| 题名 | 木糖酸脱水酶YjhG性质研究及在丁三醇体外合成中的应用 |
| 作者 | 蒋昱东 |
| 学位类别 | 硕士 |
| 答辩日期 | 2015-05 |
| 授予单位 | 中国科学院大学 |
| 授予地点 | 北京 |
| 导师 | 咸漠 |
| 关键词 | 生物合成 1,2,4-丁三醇 D-木糖酸脱水酶 酶学性质 体外酶法合成 |
| 学位专业 | 化学工程 |
| 中文摘要 | 1,2,4-丁三醇（BT）是重要的有机合成化学中间体，广泛应用于医药、烟草、化妆品、造纸、农业和高分子材料领域。BT的化学合成法存在反应条件苛刻、污染物排放严重等诸多缺点，因此，越来越多的研究者将目光转向了更为经济环保的生物合成技术。目前，已有利用木糖为底物生物法合成1,2,4-丁三醇的报道。虽然已实现了1,2,4-丁三醇的生物合成，但由于对代谢途径中关键酶的研究并不深入，且微生物体内代谢不易调控，致使1,2,4-丁三醇的产量、产率等指标不高，难以进行工程化放大。针对上述问题，本论文主要进行了以下两个方面的研究：（1）对生物合成途径中的重要酶—木糖酸脱水酶YjhG进行酶学性质研究，以期提高其在1,2,4-丁三醇合成过程的催化能力；（2）构建体外酶法合成1,2,4-丁三醇的体系，实现其体外合成，以期为扩大化生产奠定基础。 大肠杆菌yjhG基因编码的蛋白YjhG是BT生物合成途径中的关键酶，此外，它还参与许多重要的代谢途径，如乙醇生物合成途径、木糖降解途径及戊糖/葡萄糖醛酸转化途径。研究表明YjhG具有D-木糖酸脱水活性，但对该酶的性质并没有详细研究。本论文在工程大肠杆菌中构建并过表达了木糖酸脱水酶YjhG，并对其进行分离纯化。同时，通过产物检测方法研究，建立了以盐酸氨基脲检测法为基础的酶活检测方法。在此基础上，进行了木糖酸脱水酶YjhG的酶学性质研究，包括温度、pH、金属离子等对酶活的影响及动力学参数。研究发现，该酶反应的最适温度为30℃，最适pH值为8.0，在30℃附近及pH值7.0~8.5范围内均能保持较高活性。Mn2+和Mg2+对酶有激活作用，而Ni2+和Zn2+对酶有明显的抑制作用。以D-木糖酸为底物，测定该酶的Km和Vmax值分别为4.88 mM和78.62 μM l-1h-1。 体外酶反应法具有可调控、专一性强、竞争途径消耗少等优点，在重要化学品合成领域中具有广泛的实用潜力，酶法反应已应用于药物、食品添加剂等精细化学品的生产合成。本文利用基因工程手段构建重组大肠杆菌，过表达了1,2,4-丁三醇合成途径中的3种关键酶。以D-木糖酸为底物，通过研究1,2,4-丁三醇体外合成系统，成功地实现了1,2,4-丁三醇的体外酶法合成。同时，通过对3种关键酶用量的研究，实现了体外合成体系的优化。当D-木糖酸脱水酶、苯甲酰甲酸脱羧酶、醇脱氢酶加入量分别为0.5 mg/ml、0.5 mg/ml、1.0 mg/ml时1,2,4-丁三醇体外合成效果最好，产量达277.8 mg/l，为生物法合成丁三醇的工业放大奠定基础。 |
| 英文摘要 | 1,2,4-butanetriol (BT) is an important chemical intermediate in organic synthesis which is widely used in the fields of medicine, tobacco, cosmetics, paper making and polymer materials. Considering the disadvantages of chemical synthesis like harsh reaction conditions and environmental polutions, more and more researches are conducted on microbial productions. Though biosyntheses of 1,2,4-butanetriol were achieved, the yield wasn’t high enough to achieve its economic production because key enzymes in this pathway were not well studied and in vivo synthetic biology of intact microbes was difficult to mediate. To improve the biosynthesis of 1,2,4-butanetriol, two aspects were mainly studied in this article. One aspect was the enzyme characterization of D-xylonate dehydratase YjhG in order to make it work more effectively in 1,2,4-bitanetriol bioconversion. The other one was to establish an in vitro synthetic system of 1,2,4-butanetriol which would improve the industrial enlargement of 1,2,4-butanetriol.YjhG, D-xylonate dehydratase encoded by yjhG gene is a key enzyme in BT biosynthesis, furthermore, it is involved in many microbial pathways including biosynthesis of ethylene glycol, D-xylonate degradation and conversion of pentose/glucuronic acid. The protein YjhG was found to catalyze D-xylonate dehydration, but there weren’t reports about its enzymatic properties. Recombinant E. coli strain was constructed to overexpress YjhG, and then the recombinant protein was purified. Through screening of the product detection methods, the assay method of YjhG activity was constructed which based on semicarbazide derivatization. Finally, characterization of YjhG was systematically studied in vitro. This enzyme showed maximal activity at 30 ℃ and pH 8.0. Bivalent metal ions such as Mg2+ and Mn2+ activated, whereas Ni2+ and Zn2+ inhibited the activity of YjhG. Under optimalreaction conditions, the Km and Vmax values using D-xylonate as a substrate were 4.88 mM and 78.62 μM l-1h-1.In vitro enzymatic conversion has advantages including simplified process control, high specificity and low competitive consumption, which has been applied in the syntheses of fine chemicals like medicine, food additives. Recombinant E. coli strains were constructed to overexpress the key enzymes in 1,2,4-butanetriol biosynthetic pathway. 1,2,4-Butanetriol synthetic reaction was achieved in vitro by exploring 1,2,4-butanetriol cell-free system. And the system was optimized through researches on enzyme concentration. The highest 1,2,4-butanetriol concentration was 277.8 mg/l with the addition of 0.5 mg/ml D-xylonate dehydratase, 0.5 mg/ml benzoylformate decarboxylase and 1.0 mg/ml alcohol dehydrogenase. The optimized in vitro enzyme method enhanced specificity and controllability and laid the foundation for industrial enlargement of 1,2,4-butanetriol bioconversion. |
| 语种 | 中文 |
| 学科主题 | 工学 |
| 公开日期 | 2020-09-30 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.qibebt.ac.cn/handle/337004/8098]  |
| 专题 | 青岛生物能源与过程研究所_材料生物技术研究中心 |
| 作者单位 | 1.中国科学院大学 2.中科院青岛生物能源与过程研究所 |
| 推荐引用方式 GB/T 7714 | 蒋昱东. 木糖酸脱水酶YjhG性质研究及在丁三醇体外合成中的应用[D]. 北京. 中国科学院大学. 2015. |
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