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High-temperature-resistant transaldolase and preparation method thereof

A transaldolase, high-temperature-resistant technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, transferase, etc., can solve problems such as low activity and achieve good thermal stability.

Inactive Publication Date: 2011-04-13
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But only in 2004, a thermophilic transaldolase was successfully cloned and expressed from the thermophilic bacterium Methanocaldococcus jannaschii. However, although its heat resistance is remarkable, its activity is low

Method used

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  • High-temperature-resistant transaldolase and preparation method thereof
  • High-temperature-resistant transaldolase and preparation method thereof
  • High-temperature-resistant transaldolase and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: the preparation of thermophilic high temperature resistant transaldolase

[0038] The preparation method of thermophilic and high temperature resistant transaldolase comprises strain, plasmid, enzyme and culture medium, PCR amplification and construction of recombinant plasmid, induced expression of gene and purification of protein. Specific steps are as follows:

[0039]The first step is to amplify the DNA fragment according to the upstream and downstream primers and use the whole genome sequence of Thermotoga maritima as a template. After the fragment is recovered, it is double-digested with NdeI and SalI endonucleases, and then connected to the same endonuclease The enzyme-digested expression plasmid vector pET28a was transformed into Escherichia coli E.coli DH5α, the recombinant plasmid was screened, and double-digested and sequenced for verification.

[0040] The upstream and downstream primers are respectively:

[0041] Upstream 5'-GGAATTC CATATG ...

Embodiment 2

[0048] Example 2: Basic enzymatic properties of transaldolase

[0049] The enzyme reaction equation is:

[0050] Erythrose-4-phosphate + Fructose-6-phosphate → Sedoheptulose-7-phosphate + Glyceraldehyde-3-phosphate

[0051] Enzyme reaction system: 500μl, 50mM phosphate buffer (pH 10) reaction system containing 0.6mM fructose-6-phosphate, 0.5mM erythrose-4-phosphate, 10μl purified enzyme, reaction temperature 80℃, reaction time 1min .

[0052] Through the analysis and identification of transaldolase, the main enzymatic properties of the enzyme are as follows:

[0053] (1) The purified transaldolase protein electrophoresis pattern that obtains is as follows figure 1 shown.

[0054] (2) The optimal temperature of the enzyme is 80°C (such as figure 2 shown)

[0055] (3) The enzyme has a wider optimum pH, and has a high enzyme activity in the range of pH 6-10, and the optimum enzyme activity pH is 10.0 (such as image 3 shown)

[0056] (4) Under the experimental condition...

Embodiment 3

[0057] Embodiment 3: the thermostability analysis of transaldolase

[0058] Thermal stability analysis method: place the prepared transaldolase solution (1mg / ml) in water baths at 60°C and 80°C respectively, and measure it at 0, 0.5, 1, The remaining enzyme activity at 2, 4, 8, 12, 24, and 48 hours. The method for measuring enzyme activity is the same as in Example 3.

[0059] Through the thermostability analysis identification of this transaldolase, obtain following result:

[0060] The enzyme has high thermal stability, and the enzyme activity is 80% and 10% of the initial enzyme activity after being placed at 60°C and 80°C for 48 hours. At this point the activity is still as high as 33.53U / mg and 3.94U / mg.

[0061] Among them, 1 U / mg of enzyme activity represents 1 μmol of substrate that can be converted per minute per mg of protein.

[0062] In 2004, a thermophilic transaldolase was successfully cloned and expressed from the thermophilic bacterium Methanocaldococcus ja...

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Abstract

The invention relates to high-temperature-resistant transaldolase and a preparation method thereof in the technical field of biological genetic engineering. A nucleotide sequence of zymoprotein of the high-temperature-resistant transaldolase is shown in SEQ ID No.1. An amino acid sequence of the high-temperature-resistant transaldolase is shown in SEQ ID No.2. The preparation method comprises the following steps of: amplifying the transaldolase by taking a whole genome sequence of thermotoga maritima as a template according to forward and reverse primers to obtain a deoxyribose nucleic acid (DNA) fragment; carrying out double digestion by using NedI and SalI endonuclease after the fragment is recovered; connecting the fragment subjected to endonuclease digestion to an expression plasmid vector pET28a which is also subjected to endonuclease digestion by using endonuclease; transforming Escherichia coli E.coliDH5alpha; screening recombinant plasmids and transforming E.coliBL21; cultivating the transformed E.coliBL21 until the outer diameter (OD) is between 0.5 and 0.8; adding isopropyl-beta-D-thiogalactopyranoside and continuously cultivating to obtain thalli; then adding tri(hydroxymethyl) aminomethane hydrochloride (Tris-HCl) buffer solution and sufficiently and uniformly stirring; carrying out ultrasonication treatment; centrifuging disintegrated solution; and carrying out nickel column affinity chromatography on supernate containing a soluble transaldolase protein which contains induction expression to obtain the purified transaldolase protein.

Description

technical field [0001] The invention relates to an enzyme in the technical field of biological genetic engineering and a preparation method thereof, in particular to a high-temperature resistant transaldolase and a preparation method thereof. Background technique [0002] Since the first thermophilic bacterium, Thermus aquaticus, was isolated from Yellowstone National Park in the United States in 1969, more than 140 species of thermophilic bacteria of more than 70 genera have been discovered. After the first thermophilic enzyme, Taq DNA polymerase, was successfully used in DNA polymerase chain reaction (PCR) in 1985, the application of thermophilic bacteria has attracted the attention of scientists all over the world. [0003] However, the growth conditions of most thermophilic bacteria are relatively harsh, requiring strict anaerobic or extreme high temperature, etc., and the growth rate is also slow, so it is difficult to isolate a large number of thermophilic enzymes by c...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70
Inventor 钟建江黄松燕张以恒
Owner SHANGHAI JIAO TONG UNIV
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