阅读说明：本技术 一种促进紫色色杆菌中脱氧紫色杆菌素表达的方法 (Method for promoting expression of deoxyviolacein in chromobacterium violaceum ) 是由 张丽影 权春善 李容庆 马浩迪 石荟 向航宇 于 2019-09-30 设计创作，主要内容包括：本发明涉及一种促进紫色色杆菌中脱氧紫色杆菌素表达的方法,属于生物医药领域。主要技术方案为：在25-35℃条件下,将一定浓度的组氨酸脂肪醛希夫碱与已活化12小时的紫色色杆菌共同培养12-48小时,随后收集菌体表达的脱氧紫色杆菌素。本发明提供的组氨酸脂肪醛希夫碱具有与紫色色杆菌群体感应系统信号分子正己烷-L-高丝氨酸内酯极为相似五元环头部结构和长链脂肪醛尾部,可有效促进紫色色杆菌中脱氧紫色杆菌素的表达,该方法简便易行,便于推广,为解决脱氧紫色杆菌素产量极低的问题提供理论参考。(The invention relates to a method for promoting expression of deoxyviolacein in chromobacterium violaceum, belonging to the field of biological medicine. The main technical scheme is as follows: culturing histidine fatty aldehyde Schiff base with activated chromobacterium violaceum at 25-35 deg.c for 12-48 hr, and collecting deoxyviolacein expressed by thallus. The histidine fatty aldehyde Schiff base provided by the invention has a five-membered ring head structure and a long-chain fatty aldehyde tail part which are very similar to a signal molecule n-hexane-L-homoserine lactone of a chromobacterium violaceum quorum sensing system, and can effectively promote the expression of deoxyviolacein in chromobacterium violaceum.)

1. A method for promoting the expression of deoxyviolacein in Chromobacterium violaceum, comprising the steps of: co-culturing histidine fatty aldehyde Schiff base with certain concentration and activated chromobacterium violaceum at 25-35 deg.c for 12-48 hr, and collecting deoxyviolacein expressed by thallus;

the preparation method of the histidine fatty aldehyde Schiff base comprises the following steps: reacting histidine with fatty aldehyde with different chain lengths in a solvent mixed with an alkaline substance at 50-90 ℃ for 3-8 hours under the protection of nitrogen to generate histidine fatty aldehyde Schiff base;

the chemical formula of the histidine fatty aldehyde Schiff base is shown as

Wherein n is any integer between 1 and 13.

2. The method of claim 1, wherein the concentration of histidine fatty aldehyde schiff base is less than 2.5 mmol/L.

3. The method of promoting the expression of deoxyviolacein in chromobacterium violaceum of claim 1, wherein: 1.5mmol/L histidine fatty aldehyde Schiff base and the activated chromobacterium violaceum are cultured at 30 deg.c for 24 hr and the deoxyviolacein expressed in the thallus is collected.

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a method for promoting expression of deoxyviolacein in chromobacterium violaceum.

Background

Violacein is a microbial metabolite belonging to the indole derivatives and is a water insoluble bluish-black pigment. In the 19 th century, it was found that Chromobacterium violacea expressed violacein, and thereafter, various species were successively found to produce violacein. Subsequently, it was found that the purple pigment has a smaller content of deoxyviolacein in addition to violacein.

Research shows that violacein has very excellent biological activity, including broad-spectrum antibacterial property (effectively inhibiting streptococcus, bacillus, staphylococcus aureus, pseudomonas and the like); antiviral property (activity against herpes simplex virus and poliovirus infecting HeLa cells); anti-tumor cells (significantly inhibiting colon cancer HT29 cell proliferation); genotoxicity and antiprotozoal activity, etc. Deoxyviolacein is different from violacein in structure by one hydroxyl group, and the expression quantity of the pigment in thalli is smaller and is only one tenth of that of violacein, but the deoxyviolacein also has the activities of resisting tumors, gram-positive bacteria, plant pathogenic bacteria and the like. However, the two blue-violet microbial metabolites are currently extremely low in yield, and cannot be industrially produced. Therefore, a method for promoting the expression of violacein and deoxyviolacein in chromobacterium violaceum is urgently needed to solve the problem of low yield.

