【作者】 张婕；

【导师】 李多川；

【作者基本信息】 山东农业大学 ， 环境生物学， 2010， 博士

【摘要】 几丁质(chitin)是由N-乙酰-β-D-氨基葡萄糖通过β-1,4-糖苷键连接起来的直链多聚物,是最丰富的天然高分子化合物之一,广泛分布于动物、植物及微生物体内。几丁质酶可以水解几丁质产生几丁寡糖和N-乙酰氨基葡萄糖,且广泛存在于自然界的生物中。近年来,随着几丁质酶的降解产物在医药、食品、农业、环保、造纸、化工等领域的应用越来越深入,几丁质酶作为制备几丁寡糖的重要工具越来越受到人们的重视。国内外已筛选出多种几丁质酶生产菌,并将其分离提纯。但是,直接由真菌发酵产生几丁质酶在工业化生产中受多方面因素的限制,随着分子生物学技术的发展,人们已不满足于从原始产生生物中提取和利用几丁质酶,而是希望得到编码这种酶的基因并转入宿主进行高效低成本的表达。因此利用重组微生物实现几丁质酶的工业化生产、提高几丁质酶活性和产量,是目前较为理想的方法。嗜热子囊菌原变种(Thermoascus aurantiacus var. aurantiacus)是一种广泛分布的,生长上限温度较高的嗜热真菌,能够在45℃～50℃的高温下很好的繁殖生长。本研究根据真菌几丁质酶的同源保守序列设计兼并引物,通过RT-PCR和Tail-PCR等技术首次从嗜热子囊菌原变种中分离得到了一种几丁质酶基因Chit-TAA,全长DNA为1826bp,在包含一个由1191个核苷酸构成的开放阅读框,编码396个氨基酸,该基因cDNA在GenBank中的登录号为GU338053。序列比对分析表明,该蛋白属于糖苷水解酶18家族几丁质酶,其中两个序列S-GGW和DG-D-DWE非常保守。这两段保守氨基酸序列与糖基水解酶(glycosylhydrolases)18家族催化活性有关。其中不变氨基酸残基Asp和Glu为几丁质酶的催化活性位点。利用基因重组的方法构建酵母分泌型表达载体,并转化到毕赤酵母中。于MD/MM平板上筛选His+Mut+表型的酵母转化子,经过PCR检测及G418抗性筛选得到多拷贝整合子,进行甲醇诱导培养。通过检测各转化子每隔24h的蛋白表达情况来筛选高效表达目的几丁质酶的工程菌株,筛选到表达酶活性最高的菌株GS-Chit-176。在甲醇的诱导下,重组蛋白得到了高效表达,第6天的表达量最高,达到0.433mg/mL,酶活力为7.25U/mL,同时对表达的几丁质酶进行了纯化,SDS-PAGE检测该蛋白的分子量为43.9kDa。该几丁质酶的最适反应温度为60℃,最适反应pH值为8.0,70℃处理30min仍有45%的相对酶活,具有较好的热稳定性。对其降解产物的研究表明,该几丁质酶降解胶体几丁质的主要产物为几丁质二糖。而且该酶对烟草赤星病菌(Alternaria alternata)、串珠镰刀菌(Fusarium moniliforme)的菌丝生长也有一定的抑制作用,对玉蜀黍平脐蠕孢(Bipolaris maydis)的孢子萌发有一定的抑制作用。嗜热子囊菌原变种几丁质酶基因能在毕赤酵母中分泌表达,表达产物具有优良特性,这就预示了表达几丁质酶在几丁质工业和生物学领域的广阔应用前景。因此,期望能对该基因进行改造,或进一步优化表达条件,获得真正有工业应用价值的酵母工程菌株。生物几丁质酶基因一般包含信号肽序列、催化域、富含丝苏氨酸域、几丁质结合域、C端延伸域。底物结合域的存在提高了酶在底物表面的浓度,有助于酶的催化区对底物的水解。植物和细菌几丁质酶的CHBD序列与纤维素酶的结合域(CBD)序列有较高的相似性,而且纤维素和几丁质都是通过β-1,4糖苷键连接起来的直链多聚物,结构及其相似。本研究利用融合PCR技术分别在嗜热子囊菌几丁质酶Tachit的C端和N端引入嗜热毛壳菌(Chaetomium thermophilum)纤维素酶chh1、cbh2的CBD结构域,构建表达载体转化毕赤酵母中,筛选到两株高表达量的酵母工程菌株。表达的融合蛋白Tachit-CBD1和CBD2-Tachit对胶体几丁质的活性分别提高了1.6倍和0.6,对粉状几丁质的活性提高了0.9和0.3倍。而且表达的Tachit-CBD1和CBD2-Tachit同时还具有热稳定性,最适反应温度为都为60℃,50℃条件下相对稳定。最适反应pH值都为8.0,在pH5.0-8.0之间稳定。通过对Tachit催化区非保守氨基酸进行定点突变,构建了两个突变体Tachit-L129F和Tachit-V134I,通过比较Tachit-L129F的最适反应温度提高了10℃,而热稳定基本不变,最适反应pH值为降为5.0,在酸性条件下稳定；Tachit-V134I的性质研究结果表明,突变对该酶最适反应温度的影响变化不大。对其最适反应pH的测定表明,该突变酶表现出较好的嗜碱性,最适反应pH值为11,当pH值为12时,仍保持85%的酶活性。更多还原

