Method for quickly extracting carotenoid generated by in-vitro enzyme reaction

A carotene and extraction method technology, applied in the field of bioengineering, can solve the problems of low extraction amount, long time consumption, etc., and achieve the effects of increasing product extraction amount, reducing product loss, and shortening operation time

Inactive Publication Date: 2011-11-23
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims at the defects of long time-consuming and low extraction amount of carotenoid products generated by in vitro enzyme reaction, and provides a fast and efficient method for extracting carotenoids generated by in vitro enzyme reaction, that is, the denatured protein precipitation method, which shortens the Product extraction time, reduce product loss, greatly increase product extraction

Method used

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  • Method for quickly extracting carotenoid generated by in-vitro enzyme reaction
  • Method for quickly extracting carotenoid generated by in-vitro enzyme reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Extraction of in vitro reaction product of Rhodobacter sphearoides No.ATCC 17025 recombinant phytoene dehydrogenase

[0035] A rapid extraction method for generating carotenoids by enzymatic reaction in vitro, the steps are as follows:

[0036] (1) Take 200 mL of E. coli culture fluid expressing Rhodobacter sphaeroides recombinant phytoene dehydrogenase, centrifuge at 12,000 rpm for 5 minutes, and collect bacterial cells; resuspend the collected E. coli cells in 10 mL of 100 mmol / L Tris. In the HCl (pH7.9) buffer solution, the ultrasonic breaker of Sonics Company of the United States was crushed for 30 minutes (22kHz, 150W) to obtain the crude enzyme liquid of phytoene dehydrogenase;

[0037] (2) Take 400 μL of the crude phytoene dehydrogenase enzyme solution prepared in step (1) in a 1.5 mL centrifuge tube, add phytoene acetone solution, glucose, glucose oxidase and catalase, The final concentrations were 10μmol / L (phytoene), 2mmol / L (glucose), 20U / mL (gluc...

Embodiment 2

[0043] Example 2: Extraction of in vitro reaction product of Rhodobacter sphearoides No.ATCC 17025 recombinant hydroxystreptosporin synthase

[0044] Get 200 mL of E. coli culture fluid expressing rhodobacter sphaeroides recombinant hydroxystreptosporin synthase and centrifuge at 12000 rpm for 5 minutes to collect the bacterial cells; the bacterial cells are resuspended and broken as in Example 1, and 400 μL of broken liquid is added to the streptosporin acetone solution , so that the final concentration of streptosporin was 10 μmol / L, and the reaction system was adjusted to 500 μL with buffer. All the other operations and steps are the same as in Example 1.

[0045] The in vitro reaction product of the above-mentioned Rhodobacter sphaeroides recombinant hydroxystreptosporin synthase is hydroxystreptosporine, and the extraction amount is 1.748 μg.

experiment example

[0046] Experimental example: comparison of lycopene extraction methods in in vitro enzyme reaction system

[0047] Take 4 mL of the cell disruption solution described in Example 1, add 100 μL of 20 μg / mL lycopene in acetone solution, dilute to 5 mL with buffer, and divide into 9 parts at 500 μL / part after mixing, 3 parts are 1 group, a total of 3 Group; using methanol-petroleum ether extraction, acetone-petroleum ether extraction and denatured protein precipitation extraction method described in Example 1 for product extraction.

[0048] (1) Methanol-petroleum ether: Add 2.5 mL of methanol to 500 μL of the mixture and incubate at 55°C for 15 minutes, add 300 μL of petroleum ether (boiling range 30°C to 60°C) and shake for 5 minutes to extract lycopene, and centrifuge to collect the upper layer of petroleum ether.

[0049] (2) Acetone-petroleum ether: Add 2.5 mL of acetone to 500 μL of the mixture and incubate at 55°C for 15 minutes, add 300 μL of petroleum ether (boiling range...

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Abstract

The invention relates to a method for quickly extracting carotenoid generated by an in-vitro enzyme reaction, belonging to the technical field of biological engineering. The method comprises the following steps of: ultrasonically breaking the wall of a recombinant bacillus coli strain expressing a carotenoid metabolism-related enzyme, adding corresponding reaction substances, ultrasonically mixing uniformly and undergoing an enclosed oscillating reaction in a dark place; adding a 10% SDS (Sodium Dodecyl Sulfate) solution into the mixed liquor, adding an NaAc solution, oscillating, centrifuging and removing supernatant to obtain protein precipitates; and adding acetone into the protein precipitates, stirring, uniformly dispersing the precipitates, centrifuging, extracting supernatant, filtering with a filter film and detecting to obtain an acetone solution of carotenoid. According to the method, carotenoid and proteins are co-precipitated and carotenoid is concentrated from a large-volume in-vitro reaction solution into a small quantity of protein precipitates, so that the subsequent separating and purifying processes are avoided.

Description

technical field [0001] The invention relates to a rapid extraction method of carotenoids produced by in vitro enzyme reaction, belonging to the technical field of bioengineering. Background of the invention [0002] Carotenoids are a kind of edible natural pigments, which are not only commonly used coloring agents, but also have various biological activities. This type of compound is a chain or ring polyisoprene compound containing 8 isoprene units connected head-to-tail with tetraterpenes, and is insoluble in water. As a natural pigment, carotenoids are not restricted in terms of pigment types, resources, and chemical properties of pigments, and carotenoids are very strong antioxidants, which can effectively scavenge oxygen free radicals and have certain effects on disease prevention and treatment. [0003] With the development of science and technology and people's emphasis on food safety, the development of natural pigments has become a research hotspot. The carotenoid m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P5/02C07C33/02C07C29/74C07C11/21C07C7/00C12R1/19
CPCY02E50/343Y02E50/30
Inventor 肖敏张金华卢丽丽
Owner SHANDONG UNIV
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