论文总字数：16396字

摘 要

本课题首先在NCBI数据库中查找到了Candida Tenuis CBS 4435的木糖还原酶(XR)基因序列和Bacillus cereus ATCC 14579的葡萄糖脱氢酶(GDH)的基因序列,然后根据大肠杆菌的密码子偏好对基因序列进行优化并合成，再使用引物通过PCR技术扩增XR和GDH使之亚克隆于pETDuet-1，构建可以使XR和GDH同时表达的重组质粒具体为：用XR-up和XR-dn克隆XR；用GDH-up和GDH-dn克隆GDH；将XR的PCR扩增物连接到线性化质粒pETDuet-1上 NcoI 与SalI两个酶切位点之间，得到pETDuet-XR；将GDH的PCR扩增物连接到线性化质粒pETDuet-XR上BglII 与 XhoI两个酶切位点之间，这样即构成了重组质粒。下一步将构建好的重组质粒通过热激转化导入到E.coli DH5α 菌株中，并且对经过转化的E.coli DH5α进行质粒的提取，然后使用ApaI和AflII 酶进行酶切验证，并对酶切片段进行DNA测序，以此来验证质粒是否构建成功。质粒构建验证成功后将其导入大肠杆菌中，于含有氨苄青霉素的LB培养基中培养，并使用异丙基-ß-D-硫代半乳糖苷(IPTG)进行诱导，使大肠杆菌表达木糖还原酶和葡萄糖脱氢酶。经过超声破碎提取粗酶液，并通过SDS-聚丙烯酰胺凝胶电泳进行检测，通过蛋白表达的条带分布证明木糖还原酶和葡萄糖脱氢酶成功诱导表达。最后用诱导成功的湿细胞在木糖和葡萄糖的混合糖液中进行全细胞催化生产木糖醇。

关键词：木糖还原酶 葡萄糖脱氢酶 诱导表达 SDS-聚丙烯酰胺凝胶电泳 全细胞催化反应

Construction of Recombinant Escherichia Coli and Induction of Expression of Enzyme

ABSTRACT

In this paper, we first found the xylose reductase (XR) gene sequence of Candida Tenuis CBS 4435 and the glucose dehydrogenase (GDH) gene sequence of Bacillus cereus ATCC 14579 in the NCBI database, and then based on the codon preference of E. coli. The sequence was optimized and synthesized, and then XR and GDH were amplified by PCR to subclone it into pETDuet-1, and a recombinant plasmid capable of simultaneously expressing XR and GDH was constructed: XR-up and XR-dn were used to clone XR. GDH was cloned with GDH-up and GDH-dn; the PCR amplification product of XR was ligated to the linearization plasmid pETDuet-1 between NcoI and SalI to obtain pETDuet-XR; PCR amplification of GDH was performed. The extender was ligated between the BglII and XhoI restriction sites on the linearized plasmid pETDuet-XR, thus constituting the recombinant plasmid. Next, the constructed recombinant plasmid was introduced into E. coli DH5α strain by heat shock transformation, and the transformed E. coli DH5α was subjected to plasmid extraction, and then digested with ApaI and AflII enzymes, and digested. The fragment was subjected to DNA sequencing to verify whether the plasmid was successfully constructed. After successful plasmid construction, it was introduced into E. coli, cultured in LB medium containing ampicillin, and induced with isopropyl-ß-D-thiogalactoside (IPTG) to express xylose in Escherichia coli. Reductase and glucose dehydrogenase. The enzyme solution was extracted by ultrasonication and detected by SDS-polyacrylamide gel electrophoresis. The band expression of protein expression proved that xylose reductase and glucose dehydrogenase were successfully induced to express. Finally, whole-cell catalyzed production of xylitol was carried out using wet cells that induced success in a mixed sugar solution of xylose and glucose.

Keywords: Xylose reductase;Glucose dehydrogenase;Induced expression;SDS-polyacrylamide gel electrophoresis;Whole cell catalytic reaction

目 录

摘要 I

ABSTRACT II

第一章 文献综述 1

1.1木糖醇简介 1

1.2木糖醇的应用 1

1.2.1木糖醇在食品工业中的应用 1

1.2.2木糖醇在医药方面的应用 1

1.2.3木糖醇在制造业上的应用 1

1.3木糖醇的生产工艺 1

1.3.1化学合成法 1

1.3.2生物转化法 2

第二章 材料和方法 4

2.1材料 4

2.1.1菌株和质粒 4

2.1.2实验试剂和药品 4

2.1.3 培养基配方 4

2.1.4实验仪器与设备 5

2.2 实验方法 6

2.2.1查找目的基因序列 6

2.2.2构建重组质粒 6

2.2.3对重组质粒进行酶切验证 7

2.2.4进行酶的诱导表达 9

2.2.5进行SDS-聚丙烯酰胺蛋白电泳验证 10

2.2.6在纯糖液中全细胞催化生产木糖醇 10

第三章 结果与讨论 11

3.1结果 11

3.1.1 酶切验证结果分析 11

3.1.2 SDS-聚丙烯酰胺蛋白电泳分析 11

3.1.3在纯糖液中进行全细胞催化的结果分析 12

3.2讨论 13

第四 章结论与展望 15

4.1结论 15

4.2展望 15

参考文献 16

第一章 文献综述

1.1木糖醇简介

木糖醇与蔗糖都是一种天然甜味剂，它和蔗糖都具有白色结晶状的、易溶解等性质，其广泛存在于自然界中，在化工、食品、医药等行业中有很广泛的应用^[1,2]。许多水果蔬菜中都含有木糖醇，但它的含量普遍偏低，提取的成本很高。同时木糖醇用处很广，从它的甜度上来看，木糖醇可以替代蔗糖^[3]，从发热量来看，又可以媲美葡萄糖，在许多行业有着很重要的应用。

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