长温度在55–60°C之间,最适生长pH为3.0–3.5。用各种诱导培养基培养后,测到了多种糖基水解酶活性,表明该菌株为产水解酶系丰富的菌株。在双酶法制糖工艺中,目前广泛应用的是高温淀粉酶和中温糖化酶。然而高温淀粉酶的最适作用温度和pH与中温糖化酶均不相同,需要反复调整pH值和温度。同时,高温淀粉酶不具备降解生淀粉的能力,需要对生淀粉进行糊化,因此造成了生产的较高能耗和环境污染问题。利用可溶性淀粉为碳源,从Alicyclobacillus sp. A4中分离纯化了一个酸性中温α-1,4-淀粉酶(AmyA4)。AmyA4的最适pH值为4.2;最适温度为75°C;在55–75°C范围内都具有很高的活性,这与工业上应用的糖化酶性质相似;同时AmyA4还具有很强的生淀粉降解能力。在模拟双酶法制糖工艺中,以生淀粉为底物,AmyA4在配合商业化糖化酶的作用下,可以达到96.7%的水解率,表明具有很大的应用潜力。利用大麦葡聚糖为碳源,分离纯化了一个极端酸性β-1,4-葡聚糖酶(CelA4)。CelA4的最适pH为2.6,在酸性pH范围内保持稳定,对大麦β-葡聚糖,纤维素,燕麦木聚糖和甘露聚糖均具有催化活性,同时还能对胃蛋白酶有很高的抗性。在人工模拟胃液中,CelA4可以明显降低大麦豆粕饲料的粘度,具有猪饲料添加剂的应用潜力。通过基质辅助激光解吸离子飞行质谱(MALDI-TOF-MS)和全基因组序列分析,确定了CelA4的基因序列。CelA4编码715个氨基酸,与来源于Alicyclobacillus acidocaldarius第51家族的β-1,4-葡聚糖酶CelB有最高的一致性(44%)。CelA4具有三个明显的结构区,即信号肽区(0–39 AAs)、催化区(40–498 AAs)、和苏氨酸高含量区(499–715 AAs)。对全长的成熟蛋白CelA4F(40–715 AAs)和截短的葡聚糖酶CelA4T(40–498 AAs)分别进行了毕赤酵母的外源表达和性质测定。结果表明,重组的CelA4F和CelA4T具有和原酶CelA4相似的性质。在3.7L发酵罐上,截短的葡聚糖酶CelA4T获得了更高的表达量。SDS-PAGE和脱糖基化后分析表明,重组CelA4F和原酶CelA4一样都发生了截短的现象,苏氨酸高含量区对其表达量有明显作用。对来源于Alicyclobacillus sp.A4的第九家族β-1,3(4)-葡聚糖酶基因(agl9A)进行了毕赤酵母的外源表达和性质测定。Agl9A与来源于A. acidocaldarius第九家族的葡聚糖酶具有最高的氨基酸一致性(48%)。纯化的重组Agl9A最适pH为5.8,最适温度为75°C,具有钙离子依赖性,对各种中性蛋白酶包括添加到啤酒酿造的蛋白酶Neutrase 0.8L有抗性。在模拟麦芽汁糖化条件下,无论是单独添加Agl9A,还是和木聚糖酶Xyn(商业酶)同时添加均能有效降低麦芽汁的过滤速率和粘度。上述性质表明Agl9A是一个优秀的葡聚糖酶,可应用于啤酒酿造中的葡聚糖降解。对来源于Alicyclobacillus sp.A4的第十家族β-1,4-木聚糖酶(XynA4)进行了大肠杆菌的外源表达和性质测定。XynA4与来源于Geobacillus stearohermophilus第10家族的木聚糖酶有最高的氨基酸一致性(53%)。纯化的重组XynA4在pH7.0和55°C具有最佳活性,在pH值3.8–9.4范围内,能保持40%以上的最大酶活。该酶还在非常宽的pH范围(pH值2.6–12.0,>80%)内保持稳定。由于XynA4具有良好的热稳定性和pH值稳定性,并且在很宽的pH范围内具有活力,因此它具有在造纸行业中应用的潜力。鉴于脂环酸芽孢杆菌给饮料行业带来的巨大危害和对极端环境的特殊生长要求,并且其16S rDNA序列与已知全基因序列的A. acidocaldarius DSM 446一致性只有95%。因此对Alicyclobacillus sp. A4的全基因组进行了测序,并对测序结果进行了初步分析。结果表明:该菌与A. acidocaldarius DSM 446相似,有共线性。染色体基因组全长2,815,170 bp,编码2,884个基因,GC含量为46.5%。在染色体基因组编码的2,884个基因中,有2,359个基因(81.8%与A. acidocaldarius DSM 446的序列同源,平均一致性为65.4%。质粒基因组全长80,481 bp,编码88个基因,GC含量为52.1%。在质粒基因组编码的88个基因中,有58个基因(65.9%)与A. acidocaldarius DSM 446质粒pAACI01的序列同源,平均一致性为83.3%。对Alicyclobacillus sp. A4的分泌组蛋白进行了分析,结果表明有10.9%的序列被鉴定为具有明显信号肽序列。另外Alicyclobacillus sp. A4的全基因组共含有31条糖基水解酶序列,其中有6条蛋白序列具有明显的信号肽序列。更多还原

【Abstract】 Acidophilus, widely distributed in extremely acidic environments, are important microorganisms; the knowledge of this group of microorganisms is very limited however. Alicyclobacillus sp., as the typical thermalacidophilus, has attracted much attention in the recent years. Herein a thermalacidophilus, denoted as A4, was isolated from the soil close to the outflow of a hot spring in Baoshan, Yunnan, China, and was identified as Alicyclobacillus sp. by comparison of the 16s rDNA sequence with known ones. The strain showed optimal growth at 55–60°C and pH 3.0–3.5. Activities of several glycosyl hydrolyses were detected when induced by various carbon sources, suggesting that stain A4 is a producer of multiple glycosyl hydrolyses.During the double-enzymatic sugar-making process, thermophilicα-amylase and mesophilic saccharifying enzyme are widely applied. Because both enzymes have different optimum temperature and pH, the pH values and temperatures must be adjusted repeatedly. Moreover, thermophilicα-amylase has no ability to degrade raw starch, which must be gelatinized before treatment, thus causing problems such as high energy consumption and environmental contamination. Using soluble starch as carbon source, an acidα-amylase (AmyA4) was isolated and purified. AmyA4 had the optimum pH of 4.2 and optimal temperature at 75°C, showed high activity at 40?65°C, and possessed strong capacity to degrade raw starch. Under simulated double-enzymatic sugar-making conditions, AmyA4 combined with commercialized saccharifying enzyme could catalyze the hydrolysis of 96.7% substrate, and thus has great potential for application.Using barleyβ-glucan as carbon source, an extremely acidβ-1,4-glucanase (CelA4) was isolated and purified from the culture supernatant of Alicyclobacillus sp. A4. CelA4 showed optimal activity at pH 2.6, was stable in acidic