【Abstract】 Chitin as one of the most abundant natural polymeric compounds, is a linear chain polymer which is connected by N-Acetyl-β-D-glucosamine throughβ-1,4-glucosidic bond. Chitinase is widely spreading in animals, plants and microbes, and has a function of hydrolyzing chitin into chitin oligosaccharide and N-Acety glucosamine. Recently, as the wide application of degradation products from chitinase in medicine, food, agriculture, environmental protection, paper making, and chemistry, chitinase receives much attention as an important tool for chitin oligosaccharide.Many organisms capable of producing chitinase were screened, isolated, and purified both at home and abroad. However, Chitinases from fungal fermentation have many limitations in commercial process. Following the development of Protocols in Molecular Biology, induction, production and use of chitinase from microorganism is not met to the requirements of human. The process of encoding for the enzyme and expressed in the source at a high effective and low cost way, is undertaking. So, it’s ideal that commercial process of chitinase, improved the activity and enhanced the production can be achieved using a method of recombinant microorganisms.Thermoascus aurantiacus var. aurantiacus is a wide distributed thermophilic fungus and a higher growth upper-limitation temperature, which can grow well up to 40-50 degrees. Degenerate primers based on the conserved sequences of the fungi chitinase and a chitinase gene named Chit-TAA was isolated for the first time from T. aurantiacus var. aurantiacus through RT-PCR and Tail-PCR. The full-length of DNA 1824bp contained an ORF of 1191bp encoding 396 amino acids. Accession number of the nucleotide sequence is GU338053 in the GenBank database. Sequence analysis of the deduced amino acid sequence revealed high homology with the catalytic domains of the Glycoside hydrolase family 18, contained 2 conserved motifs which were related with catalytic activity of chitinase. Among these conserve sequences, the invariant carboxylic amino acid residue Asp and Glu have been proved essential in the acid-base catalytisis of glycoside hydrolase of family 18.Chit-TAA was expressed in Pichia pastoris GS115. Gene recombination was used to construct the expression plasmid and then the plasmid was transformed into P. pastoris GS115. After screening for His+Mut+ transformants on MD and MM plates, PCR analysis of Pichia integrants and G418 screening determined the multicopy integrants to induce by methanol. These integrants were used to analyze expression levels every 24 hours of interest protein and determine the engineering strains with high expression level, such as GS-Chit-176.After six days induction using methanol the transformant strain had the highest efficient production and the expressing level was 0.433mg/mL. The activity of expressed protein was 7.25U/mL. The molecular mass of a single band of the enzyme was estimated to be 43.9 kDa. The optimum temperature of the enzyme was 60℃and its maximal activity was at pH 8.0. The enzyme was thermostable and could still keep 45% activity at 70℃after 30 min. The product was mainly chitobiose from the hydrolyzation of colloidal chitin by chitinases. To some extent, the chitinase revealed antifungal activity against the growthing of Alternaria alternata and Fusarium moniliforme and the sporegermination of Bipolar is maydis.The gene Chit-TAA was expressed in P. pastoris and the expression products had the activity of Chitinase. It implied the expression chtinase could be applied widely for the chitin industry and biological fields. Therefore, we hope to reconstruct the gene or optimize the expression condition to obtain the yeast engineering strains suitable for industrial application.Chitinase comprises signal peptide coding sequence, catalysis domain (CD), serine/threorine domain, Chitinase binding domain (BD), and c-terminal extension region. The existence of BD increases the concentration of enzymes on the surface of substrate, which helps CD hydrolyze substrate. CHBD of plants and bacterium is pretty similar to’CBD of cellulases; and cellulos as well as chitin are linear chain polymer connected throughβ-1,4-glucosidic bond, which show very similar structure between each other. This study introduced CBD of Cellulase gene chhl and cbh2 from Chaetomium thermophilum to C-and N-Tachit from T. aurantiacus var. aurantiacus, separately, via fusion PCR. The gene was expressed in P. pastoris. Two high expression strains were gotten from screening. The fused protein Tachit-CBD1 and CBD2-Tachit increased activity for colloidal chitin by 1.6-and 0.6-fold, separately, and by 0.32-and 0.9-fold, separately, for powdery chitin and they also had the thermostability. The optimum temperatures were both 60℃, and their maximal activity were both at pH 8.0.They kept relative stability at 50℃and the pH in the range of 5.0 to 8.0.Two mutants Tachit-L129F and Tachit-V134I were constructed by site-direct mutagenesis to amino acid in the CD of Tachit. The optimum temperature of Tachit-L 129 F was increased by 10℃, while the thermostable was almost the same. The optimum pH was decreased to 5.0, and kept stable under acid condition. The results of Tachit-V134I showed little effect of mutation on the optimum temperature of the enzyme. The mutant showed pretty basophilia, the maximal activity was at pH 11, and still kept 85% activity at pH 12.更多还原