Caffeic acid O-methyltransferase (COMT3) in Morus notabili and application thereof

A technology of oxymethyltransferase and caffeic acid, which is applied in the biological field, can solve the problems of inability to obtain a large amount of extraction and low content, and achieve the effect of high enzyme activity and good application prospects

Pending Publication Date: 2020-10-30
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chuan Mulberry contains melatonin, but its content is e

Method used

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  • Caffeic acid O-methyltransferase (COMT3) in Morus notabili and application thereof
  • Caffeic acid O-methyltransferase (COMT3) in Morus notabili and application thereof
  • Caffeic acid O-methyltransferase (COMT3) in Morus notabili and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, Chuanmulberry caffeic acid oxymethyltransferase gene COMT3 clone

[0026] The primers for amplifying the caffeic acid oxymethyltransferase gene COMT3 were designed according to the Morus.COMT3 caffeic acid oxymethyltransferase gene (Morus013036) reported on the Morus.COMT3 datebase. COMT3 upstream primer is: 5'-gg ggtacc atggcatccccattggagct-3' (SEQ ID NO.1), the downstream primer of COMT3 is: 5'-cg gaattc ttaaagaactccataaccc-3' (SEQ ID NO. 2). The cDNA of Chuan Sang was used as a template, and the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 were used as primers for PCR amplification to obtain the full length of the COMT3 gene. The amplified product was subjected to agarose gel electrophoresis, and the results were as follows: figure 1 shown. The target gene was recovered and then ligated with the pMD19-T vector to obtain the pMD19-COMT3 vector, and then the pMD19-COMT3 vector was transferred into E.coli.Trans1-T1 competent cells, and the positive ...

Embodiment 2

[0027] Example 2, Construction and Prokaryotic Expression of the Caffeic Acid Omethyltransferase Gene COMT3 Recombinant Vector of Chuanmulberry

[0028] The target gene COMT3 and the Pcold-tf empty plasmid obtained by cloning in the example were double-digested with KpnI and EcoRI respectively, and the recovered product was used as T4 DNA ligase was used to connect them to obtain the recombinant plasmid Pcold-tf-COMT3, which was transformed into Escherichia coli DH5ɑ competent cells, and the correct identified Pcold-tf-COMT3 was sent to Huada Gene Company for sequencing. The sequences obtained by the second sequencing were consistent to obtain the correct sequence.

[0029] Extract the Pcold-tf-COMT3 plasmid, transfer it into the expression strain B21(DE3), add 450μl LB medium containing Amp resistance to a 1.5ml centrifuge tube, and expand the culture into the test tube at a ratio of 1:100, 28°C, 220rpm Shaker culture to OD 600 =0.6, take 1ml of the bacterial solution and sa...

Embodiment 3

[0031] Example 3, concentration and activity of recombinant caffeic acid oxymethyltransferase COMT3

[0032] Take the supernatant induced by the Pcold-tf-COMT3 plasmid strain for 8 hours and purify it through a nickel column, and then use UPLC to measure the amount of the product of the enzymatic reaction to represent the enzyme activity. The amount is a specific activity, that is, after 1U of the induced bacterial solution was shaken for 8 hours, 5ml of the supernatant was purified and eluted with 10ml of imidazole at a concentration of 100mM. The concentration of transferase is 0.089mg / ml;

[0033] With 5-hydroxytryptamine at a concentration of 0.2μM as a substrate, caffeic acid oxymethyltransferase with an enzyme activity of 0.0831U was reacted for 20min at a temperature of 37°C and a pH of 5.4, and then identified by UPLC-MS / MS. Such as image 3 As shown, the results showed that the total amount of 5-methoxyl-tryptamine generated was 1.66nmol (317ng).

[0034] With N-ac...

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Abstract

The invention discloses COMT3 in Morus notabili and application thereof. The COMT is prepared by coding 374 amino acids with a COMT3 gene having a full length of 1125 bp and a PI value of 4.98. The Morus notabili COMT3 gene is cloned, then a prokaryotic expression system is constructed, enzyme activity determination is carried out after recombinant expression, the activity of the COMT is measuredto be high, and therefore, the Morus notabili COMT gene can be used as a target gene of engineering bacteria, and the Morus notabili COMT3 can also be used for catalyzing conversion of 5-hydroxytryptamine into 5-methoxy-tryptamine or conversion of N-acetyl-5-hydroxytryptamine into melatonin in vitro and has good application prospects.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to caffeic acid oxymethyltransferase COMT3 from mulberry, and also relates to the application of the caffeic acid oxymethyltransferase COMT3 from mulberry. Background technique [0002] Melatonin, scientific name N-acetyl-5-methoxy-tryptamine, is an indoleamine, a metabolite of tryptophan. It is known that it widely exists in human body, animal body and plant body, and its physiological functions are very extensive. Because it was first discovered in the pineal gland of the human body, it is also called pineal gland. Subsequent studies have found that it is widely present in various parts of the human body, and its content is very small, only pg (1×10 -12 g) / mL level. The currently known physiological functions of melatonin mainly include regulating the circadian rhythm of organisms and relieving sleep disorders; anti-oxidation, melatonin itself and its metabolites not only have stro...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12P17/10
CPCC12N9/1007C12N15/70C12P17/10C12Y201/01068
Inventor 赵爱春郑莎朱映雪范伟刘长英张帅向仲怀
Owner SOUTHWEST UNIVERSITY
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