N-acetyl-5-hydroxytryptamine O-methyltransferase (ASMT12) in Morus notabili and application thereof

A technology of serotonin oxygen methyl and transferase, applied in the direction of transferase, application, recombinant DNA technology, etc., can solve the problems of not being able 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 extremely low and cannot be extracted in large quantities

Method used

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  • N-acetyl-5-hydroxytryptamine O-methyltransferase (ASMT12) in Morus notabili and application thereof
  • N-acetyl-5-hydroxytryptamine O-methyltransferase (ASMT12) in Morus notabili and application thereof
  • N-acetyl-5-hydroxytryptamine O-methyltransferase (ASMT12) in Morus notabili and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1, N-acetyl-5-hydroxytryptamine oxygen methyltransferase gene ASMT12 clone

[0026] According to the Morus.ASMT12 N-acetyl-5-hydroxytryptamine oxygen-methyltransferase gene (Morus015040) reported on the Morus.ASMT12 database, the primers for cloning the N-acetyl-5-hydroxytryptamine oxygen-methyltransferase gene ASMT12 were designed, and the upstream primers of ASMT12 were :5'-cg ggatcc atgctgtccttcataagttccatgt-3' (SEQ ID NO.1); the downstream primer of ASMT12 is: 5'-acgc gtcgac tcatttaagcaattcataaac-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. The amplified products were subjected to agarose gel electrophoresis, and the results were as follows: figure 1shown. The full-length ASMT12 gene was amplified, the recovered product was ligated with the pMD19-T vector, and transformed into E.coli.Trans1-T1 competent cells, and the obtained positiv...

Embodiment 2

[0027] Example 2. Construction and prokaryotic expression of the ASMT12 recombinant vector of N-acetyl-5-hydroxytryptamine oxygen methyltransferase gene ASMT12 in Chuanmulus. The target fragment of the ASMT12 gene cloned in Example 1 and the Pcold-tf empty plasmid were double-digested with BamHI and SalI, respectively. , recovering the product with T 4 Connect it with DNA ligase to obtain the recombinant vector Pcold-tf-ASMT12, transform the obtained recombinant vector Pcold-tf-ASMT12 into Escherichia coli DH5ɑ competent cells, and send the correct identified Pcold-tf-ASMT12 to Huada Gene Company Sequencing, the sequencing results were consistent with the sequence obtained by the first sequencing so that the correct sequence was obtained.

[0028] Extract the Pcold-tf-ASMT12 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, 220r...

Embodiment 3

[0030] Example 3, concentration and activity detection of recombinant N-acetyl-5-hydroxytryptamine oxygen methyltransferase ASMT12

[0031] Take the supernatant induced by the strain containing Pcold-tf-ASMT12 plasmid 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 indicate the enzyme activity. The unit of enzyme activity is defined as the substance that generates 1 nmol per minute The amount is a specific activity, that is, after shaking 1U of the induced bacterial solution for 8 hours, 5ml of the supernatant is purified, and then eluted with 10ml of imidazole at a concentration of 100mM. After the liquid is eluted, the N-acetyl- The concentration of 5-hydroxytryptamine oxygen methyltransferase is: the concentration of N-acetyl-5-hydroxytryptamine oxygen methyltransferase ASMT12 is 0.064mg / ml.

[0032] Using 5-hydroxytryptamine at a concentration of 0.05 μM as a substrate, reacted with N-ac...

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Abstract

The invention discloses ASMT12 in Morus notabili and application thereof. The ASMT12 in Morus notabili is obtained by coding 370 amino acids with an ASMT12 gene having a full length of 1110 bp and a PI value of 6.57. The Morus notabili ASMT12 gene is cloned, then a prokaryotic expression system is constructed, enzyme activity determination is carried out after recombinant expression, the activityof the N-acetyltryptamine O-methyltransferase is measured to be high, and therefore, the Morus notabili ASMT12 gene can be used as a target gene of engineering bacteria, can also be used for catalyzing conversion of N-acetyl-5-hydroxytryptamine into melatonin or catalyzing conversion of 5-hydroxytryptamine into 5-methoxy-tryptamine in vitro, and has good application prospects.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the Chuanmulus N-acetyl-5-serotonin transferase SNAT5, and also relates to the application of the Chuanmulus N-acetyl-5-serotonin transferase SNAT5. 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 f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12P17/10
CPCC12N9/1007C12N15/70C12P17/10C12Y201/01004
Inventor 赵爱春郑莎朱映雪刘长英张帅向仲怀
Owner SOUTHWEST UNIVERSITY
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