【作者】 马延和；

【导师】 陶文沂；

【作者基本信息】 江南大学 ， 发酵工程， 2005， 博士

【摘要】 β-甘露聚糖酶是第二大半纤维素酶类,在食品、医药、纺织、洗涤剂、造纸、饲料、石油开采等方面具有广泛的应用前景,碱性β-甘露聚糖酶具有特殊的应用优势,从碱湖嗜碱微生物资源中发掘新的碱性β-甘露聚糖酶具有很大的发展潜力。本文从分析我国碱湖微生物资源入手,进行了新型的碱性甘露聚糖酶产生菌的筛选、发酵条件优化、酶学表征、基因克隆、嗜碱机制以及工业应用评价等方面的研究,为我国工业用酶的开发提供新的思路与途径。论文取得的主要结果如下:(1)应用分子生态学的方法评估了我国碱湖中细菌的多样性,发现了碱湖环境中存在革兰氏阴性菌中变形菌纲α和β两个亚群及革兰氏阳性菌4个新的分类单位。在此基础上对来自我国内蒙、西藏等碱湖样品进行了嗜碱菌的分离培养与系统发育学分析,获得了嗜碱细菌106株,其最适生长pH范围集中在9-10之间,完成了其中32株嗜碱菌的系统发育学分析,有21株菌的16S rDNA序列同源性低于97%,可能是新的分类单位。分离获得的菌株N10是变形菌纲γ-3亚群中的成员,做为一个新属的模式菌,定名为淀粉水解嗜碱单胞菌(Alkalimonas amylolytica)。首次证明了Halomonas和BacillusrRNA第7类群是碱湖中主要的多糖水解酶产生者。从上述嗜碱菌中筛选获得了22株β-甘露聚糖酶产生菌,其中嗜碱菌N16-5能够高水平产生碱性甘露聚糖酶,经鉴定表明该菌属于Bacillus属的一新种,定名为解甘露聚糖芽孢杆菌Bacillus mannanolyticus sp.nov.。(2)研究了嗜碱菌N16-5产碱性甘露聚糖酶的营养及环境条件的优化及放大工艺,在优化条件下该菌株在250L和1000L罐中产酶水平可达470U/mL;该菌株发酵液粗酶的最适反应pH为9.5-10.0,最适反应温度为70℃,水解魔芋粉和槐豆胶的产物以寡糖为主。(3)对菌株Nl6-5的β-甘露聚糖酶进行了分离纯化与表征,获得了三种不同分子量的β-甘露聚糖酶M1、M2和M3,其分子量分别为51kD、38kD和34.7kD,最适作用pH分别为9.0、10.0和10.0。其中,M1和M3在pH10.0条件下最为稳定,而M2在pH8.0-10.0范围内稳定。上述三种酶的最适作用温度皆为70℃,对大部分金属离子不敏感,对魔芋葡萄甘露聚糖底物的Km值分别为2.9、1.7和12.5mg/mL。。(4)克隆了菌株N16-5的碱性甘露聚糖酶A(M1)基因,基因片段全长为1479 bp,编码493个氨基酸残基,推断分子量为54215 Da,属于糖苷水解酶家族5的A8亚族,更多还原

