【作者】 王娇；

【导师】 刘万顺；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2011， 硕士

【摘要】 壳寡糖具有许多独特的生理活性及功能性质,如增强机体免疫力、抗肿瘤、抑菌等,在医药、农业、食品等领域有着广泛的应用。壳聚糖酶是专一性降解壳聚糖的水解酶,可用于功能性壳寡糖的制备。本研究的主要目的是构建高效表达壳聚糖酶的工程菌株,以解决目前壳聚糖酶产量低、成本高等问题,同时为壳寡糖的制备提供有效途径。Microbacterium sp.是本实验室已筛选得到的产壳聚糖酶菌株,在此基础上,本论文报道了Microbacterium sp.壳聚糖酶的分离纯化、壳聚糖酶基因在解脂耶氏酵母中的表达及重组酶的性质等研究。采用硫酸铵盐析、DEAE-Sepharose Fast Flow离子交换层析、Sephacryl S-100凝胶层析相结合的方法对Microbacterium sp.粗酶液进行了分离纯化,得到电泳纯壳聚糖酶B,纯化倍数为9.9,回收率为20.5%。SDS-PAGE凝胶电泳显示单一条带,其相对分子质量约为33.8 kDa。酶解产物质谱分析表明壳寡糖主要为四糖、五糖,部分二糖及三糖,无单糖,说明此酶为内切酶。酶N端氨基酸序列为ETAGTVDLDAPVQKDT。根据酶N端测序结果及已注册壳聚糖酶序列设计引物,以Microbacterium sp.基因组为模板扩增得到约1000 bp特异性片段,序列分析表明含有完整的壳聚糖酶编码基因,其开放阅读框为801 bp,编码266个氨基酸残基,属于糖苷水解酶46号家族。该基因前26个氨基酸为信号肽,其N端推导序列与测序结果一致。壳聚糖酶基因表达采用解脂耶氏酵母表达体系。目的基因与表达载体pINA1317经限制性内切酶SfiⅠ、KpnⅠ双酶切后,连接,构建重组载体。重组载体NotⅠ酶切线性化后转化Yarrowia lipolytica Polh。采用YNB平板筛选阳性转化子。菌落PCR表明目的基因整合入酵母基因组中。转化子接种于PPB培养基（蔗糖2 g,酵母粉0.132 g,氯化铵0.132 g,磷酸二氢钾0.032 g,无水硫酸镁0.0132 g,硫胺素0.0334 mg,溶于100 ml pH 7.0 100 mM柠檬酸盐缓冲液）,28℃培养120 h,发酵上清液酶活力可达10.4 U/ml,是Microbacterium sp.上清液酶活力的3.15倍。转化子粗酶液经DEAE-Sepharose Fast Flow离子交换层析得到电泳纯重组壳聚糖酶。SDS-PAGE及Western blotting鉴定了重组蛋白的表达,重组酶相对分子量偏大,为41kDa,可能与酵母表达体系的糖基化有关。酶学性质研究表明重组酶最适反应温度45℃,最适反应pH为5.6,缓冲体系为0.2 M乙酸盐缓冲液。重组酶在pH 5.0-7.0、25℃以下具有较好的稳定性。Na⁺对酶活力有一定的促进作用;Ag⁺、Fe³⁺具有较强的抑制作用,其中Fe³⁺（5 mM、10 mM）、Ag+（10 mM）完全抑制酶活力。以壳聚糖为底物,重组酶的Km值为0.926 mg/ml,Vmax为6.15 U/ml。酶解产物主要为三糖、五糖,降解专一性较好。本研究通过构建相关表达载体及工程菌株,实现了壳聚糖酶在解脂耶氏酵母中的分泌表达,并对表达蛋白进行了相关的生物活性鉴定,为壳聚糖酶的工业化生产奠定了理论基础。更多还原

【Abstract】 Chitooligosaccharides(COSs), with various functional properties such as immunity-enhancing, antitumor activity and antimicrobial activity, have become a remarkable resource for development of biomedicine, agriculture, and food industry. Chitosanase, which catalyze the hydrolytic degradation of chitosan, can be used for preparation of functional COSs. The purpose of this study is to investigate the expression of chitosanase gene in yeast to resolve the problem of chitosanase such as high cost and unavailability in bulk quantities. The study will provide a more efficient way for COSs production.Microbacterium sp. that can express chitosanase was isolated previously. Purification, expression in Yarrowia lipolytica and characterization of the recombinant chitosanase are reported in this work.The chitosanase from Microbacterium sp. is purified 9.9 folds by a procedure of ammonium sulfate precipitation, DEAE-sepharose Fast Flow ionexchange and Sephacryl S-100 gel filtration chromatography with a recovery of 20.5%. The molecular weight is estimated to be 33.8 kDa by SDS-PAGE. The reaction products analyzed by LC–MS/MS show that chitosan can be degraded into different lengths of oligosaccharides, chitotetraose and chitopentaose are detected as the major products with some levels of chitobiose and chitotriose. The product such as glucosamine cannot be detected indicating that the mode of chitosanase B is endo–type. The N-terminal amino acid sequence is determined to be ETAGTVDLDAPVQKDT.The primers are designed according to N-terminal amino acid sequence and chitosanase gene registered. A 1000-length fragment encoding the chitosanase gene is obtained using the genomic DNA of Microbacterium sp. as a template. The sequence contains one open reading frame of 801 bp encoding 266 animo acids with a signal peptide of 26 amino acids. The deduced N-terminal amino acid sequence of the chitosanase gene, which is classified into glycoside hydrolase family 46, is completely identical to the result sequenced previously.Yarrowia lipolytica is chosen as the host for chitosanase expression. The amplified fragments are inserted into Sfi I–Kpn I sites of the vector pINA1317, resulting the recombinant plasmid. The expression plasmid is transformed to Y.lipolytica Polh competent cell after linearized with restriction enzyme Not I. The transformants are selected on YNB–N5000 plates without uracil. The colony PCR shows that the chitosanase gene is integrated into the genomic DNA of Y.lipolytica. After positive transformant is grown in PPB medium at 28℃for 120 h, the chitosanase activity in the supernatant of the culture is determined to be 10.4 U/ml, which is about 3.15–fold compared to that of Microbacterium sp.Purification of the recombinant chitosanase is achieved by DEAE–Sepharose Fast Flow ionexchange chromatography. The recombinant protein is demonstrated by western blotting. The molecular mass analyzed by SDS-PAGE is 41 kDa, which is larger than native chitosanase. It’s probably that the protein modification is different in Y.lipolytica Polh, such as N-glycosylation, signal peptide cleavage and other modifications of the propreprotein. The optimal temperature for recombinant chitosanase is 45℃and the optimal pH is 5.6 (0.2 M acetate buffer). The chitosanase activity is stable in the pH range of 5.0-7.0 and under 25℃. Na+ has an activating effect on enzyme activity. Ag+(10 mM) and Fe3+ (5 mM, 10 mM) can completely inhibit activity. The apparent Km and Vmax values of the recombinant chitosanase with souble chitoan as substrate are 0.93 mg/ml and 6.15 U/ml, respectively. Chitotriose and chitopentaose are detected as the major products.The chitosanase gene is successfully expressed in Y.lipolytica through the construction of expression vector and engineering strains, making an important step towards the production of chitosanase on industrial scale.更多还原