通过化学补充来指导糖合成酶的进化
Hening Lin1, Haiyan Tao, Virginia W Cornish
1Department of Chemistry, Columbia University, New York, New York 10027, USA.
Journal of the American Chemical Society
|November 19, 2004
概括
我们开发了化学补充,一种酵母三杂交试验,以增强酶进化. 这种方法成功地改善了糖合成酶的活性,证明了它对碳水化合物活性酶的定向进化的有用性.
科学领域:
- 生物化学 生物化学
- 酶学 是一种酶学.
- 合成生物学 合成生物学
背景情况:
- 酶催化对于碳水化合物合成至关重要.
- 定向进化需要强大的测试来选酶变体.
- 化学补充是一种用于酶进化的新型酵母三混合试验.
研究的目的:
- 将化学补充应用于糖合成酶的定向演变.
- 为了增强来自Humicola insolens Cel7B.的糖合成酶的活性.
- 为了证明化学补充对新类酶的多功能性.
主要方法:
- 使用酵母三杂交试验 (化学补充).
- 将Cel7B E197A突变体的糖合成酶活性与LEU2记者基因联系起来.
- 在Glu197位置进行和突变发生,并为提高活性进行选择.
主要成果:
- 通过LEU2报告员成功将糖合成酶活性与细胞生长联系起来.
- 隔离了一种Cel7B E197S变体,其糖合成酶活性增加了5倍.
- 验证的化学补充作为糖合成酶进化的有效平台.
结论:
- 化学补充是一种多功能平台,用于指导糖合成酶的进化.
- 这种测定有助于发现用于碳水化合物合成的增强酶.
- 这种方法是一般的,可以很容易地适应其他酶家族和反应.
相关概念视频
Chemical and Solubility Equilibria
The free energy change associated with dissolving a solute in a liter of solvent is called the free energy of a solution, ΔGsolution. The overall ΔGsolution is expressed as the balance of ΔGinteraction against the always-favorable free-energy of mixing, ΔGmixing. Solution formation is favorable if ΔGsolution is less than zero, whereas it is unfavorable if ΔGsolution is greater than zero. In short, for a solution to form and complete dissolution to take place, the Gibbs energy change must be...
Acid Halides to Alcohols: Grignard Reaction
Organomagnesium halides, commonly known as Grignard reagents, convert acid halides to tertiary alcohols. The reaction requires two equivalents of the Grignard reagent and proceeds via a ketone intermediate.
Grignard reagents are a source of carbanions and function as nucleophiles. The mechanism begins with the nucleophilic attack by the carbanion at the carbonyl carbon of the acid halide to form a tetrahedral intermediate. Next, the carbonyl group is re-formed, and the halide ion departs,...
Grignard reagents are a source of carbanions and function as nucleophiles. The mechanism begins with the nucleophilic attack by the carbanion at the carbonyl carbon of the acid halide to form a tetrahedral intermediate. Next, the carbonyl group is re-formed, and the halide ion departs,...
Introduction to Chemical Reactions
All chemical reactions begin with a reactant, the general term for one or more substances entering the reaction. Sodium and chloride ions, for example, are the reactants in the production of table salt. One or more substances produced by a chemical reaction are called the product. Chemical reactions follow the law of conservation of mass, which means that matter cannot be created nor destroyed in a chemical reaction. The components of the reactants—the number of atoms and the elements—are all...
Types of Chemical Reactions: Exchange and Reversible
An exchange reaction is a chemical reaction in which both synthesis and decomposition occur, chemical bonds are both formed and broken, and chemical energy is absorbed, stored, and released.
A special kind of exchange reaction is the oxidation-reduction reaction, or the redox reaction. These reactions involve the transfer of electrons from one compound to another. The electrons in these reactions commonly come from hydrogen atoms, which consist of an electron and a proton. A molecule gives up a...
A special kind of exchange reaction is the oxidation-reduction reaction, or the redox reaction. These reactions involve the transfer of electrons from one compound to another. The electrons in these reactions commonly come from hydrogen atoms, which consist of an electron and a proton. A molecule gives up a...
Effects of Chemicals: Overview
Drugs, encompassing various chemical compounds from natural sources, lab synthesis, or genetic engineering, elicit different biological responses in living organisms. Some of these responses are desirable or therapeutic, while others are undesirable. The primary goal of administering a drug is to achieve a therapeutic effect, that is, to address a specific disease or health condition. Any concurrent effects outside of this therapeutic outcome are considered undesirable. These undesirable...
Production of Pharmaceuticals
Industrial insulin production uses genetically engineered E. coli expressing a proinsulin gene controlled by a tryptophan promoter and containing a methionine linker for later cleavage. The cells also carry ampicillin resistance for selective growth. Seed cultures are stored at −80 °C and production begins by thawing a small amount to inoculate starter cultures, which are progressively scaled to a 50,000-L bioreactor. In the bioreactor, E. coli grow in nutrient-rich media under sterile, tightly...


