DEVELOPMENTS OF ENZYME SYSTEMS TOLERANT TO ARTIFICIAL CONDITIONS AND ANALYSIS OF THEIR FUNCTIONS

耐人工条件酶系统的研制及其功能分析

• 批准号: 10555288
• 负责人: KUNUGI Shigeru
• 金额: $ 4.1万
• 依托单位: KYOTO INSTITUTE OF TECHNOLOGY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10555288/
• 关键词: Tolerant Enzyme; High Pressure; High Temperature; Induction; Artificial Conditions; Screening Procedures; Organic Solvents

Developments of enzyme systems which can be used in rather hard artificial conditions such as high temperature, high salt conditions, high contents of organic solvents, and high pressure, are necessary to the wider applications of enzymatic processes into chemoenzymatic applications.In this project two extreme artificial conditions, namely solvent-tolerance and pressure-tolerance, are chosen as the targets and in order to obtain microbilas which produce these tolerant enzymes convenient screening procedures are to be investigated. In addition to this, chemical modification methods and amino-acid replacements are also applied to produce the aimed enzyme systems.Namely, direct observations of microbilas through micrographic methods under high pressure conditions were made possible by investing pressure micrgoraph cell and a very convenient method of pressure-tolerance screening was proposed by use of a commercially available high-pressure pressing apparatus.For the chemical modifications, high pressure-induced partial refolding of enzyme proteins was utilized to introduce the midifier reagent into the depth of the protein molecules, which is original to the investigator's laboratory.The functional analysis of the obtained enzymes under artificial conditions were further performed by use of various spectrophotometric methods, such as 4th derivative absorbance, FTIR, fluorescence, and high pressure electrophoresis, both in situ and ex situ manners.

发展能在高温、高盐、高有机溶剂和高压等较难的人工条件下使用的酶系统，对于酶过程在化学酶中的广泛应用是必要的。本项目选择耐溶剂和耐压力两种极端的人工条件作为目标，为了获得产生这些耐受酶的微生物，需要进行简便的筛选程序。此外，还采用了化学修饰和氨基酸取代的方法来制备目标酶系统。通过引入压力微管，实现了在高压条件下通过显微照相方法直接观察微球，并利用市售的高压加压设备提出了一种非常方便的耐压筛选方法。在化学修饰方面，利用高压诱导酶蛋白的部分复性，将调节剂引入到蛋白质分子的深处，这是本实验室独创的。进一步利用各种分光光度方法对所获得的酶在人工条件下进行功能分析。如四阶导数吸光度、FTIR、荧光和高压电泳法，包括原位和非原位方法。

项目成果

S. Kunugi et al.: "Pressure-assisted cold-denaturation of carboxypeptidase Y"Bulletin of the Chemical Society Japan. 72 (12). 2803-2806 (1999)

S. Kunugi 等人：“羧肽酶 Y 的压力辅助冷变性”日本化学会通报。

S. Kunugi 他: "Studies on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhabitor and thermolysin"European Journal of Biochemistry. 259. 815-820 (1999)

S. Kunugi 等：“链霉菌蛋白质金属蛋白酶抑制剂和嗜热菌蛋白酶复合物的形成和稳定性研究”欧洲生物化学杂志 259. 815-820 (1999)。

G.-Q. Tang 他: "Salt Effects on Fluorescence spectal Shifts of DNA-bound Hoechst 33258 and reaction Volumes of the Minor Groove Binding"Bulletin of the Chemical Society Japan. 72(5). 1129-1137 (1999)

G.-Q. Tang 等人：“盐对 DNA 结合的 Hoechst 33258 的荧光光谱变化和小凹槽结合的反应体积”日本化学学会公报 72(5) (1999)。

S. Kunugi et al.: "Single-point amino acid substitutions at the 119th residue of thermolysin and their pressure-induced activation"FEBS Letters. 462 (3). 231-235 (1999)

S. Kunugi 等人：“嗜热菌蛋白酶第 119 个残基处的单点氨基酸取代及其压力诱导的激活”FEBS Letters。

G.-Q.Tang 他: "Salt Effects on Fluorescence spectral Shifts of DNA-bound Hoechst 33258 and Reaction Volumes of the Minor Groove Binding"Bulletin of the Chemical Society Japan. 72(5). 1129-1137 (1999)

G.-Q.Tang 等人：“盐对 DNA 结合的 Hoechst 33258 的荧光光谱变化和小凹槽结合的反应体积的影响”日本化学学会公报 72(5) (1999)。