摘要氨基葡萄糖(Glucosamine，GlcN)又被称为氨基葡糖或是葡糖胺，是葡萄糖的一个 羟基被氨基庖代后形成的化合物。乙酰氨基葡萄糖(GlcNAc)也就是葡糖胺和其乙酰化 的衍生物，已经被广泛的用于食品，化妆品和制药工业等相关方面。目前，氨基葡萄 糖主要是以酸水解虾蟹类、贝壳类等外骨骼中几丁质的方法获得。81703

为构建 GlcN 基因工程菌株奠定良好基础，本人参与耐氨糖菌株的筛选工作，即 在基础培养基中添加氨基葡萄糖使其浓度达到 40 g/L，发现这株菌株能够正常生长， 从而挑取培养，离心得菌体。实验通过对这株耐氨糖菌株基因组的提取以及该菌氨基 葡萄糖合成酶基因的克隆和纯化为构建氨基葡萄糖基因工程菌株奠定基础。

实验结果表明：在高浓度氨糖环境中筛选得到一株耐氨糖菌株，筛选出的菌株在 培养温度为 37℃，蛋白胨浓度为 15 g/L，葡萄糖浓度为 30 g/L，初始 pH 为 7 的 LB 基础培养条件下正常存活。该菌生产氨基葡萄糖的条件为生长温度：最适 28~35℃， 最高 33~39℃；不能在含有乙酸钙的培养基上生长，但可以在含有碳酸钙的培养基上 生长。对该菌 16SrDNA 序列进行了测序，序列在核糖体数据库上比对，结果表明该 菌属别为欧文氏杆菌(Erwinia tasmaniensis sp。)。对该菌株的基因组进行提取，用提取 的基因组按照对应的两种引物进行 PCR 克隆过程，得到大小为 1830 bp 的 glmS 片段， 将片段和空质粒 PET28a 进行双酶切后通过连接试剂进行连接，得到构建完成的重组 质粒，最终转化到宿主菌中，为构建基因工程菌株奠定优良的基础。

毕业论文关键词：氨基葡萄糖；筛选鉴定；耐受；欧文氏杆菌；发酵

Abstract Glucosamine (GlcN), an amino sugar, is a compound derived from substitution of a hydroxyl group of a glucose molecule with an amino group。 GlcN and its acetylated derivative, N-acetylglucosamine (GlcNAc), have been widely used in food, cosmetics, and pharmaceutical industries and are currently produced by acid hydrolysis of chitin (a linear polymer of GlcNAc) extracted from crab and shrimp shells。

Lay a good foundation for the construction of GlcN genetically engineered strains, Himself involved in ammonia sugar resistant strains screening, which is added in the basal medium amino glucose concentrations reach 40 g/L, found that the strains to normal growth, so as to pick, centrifugal bacteria。 Through the extraction of its genome and the bacterium glucosamine synthetase gene cloning and purification of foundation for construction of glucosamine gene engineering strains。

The physiological and biochemical experiments results showed that: The strain in the culture temperature 35℃, peptone concentration of 15 g/L, 30 g/L glucose concentration, initial pH of 7 LB culture under the condition of the bacteria can live。The optimal temperature is 28 ~ 35℃, the highest temperature is 33 to 39 ℃;Can not grow on  a medium containing calcium acetate, but it can be  grown in a medium containing    calcium

carbonate。Sequencing of 16S rDNA sequences and do the comparison on the Ribosomal Database。 The results show that the genus Erwinia tasmaniensis sp。 Filter in the high concentration of ammonia sugar environment to get one of ammonia sugar resistant strain, the strain of the genome is extracted, using extracted genome in accordance with the corresponding two kinds of primers for PCR cloning process, get the size of 1830 bp glmS fragments, fragments and empty plasmid PET28a by connecting after double enzyme reagent to connect。Eventually transform host bacterium, building engineering strains。

Key words: Glucosamine; Screening and Identification; Tolerance; Erwinia tasmaniensis sp。; fermentation

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