Recombinant Escherichia coli zjut-ho1 and its application in the preparation of biliverdin

A technology for recombining Escherichia coli and biliverdin, applied in bacteria, enzymes, microorganism-based methods, etc., can solve the problems of high price, low yield, difficult industrial application, etc., and achieve short culture period, high activity, and yield. improved effect

Active Publication Date: 2022-04-19
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The extraction method is to extract biliverdin directly from salmon bile or emu eggshell, but the problem is that the source of raw materials is insufficient and it is difficult for industrial application; the chemical conversion method is prepared by dehydrogenating bilirubin, but there are two problems in this method: ①Bilirubin with high price is needed as a substrate, and the source of raw materials is difficult; ②Bilirubin is extracted from the bile of mammals, and needs to be under acidic conditions, which makes bilirubin form multiple isomers, resulting in the product bile The yield and purity of chlorophyll IXα are relatively low; there are also reports on the preparation of biliverdin by the chemical oxidation heme method, which also produces more isomers with a lower yield; A patent from cyanobacteria recombines the HO-1 gene from cyanobacteria into yeast cells, expresses HO-1, and then uses heme as a substrate to produce biliverdin through whole cell biotransformation. The advantage of this method is that there are few isomers , but the patent does not report the yield
It can be seen that there is currently a lack of economical and effective biliverdin preparation methods

Method used

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  • Recombinant Escherichia coli zjut-ho1 and its application in the preparation of biliverdin
  • Recombinant Escherichia coli zjut-ho1 and its application in the preparation of biliverdin
  • Recombinant Escherichia coli zjut-ho1 and its application in the preparation of biliverdin

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Experimental program
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Effect test

Embodiment 1

[0035] Example 1 Construction of Gene Recombinant Escherichia coli Expressing HO-1

[0036] The recombinant Escherichia coli zjut-ho1 strain expressing HO-1 of the present invention is constructed according to the following steps:

[0037] (1) After extracting the total RNA of Synechocystis PCC 6803 with "Trizol Univer Total RNA Extraction Reagent", use the "AMV First-Strand cDNA Synthesis Kit" to synthesize the first-strand cDNA by RT-PCR.

[0038] (2) Using the cDNA obtained in step (1) as a template, design primers P1 and P2, introduce restriction endonucleases NdeI and XhoI restriction sites, and amplify the gene (ho1) sequence of HO-1 by PCR. "SanPrep Column PCR Product Purification Kit" is used for purification to obtain the sequence of the target gene ho1. The nucleotide sequence of the gene ho1 is shown in SEQ ID NO:1.

[0039] The primers P1 and P2 are respectively:

[0040] P1: 5′-GGGAATTCCATATGAGTGTCAACTTAGC-3′

[0041] P2: 5'-CCGCTCGAGCTAGCCTTCGGAG-3'.

[0042]...

Embodiment 2

[0059] Embodiment 2: the preferred condition of recombinant Escherichia coli zjut-ho1 strain expressing HO-1

[0060] On the basis of obtaining the Escherichia coli zjut-ho1 strain capable of expressing HO-1 in Example 1, the culture conditions for expressing HO-1 of the strain were optimized, including the type of medium, the time of adding the inducer, and the time of induction culture , the concentration of the inducer added, and the temperature of the induced culture. After optimization, the expression level of HO-1 is significantly improved. The preferred zjut-ho1 strain expresses HO-1 and the process steps are as follows:

[0061] (1) The zjut-ho1 strains that were freeze-dried or glycerol-frozen preserved were inoculated in LB slant medium containing 50 μg / mL kanamycin, and cultured at a constant temperature of 37° C. for 24 hours to obtain slant bacteria; the LB slant culture The final base concentration is composed of: yeast extract powder 5g / L, peptone 10g / L, NaCl 1...

Embodiment 3

[0065] Example 3 Escherichia coli zjut-ho1 is applied to biotransformation of heme to prepare biliverdin

[0066] On the basis of Example 2, the zjut-ho1 bacterium expressing HO-1 was cultivated for the bioconversion of heme to prepare biliverdin. The specific steps are as follows:

[0067] (1) According to the method of embodiment 2, the zjut-ho1 culture fluid (OD) cultivated through HO-1 expression 600 =1.40~1.65), add 2g / L hemin aqueous solution to make the final concentration of hemin 200mg / L, and continue to cultivate at 37°C, 180r / min constant temperature shaking shaker for 5h. The preparation method of the hemin aqueous solution is: 100mg of hemin is slightly heated and dissolved in 50mL with a mass concentration of 0.25% Na 2 CO 3 Aqueous solution, that is, 2g / L hemin aqueous solution.

[0068] (2) The reaction system in step (1) was centrifuged at 1000 g for 10 min to collect the thalline, and the thalline was dark green at this time. The bacteria were suspended i...

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Abstract

The invention discloses a recombinant Escherichia coli zjut-ho1 and its application in the preparation of biliverdin, which is preserved in the Guangdong Microbial Culture Collection Center, with a preservation number of GDMCC No: 60372 and a preservation date of May 18, 2018. Address: 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City, Zip Code: 510075. The recombinant Escherichia coli expresses HO-1 with intracellular activity, and the recombinant Escherichia coli can be used as the HO-1 enzyme source, and hemin is used as the substrate to prepare biliverdin by biotransformation, which improves the production of biliverdin. way. Compared with the existing similar technologies, the present invention expresses ho1 from prokaryotic organisms in Escherichia coli of prokaryotic organisms, which has the advantages of high HO-1 activity and short culture period, and uses hemin as a substrate to make The yield of biliverdin is greatly improved, which is 2.6 times that of the recombinant Escherichia coli whole cell synthesis method.

Description

(1) Technical field [0001] The invention relates to a gene recombinant Escherichia coli strain expressing heme oxygenase-1 derived from algae, and the application of the strain in preparing biliverdin by biotransforming heme. (2) Background technology [0002] Biliverdin (biliverdin, CAS: 114-25-0) is a linear tetrapyrrole ring substance obtained by hydrolysis of heme by heme oxygenase (hemeoxygenase-1, HO-1) (reaction formula see figure 1 ), named for its dark green color. In vertebrate cells, under the hydrolysis of HO-1, heme opens at the α-methylene bridge to form biliverdin IXα isomers. Therefore, biliverdin generally refers to biliverdin IXα. [0003] Biliverdin is not only an intermediate metabolite of the heme metabolic circulatory system, but more importantly, it can initiate a series of physiological effects such as anti-inflammation, anti-oxidation and immune regulation, such as improving liver function and reducing alanine aminotransferase , Alleviate the ische...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P17/16C12R1/19
CPCC12N9/0083C12P17/165C12Y114/99003
Inventor 梅建凤赵文渊应国清易喻陈建澍张彦璐
Owner ZHEJIANG UNIV OF TECH
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