【Abstract】 β-mannanase has extensive applications in the food, pharmaceutical, textile,detergent, paper-making, feedstock and petroleum industry. Alkaline β-mannanases provideobvious advantages for the applications in the processes that demand extreme conditions,such as in laundry detergents, paper pulp bleaching and hydraulic fracturing of oil well. Itwill be with great potential to discovery the alkaline β-mannanase from alkaliphilicmicroorganisms in soda lakes. This study describes the alkaliphilic resources in soda lakes ofChina, isolation and identification of alkaliphiles producing mannanase, optimization offermentation conditions, purification and properties of the mannanase, cloning and expressionof the mannanase gene, alkaline adaptation of the mannanase and evaluation ofbiotechnological potential of the mannanase.(1)The bacterial diversity of the soda lakes in China was investigated usingculture-dependent and culture-independent approaches. Phylogenetic analysis of 16S rRNAgene sequences cloned showed the presence of members of the α and β subdivision ofProteobacteria, which were not found previously among cultivated soda lake isolates, and thepresence of novel taxa, which have not been recognized before. Some 106 alkaliphilic isolateswere got under aerobic conditions and grew optimally between pH 9 and 10. Thephylogenetic analysis of 16S rDNA sequences from thirty-two isolates revealed that over 60%of soda lake isolates represent potentially novel species or genera (<97% sequence similarity).Of them, strain N10 was differentiated from currently recognized genera and proposed as thetype species of the new genus Alkalimonas within the gamma subdivision of theProteobacteria, named Alkalimonas amylolytica sp. nov. The members of Halomonas andBacillus rRNA group 7 were identified for the first time as the main polysaccharide-hydrolaseproducers in soda lakes. Twenty-two alkaliphilic isolates, exhibiting extracellular mannanaseactivity at pH 10, were screened from all of soda lake isolates. The results of polyphasictaxonomy revealed that β-mannanase producing strain N16-5 represented a new specieswithin the genus Bacillus, named Bacillus mannanolyticus sp. nov.(2)The effects of nutritional and environmental factors on the production of alkalineβ-mannanase by N16-5 were investigated and optimized, the enzyme activity can reach 470U/mL in 250L and 1000L fermentor. The pH and temperature optima of the crude mannanaseof strain N16-5 were 9.5 and 70 oC, respectively. The hydrolysates of Konjac powder andlocust bean gum by this enzyme were a series of oligosaccharides.(3)Three extracellular β-mannanase (M1, M2, M3) were purified to homogeneity fromthe culture broth of strain N16-5. Their molecular weights were estimated to be 51, 38 and 35kD by SDS-PAGE, respectively. These enzymes exhibited maximal activity at pH 9.0 and70oC (M1) and pH 10.0 and 70oC (M2 and M3), and the enzymes were stable at the range ofpH 8.0 to 10.0. The enzymes were resistant to some metals and surfactants. The Km values ofthe three β-mannanases for konjac β-glucomannan were 2.9, 1.7 and 12.5 mg ml-1,respectively.(4)The gene of alkaline β-mannanase A(M1) was cloned from the genome DNA ofstrain N16-5, the full length of the gene is 1479bp encoding 493 amino acids residue with adeduced molecular weight 54215 Da. M1 belongs to glycosyl hydrolase Family 5, SubfamilyA8. M1 gene is the firstly reported alkaline β-mannanase gene which belongs to Family 5.The amino acid sequence of strain N16-5 ManA deduced from the manA ORF showed highhomology to family 5 β-mannanases: 59% to ManG of Bacillus circulans, 42% to ManA ofThermobifida fusca, 36% to ManA of Vibrio sp. MA-138 and 35% to ManA of Streptomyceslividans 66. No significant similarity was found to the alkaline β-mannanases from Bacillussp AM001(19%), only one extensively characterized among alkaline β-mannanases reportedto date. The manA has been expressed extracellularly in Pichia pastoris. The recombinantstrain secreted 100.8 U/ml of active ManA after 96 h of growth in a complex medium, and theratio of extracellular enzyme is 73%.(5)The analysis of the amino acid sequence of M1 showed that M1 has high content ofA and G. Its C-terminal region (less than 160aa) showed no significant relevant with itsalkaline-adaptation characteristics by deletion mutation analysis. A pH-acid-shifting mutantwas obtained by Error-prone PCR technique. The optimal pH of the mutant is shift down from9.5 to 8.5, and it shows no activity at pH 10.0, while the wild type M1 retains 75% activity atpH 10.0. Compared the nucleotide sequence of the mutant with that of the wild type, threesites of the nucleotide acid sequence changed, which lead to two amino acid residues changed:133rd amino acid residue, Ala, was substituted by Val, 327th amino acid residue, Thr, wassubstituted by Ala. Only Ala-133→Val mutation was responsible for the shift of optimal pH,indicating that the Ala-133 residue was essential for catalytic activity of the β-mannanase inalkaline conditions.(6)The conditions for the products of enzymatic hydrolysis from konjac polysaccharidewere optimized and the hydrolysate were analyzed. The enzyme efficiently hydrolyzed konjacpolysaccharide producing a series of manno-oligosaccharides. The contents of mannobiose,mannotriose, and mannotetraose in the hydrolysate were 14%, 25% and 21.7%, respectively.In pilot-scale experiment of 10 M3 tank, the rate of substrate hydrolysis can reach more than90%, oligomer recovery rate more than 80% and the 1 to 6mer range is from 60% to 80%.The alkaline β-mannanase from the strain N16-5 was effective to degrade thepolysaccharide in hydraulic fracturing fluids within a certain pH and temperature range. Theresidual viscosity and percent residue-after-break of the fracturing fluids were reduced to lessthan 10 mPa.s and about 6-7 %. It suggested the β-mannanase, as a alkaline and thermostableenzyme breaker, can be advantageously employed during enhancing oil recovery operations.更多还原