The expression of violacein and deoxyviolacein is regulated by the CviI/CviR quorum sensing system in Chromobacterium violaceum. The main signal molecule of the system is N-hexane-L-homoserine lactone (HHL). The molecular structural formula is as follows.

After the HHL signal molecule is combined with the receptor protein CviR, the intracellular domain of the receptor protein is phosphorylated, and then a downstream signal path is activated, so that the expression of the deoxyviolacein is realized. If the signal molecule intensity can be enhanced in the extracellular environment, the signal of the whole channel is expected to be enhanced, and the expression quantity of the deoxyviolacein is increased.

Disclosure of Invention

In order to solve the problems in the prior art, the invention designs and synthesizes histidine fatty aldehyde Schiff base, which comprises histidine heptanal, histidine octanal, histidine decanal and histidine dodecanal. The compounds show excellent biological activity in promoting the expression of deoxyviolacein in chromobacterium violaceum. The invention provides a method for promoting expression of deoxyviolacein in chromobacterium violaceum, which effectively improves the yield of the deoxyviolacein and provides a theoretical basis for realizing industrial mass production of the deoxyviolacein.

The technical scheme of the invention is as follows: a method for promoting the expression of deoxyviolacein in Chromobacterium violaceum, comprising the following steps:

co-culturing histidine fatty aldehyde Schiff base with certain concentration and activated chromobacterium violaceum at 25-35 deg.c for 12-48 hr, and collecting deoxyviolacein expressed by thallus;

the preparation method of the histidine fatty aldehyde Schiff base comprises the following steps: reacting histidine with fatty aldehyde with different chain lengths in a solvent mixed with an alkaline substance at 50-90 ℃ for 3-8 hours under the protection of nitrogen to generate histidine fatty aldehyde Schiff base;

the chemical formula of the histidine fatty aldehyde Schiff base is shown as

Wherein n is any integer between 1 and 13;

further, the concentration of the histidine fatty aldehyde Schiff base is less than 2.5 mmol/L.

Further, the optimal expression method is as follows: 1.5mmol/L histidine fatty aldehyde Schiff base and the activated chromobacterium violaceum are cultured at 30 deg.c for 24 hr and the deoxyviolacein expressed in the thallus is collected.

The invention has the following beneficial effects:

the histidine fatty aldehyde Schiff base provided by the invention has a five-membered ring head structure and a long-chain fatty aldehyde tail part which are very similar to a signal molecule n-hexane-L-homoserine lactone of a chromobacterium violaceum quorum sensing system, and can effectively promote the expression of deoxyviolacein in chromobacterium violaceum.

Drawings

FIG. 1 histidine n-heptanal Schiff base¹H nuclear magnetic spectrum;

FIG. 2 histidine n-octanal Schiff base¹H nuclear magnetic spectrum;

FIG. 3 histidine-M-decanal-Schiff base¹H nuclear magnetic spectrum;

FIG. 4 histidine dodecanal Schiff base¹H nuclear magnetic spectrum;

FIG. 5 deoxyviolacein mass spectrum;

FIG. 6 is a diagram of the ultraviolet full wavelength spectrum of deoxyviolacein;

FIG. 7 is a graph of a deoxyviolacein standard curve;

FIG. 8 is a sequence chart of loading of histidine fatty aldehyde Schiff base;

FIG. 9 is a graph showing 12 hours of co-culture of histidine fatty aldehyde Schiff base with Chromobacterium violaceum;

FIG. 10 effect of histidine fatty aldehyde Schiff base on the amount of deoxyviolacein expressed (24 hours).

Detailed Description

The present invention will be further described with reference to specific examples, but the starting materials for the present invention are commercially available unless otherwise specified.