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High-stereoselectivity methionine adenosyltransferase as well as preparation method and application thereof

A methionine adenosine, stereoselective technology, applied in the field of protein and enzyme engineering, can solve problems such as no reports, and achieve the effects of convenient operation, reduced production cost and good stability

Active Publication Date: 2021-05-07
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report to obtain the purer product (S,S)-SAM by enzymatic method.

Method used

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  • High-stereoselectivity methionine adenosyltransferase as well as preparation method and application thereof
  • High-stereoselectivity methionine adenosyltransferase as well as preparation method and application thereof
  • High-stereoselectivity methionine adenosyltransferase as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: the preparation of a kind of highly stereoselective methionine adenylyltransferase (SxMAT)

[0049] 1. Experimental method

[0050] Specific steps are as follows:

[0051] 1) extracting the genome of Streptomyces xinghai;

[0052] After culturing Streptomyces xinghai in potato medium for 5-8 days, use a genome extraction kit to extract the genome (gDNA) of Streptomyces xinghai, dilute it 10 times and store it at -20°C for later use;

[0053] 2) using the genome in step 1) as a DNA template, using PCR amplification technology to obtain the gene sequence of the methionine adenylyltransferase in Streptomyces xinghai, which is the metK target fragment;

[0054] Design upstream and downstream primers containing NdeI and XhoI restriction sites respectively, use the genome (gDNA) extracted in step 1) as a DNA template, perform PCR amplification according to Table 1 and Table 2, to obtain the purpose of the MAT gene sequence metK fragment.

[0055] Table 1 The...

Embodiment 2

[0072] Embodiment 2: HPLC and LC-MS detect the biological activity of SxMAT

[0073] 1. Experimental materials

[0074] SxMAT, Tris-HCl buffer solution, L-Met, ATP, metal ion K prepared by the method in Example 1 + -Mg 2+ / Co 2+ 、TCA

[0075] 2. Experimental method

[0076] The reaction was carried out in 100 mM Tris-HCl buffer, pH 8.5, in a total volume of 500 μL, which contained SxMAT (6 mg / mL), substrates L-Met (5 mM) and ATP (5 mM), metal ion K + (150mM)-Mg 2+ (20mM) / Co 2+ (10mM), at the same time set up negative control groups containing only ATP, only L-Met, only SxMAT, only ATP and L-Met. TCA was inactivated, centrifuged at 12000 rpm / min for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane for HPLC and LC-MS analysis.

[0077] 3. Experimental results

[0078] by such as image 3 It can be seen that the results show that only the experimental group has product peaks, while the negative control group has no product peaks. Figure 4 It ...

Embodiment 3

[0079] Example 3: Determination of the influence of temperature on the activity of SxMAT and the thermal stability of SxMAT

[0080] 1. Experimental materials

[0081] SxMAT, Tris-HCl buffer solution, L-Met, ATP, metal ion K prepared by the method in Example 1 + -Mg 2+ 、TCA

[0082] 2. Experimental method

[0083] Determination of the optimum reaction temperature of SxMAT: The reaction was carried out in 100mM, pH 8.5 Tris-HCl buffer in a total volume of 500μL, which contained SxMAT (6mg / mL), substrate L-Met (5mM) and ATP ( 5mM), metal ion K + (150mM)-Mg2 + (20mM), each reacted at 130rpm / min in a shaker at 25°C, 35°C, 45°C, 55°C, 65°C, 75°C, and 85°C for 2h, and then added 150μL of 12% TCA was inactivated, centrifuged at 12000rpm / min for 10min, and the supernatant was taken to pass through the membrane for HPLC analysis.

[0084] Determination of SxMAT thermal stability: The reaction was carried out in 100mM Tris-HCl buffer, pH 8.5, with a total volume of 500μL, which con...

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Abstract

The invention discloses high-stereoselectivity methionine adenosyltransferase as well as a preparation method and application thereof. The preparation method comprises the following steps: extracting a genome of streptomyces xinghaiensis; by taking a genome as a DNA (Deoxyribose Nucleic Acid) template and adopting a PCR (Polymerase Chain Reaction) amplification technology, obtaining a gene sequence of methionine adenosyltransferase in the streptomyces xinghaiensis, namely a metK target fragment; carrying out double enzyme digestion on the metK target fragment and an expression vector pET-32a (+), and then connecting to construct a recombinant plasmid; transforming the recombinant plasmid into competent cells of escherichia coli BL21 by adopting a thermal activation method, and carrying out inducible expression by virtue of isopropyl-beta-D-thiogalactoside, so as to obtain SxMAT. According to the invention, high-stereoselectivity methionine adenosyltransferase is subjected to denaturation and renaturation later to obtain high-purity SxMAT, the SxMAT is good in stability when the temperature is 25-55 DEG C and the pH value is 8-10.5, the optical purity (ee) of the product is higher than 90%, the yield is 80%, and the in-vitro double-enzyme cascade reaction is constructed by combining the SxMAT and the fluorinase, so that the understanding of people on a fluorination pathway in streptomyces is facilitated, and the application of fluorinated natural products is derived.

Description

technical field [0001] The invention belongs to the technical field of protein and enzyme engineering, and in particular relates to a highly stereoselective methionine adenosyltransferase and its preparation method and application. Background technique [0002] S-adenosyl-L-methionine, namely S-adenosylmethionine, commonly used abbreviations include SAM, SAM-e and AdoMet. In organisms, S-adenosylmethionine uses L-methionine (L-Met) and adenosine triphosphate (ATP) as substrates, and is also called S-adenosyl- Formed under the catalysis of L-methionine synthase (MAT, EC 2.5.1.6). In this reaction, S-adenosyl-L-methionine and tripolyphosphate are first produced; subsequently, the resulting tripolyphosphate is asymmetrically hydrolyzed to produce pyrophosphate (ppi) and orthophosphate (pi) . S-adenosyl-L-methionine widely exists in animals, plants and microorganisms, participates in more than 40 biochemical reactions in organisms, and is an important physiologically active s...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12P13/12C12P19/40
CPCC12N9/1085C12N15/70C12P13/12C12P19/40C12Y205/01006Y02A50/30
Inventor 马龙任思羽程新宽满淑丽
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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