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Enzyme with function of acyltransferase and application thereof

An acyltransferase and acyl donor technology, which is applied in the fields of genetic engineering and biopharmaceuticals, can solve the problems of unreported research on modification steps, and achieve the effects of improving bioavailability, enhancing drug efficacy, and improving water solubility.

Active Publication Date: 2020-05-22
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] As a class of drug lead compounds with great application potential, Macrolactins have attracted great attention. Macrolactins belong to polyketide compounds, and its skeleton structure Macrolactin A is catalyzed by polyketide synthases (Polyketide synthases, PKSs). However, the subsequent modification steps have not been reported

Method used

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  • Enzyme with function of acyltransferase and application thereof
  • Enzyme with function of acyltransferase and application thereof
  • Enzyme with function of acyltransferase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Cloning of embodiment 1 acyltransferase gene

[0039] 1. Genomic DNA extraction

[0040] Inoculate marine Bacillus B-9987 in 10mL LB liquid medium, culture overnight at 37°C, collect the bacteria by centrifugation, discard the supernatant; wash twice with 1mL of STE buffer; add 500μL of 3-5mg prepared with STE buffer / mL lysozyme solution, carefully and fully suspend the bacteria, in a 37°C water bath for 30 minutes, until the cells become translucent; add 250 μL of 3% SDS, mix gently up and down, continue to 37°C water bath until clear; add 1 / 10 volume After adding 200 μL of phenol:chloroform:isoamyl alcohol (25:24:1) to 3M NaAc (pH=4.8), repeatedly invert the centrifuge tube several times, centrifuge at 12,000rpm for 10min; carefully pipette the supernatant, repeat Extract with phenol: chloroform: isoamyl alcohol until the middle layer is free of protein impurities, transfer the supernatant, add an equal volume of isopropanol (or 2 times the volume of absolute ethano...

Embodiment 2

[0049] Embodiment 2: In vitro enzyme activity detection of BmmI protein

[0050] In vitro enzyme activity reaction system: (100μL)

[0051] 1M Tris-HCl buffer (pH8.5): 1.5μL

[0052] 0.5M MgCl 2 :0.12μL

[0053] 500μM Macrolactin A: 3μL

[0054] 10mM Succinyl-CoA: 1.5μL

[0055] 37.54mM BmmI protein: 0.8μL

[0056] wxya 2 O: 23.08μL

[0057] Reaction conditions: 30°C, 2 hours. After the reaction, add 30 μL of acetonitrile to terminate the reaction, centrifuge at 13,000 rpm for 20 minutes, discard the precipitate, and remove the protein in the reaction solution. The resulting supernatant was subjected to HPLC detection.

[0058] HPLC detection: use reverse phase C18column (specification: 150×4.6mm, 5μm); column temperature is 30°C; elution condition: 0-5min equilibrium: 55% A phase (ddH 2 O+0.1% formic acid) and 45% B phase (acetonitrile+0.1% formic acid); 5-15min linear elution: 55-30% A phase and 45-70% B phase; 15-25min isocratic elution: 0% A phase and 100% B phas...

Embodiment 3

[0061] Embodiment 3: BmmI acyltransferase to the transacyl effect of macrolide compound

[0062] When using Acetyl-CoA as the acyl donor, when replacing Succinyl-CoA in the enzyme reaction system in Example 2 with Acetyl-CoA, incubate at 30°C for 2h, and perform HPLC detection and HRMS analysis after the reaction is terminated ( Figure 4 B, Figure 5 , 6B), from the MS spectrum, the product [M+NH 4 ] + is 462.29, [M+Na] +It is 467.24, and the molecular weight is consistent with that of the expected target product. It was confirmed that acetylated Macrolactin A was also obtained. Thus it is proved that the acyltransferase of the present invention has a good application prospect.

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Abstract

The invention provides an enzyme with an acyl transfer function and application thereof. Acylation modification can be performed on macrolides and chloramphenicol. The acyltransferase has the amino acid sequence of SEQ ID NO: 1. According to the application of the acyltransferase, acyl groups of acyl donors are combined to the macrolides and the chloramphenicol. The enzyme with the acyl transfer function can improve the pharmacological activity of the macrolides through the acylation reaction of the macrolides, thereby improving the bioavailability, enhancing the efficacy, reducing the side effects, and providing a new strategy for the drug development of the macrolides.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and biopharmaceuticals, and specifically relates to an enzyme with acyl transfer function and its application. Background technique [0002] The acylation modification of drugs can usually change the physical and chemical properties and improve the biological efficacy. In drug synthesis, acylation reaction is often used to protect hydroxyl and amine groups; and acyl groups can be converted into other groups through oxidation, reduction, addition, rearrangement and other reactions. Therefore, acyltransferase as a tool enzyme has become a research hotspot. [0003] Macrolactins are a series of 24-membered macrolide compounds with biological and pharmacological activities such as antibacterial, antiviral, antitumor, antiinfective and antiaging. At the earliest, the Fenical research group isolated Macrolactins A-F from a strain of deep-sea bacteria in 1989. Studies have shown that Macro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P17/08C12P13/02C12R1/19
CPCC12N9/1025C12N15/70C12P17/08C12P13/02C12P19/58
Inventor 李文利肖菲刘扬
Owner OCEAN UNIV OF CHINA