conditions, showed catalytic activity towards various substrates including barleyβ-glucan, cellulose, oat spelt xylan and mannan, and had high resistance to pepsin. Under simulated gastric conditions, CelA4 can effectively reduce the viscosity of soybean-barley meal and has potential to be a candidate of pig feed additives. The gene sequence of CelA4 was determined by the analysis of MALDI-TOF-MS and whole genome sequence. CelA4 coded 715 AAs, and its deduced amino acid sequence shared the highest identity (44%) with an endo-β-1,4-glucansae from Alicyclobacillus acidocaldarius that belongs to family 51 of the glycoside hydrolases. CelA4 has three domains, namely the signal peptide (0–39 AAs), catalytic doamin (40–498 AAs), and threonine-rich region (499-715 AAs). Furthermore, the genes coding for full-length CelA4F (40–715 AAs) and truncated CelA4T (40–498 AAs) were also expressed in Pichia pastries. Biochemical characteristics of CelA4T and CelA4F were similar with native CelA4, but CelA4T achieved higher throughout than CelA4F did in 3.7 L fermentor. Based on the analysis of deglycosylation and SDS-PAGE, CelA4F was determined to be truncated by itself as native CelA4 did, and the threonine-rich region might have a significant effect on enzyme expression. The gene encoding a GH 9 glucanase (Agl9A) from Alicyclobacillus sp. A4 was cloned and expressed in P. pastries. The deduced amino acid sequence of Agl9A shared the highest identity (48%) with an endo-β-1,4-glucansae from A. acidocaldarius that belongs to family 9 of the glycoside hydrolases. The purified recombinant Agl9A had optimal pH at 5.8 and temperature optimumat 65°C, was Ca2+-dependent, and showed strong resistance to various neutral proteases including Neutrase 0.8L (Novozymes), a protease widely added to the mash. Under simulated mashing conditions, addition of Agl9A alone or in combination with a commercial xylanase can both effectively reduce the filtration rate and viscosity. These characteristics indicate that Agl9A is a good candidate to improve glucan degradation in the malting and brewing industry.The gene coding for a GH 10 xylanase (XynA4) from Alicyclobacillus sp. A4 was cloned and expressed in Escherichia coli. The deduced amino acid sequence of XynA4 shared the highest identity (53%) with an endo-β-1,4-xylanase from Geobacillus stearohermophilus that belongs to family 10 of the glycoside hydrolases. Purified recombinant XynA4 exhibited maximum activity at 55°C and pH 7.0, had broad pH adaptability (>40% activity at pH 3.8?9.4) and stability (retaining >80% activity at pH 2.6?12.0). The superior properties, such as good thermostability and pH stability and adapatability over a broad pH range, make XynA4 promising for application in the paper industry.Considering the harm to beverage caused by Alicyclobacillus sp., its special demand for extreme environment, and low 16S rDNA sequence identity (only 95%) with A. acidocaldarius DSM 446 (whole genome sequenced), the whole genome sequencing of Alicyclobacillus sp. A4 and elementary analysis was carried out. Results showed that the genome of Alicyclobacillus sp. A4 consisted of a chromosome genome and a plasmid genome, and shared near genomic synteny with A. acidocaldarius DSM 446. The chromosome genome was 2,815,170 bp in length, coded 2,884 genes, and the GC content was 46.5%. Among the 2,884 coding genes, 2,359 (81.8%) genes were identified to be homologous with A. acidocaldarius DSM 446, and the average identity was 65.4%. The plasmid genome length was 80,481 bp, coding 88 genes, with 52.1% GC content. Fifty-eight genes (65.9%) were identified to be the homologues of pAACI01 from A. acidocaldarius DSM 446 with average identity of 83.3%. The secretion of histone proteins was also analyzed, and 10.9% of the sequences were identified with signal peptides. In addition, the genome of Alicyclobacillus sp. A4 contained 31 glycosyl hydrolyse-encoding sequences, and 6 of them had signal peptides.更多还原