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Tryptophan decarboxylase (TDC) in Morus notabili and application thereof

A technology of mulberry tryptophan decarboxylase and tryptophan decarboxylase, which is applied in the field of bioengineering, can solve the problems of low content and inability to extract large quantities, and achieve good application prospects

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

AI Technical Summary

Problems solved by technology

[0004] Chuan Mulberry contains melatonin, but its content is extremely low and cannot be extracted in large quantities

Method used

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  • Tryptophan decarboxylase (TDC) in Morus notabili and application thereof
  • Tryptophan decarboxylase (TDC) in Morus notabili and application thereof
  • Tryptophan decarboxylase (TDC) in Morus notabili and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, Chuan Mulberry tryptophan decarboxylase gene TDC cloning

[0028] According to the Morus.TDC tryptophan decarboxylase gene (Morus002214) reported on the Morus.TDC database, design primers for amplification, and the TDC upstream primer is: 5'-gg ggtacc atgggtagccttggtttt-3' (SEQ ID NO.1), TDC downstream primer: 5'-acgc gtcgac ttatacacttctgagcac-3' (SEQ ID NO.2), Morus RNA was extracted and reverse transcribed into cDNA. Then with the synthesized cDNA as a template, the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are used as primers for PCR amplification, and the amplified products are detected by electrophoresis, and the results are as follows: figure 1 shown. The target fragment was recovered, and the recovered product was ligated with the pMD19-T vector, and transformed into E.coli.Trans1-T1 competent cells. The obtained positive clones were sent to Huada Gene Company for sequencing. The results showed that the nucleotides encoded by the full-lengt...

Embodiment 2

[0029] Example 2, construction and prokaryotic expression of Mulberry tryptophan decarboxylase gene TDC recombinant vector

[0030] The PCR product amplified in Example 1 was double-digested with KpnI and SalI, and the Pcold-tf plasmid was double-digested with KpnI and SalI at the same time, the TDC gene target vector and the Pcold-tf plasmid expression cassette were recovered respectively, and the recovered product was recovered with T 4 The recombinant expression vector Pcold-tf-TDC was obtained by DNA ligase, and the obtained recombinant expression vector was sent to Huada Gene Company for sequencing. The sequencing result was consistent with the sequence obtained by the first sequencing, thus obtaining the correct sequence.

[0031] Extract the Pcold-tf-TDC plasmid, transfer it into the expression strain B21(DE3), add 450μl of LB medium containing Amp resistance to a 1.5ml centrifuge tube, expand the culture into the test tube at a ratio of 1:100, 28°C, 220rpm Shaker cultu...

Embodiment 3

[0033] Embodiment 3, recombinant tryptophan decarboxylase TDC concentration and activity detection

[0034] Take the supernatant induced by the strain containing Pcold-tf-TDC plasmid for 8 hours and purify it through a nickel column, and then use UPLC-MS / MS to measure the amount of the product of the enzymatic reaction to indicate the enzyme activity, and the enzyme activity unit is defined as per minute The amount of substance that generates 1nmol is a specific activity, that is, after shaking 1U of the induced bacterial solution for 8 hours, 5ml of the supernatant is purified and eluted with 10ml of imidazole at a concentration of 100mM. The concentration of tryptophan decarboxylase is: 0.159mg / ml.

[0035] Use the concentration of 2.0μM L-tryptophan as the substrate, use tryptophan decarboxylase with the enzyme activity of 0.096U to catalyze the reaction at a temperature of 28°C and a pH of 6.5 for 20min, and then use UPLC-MS / MS to identify the results. Such as image 3 A...

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Abstract

The invention discloses a TDC in Morus notabili and application thereof. The amino acid sequence of the TDC in Morus notabili is as shown in SEQ ID NO. 4; after a Morus notabili TDC gene is cloned, aprokaryotic expression system is constructed, then enzymatic determination is conducted, and high TDC activity is determined; and thus, the Morus notabili TDC can be used as a target gene of engineering bacteria. The Morus notabili TDC can also be used as a catalyst for catalyzing conversion of L-tryptophan into tryptamine in vitro, so the Morus notabil TDC has good application prospects.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to the mulberry tryptophan decarboxylase TDC, and also relates to the application of the mulberry tryptophan decarboxylase TDC. 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 strong free radical scavenging ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12P17/10
CPCC12N9/88C12N15/70C12P17/10C12Y401/01028
Inventor 赵爱春郑莎朱映雪曹博宁刘长英张帅向仲怀
Owner SOUTHWEST UNIV
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