Diaphorase mutant with high thermal stability, diaphorase mutant gene with high thermal stability and preparation method of diaphorase mutant

A technology with high thermal stability and diaphorase, applied in the field of bioengineering, can solve the problems of high price, low output, inconvenient large-scale production, etc., achieve the effect of wide application range of pH and reduce production cost

Active Publication Date: 2020-09-15
北京达成生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the extraction of diaphorase from the myocardium of animals is not only complicated to operate, but also has low yield, which is not convenient for large-scale production.
At present, diaphorase in my country mainly relies on imports, which is expensive, and the existing diaphorase has poor thermal stability, and it is not conducive to accurately determine the content of β-hydroxybutyrate when used in a β-hydroxybutyrate assay kit.

Method used

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  • Diaphorase mutant with high thermal stability, diaphorase mutant gene with high thermal stability and preparation method of diaphorase mutant
  • Diaphorase mutant with high thermal stability, diaphorase mutant gene with high thermal stability and preparation method of diaphorase mutant
  • Diaphorase mutant with high thermal stability, diaphorase mutant gene with high thermal stability and preparation method of diaphorase mutant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1 has the design of the diaphorase mutant gene of high thermostability

[0062] Based on the amino acid sequence of the diaphorase mutant with high thermostability of the present invention, the sequence is optimized according to the codon preference of Escherichia coli to obtain a mutant gene of diaphorase with high thermostability, which has high thermostability The amino acid sequence of the positive diaphorase mutant is shown in SEQ ID NO.1, and the nucleotide sequence of the optimized diaphorase mutant gene is shown in SEQ ID NO.2.

[0063] The sequence structure of said SEQ ID NO.1 is as follows:

[0064] MAKVLYITAHPHDDTQSYSMAVGKAFIETYKQVHPDHEVIHLDLYKEYIPEIDDVDVFSGWGKLRSGQSFEQLSAEEKTKVGRMNELCDQFISADKYVFVTPMWNFSFPPVLKAYIDAVAVADKTFKYTAQGPIGLLTDKKALHIQARGGFYSEGPAAQMEMGHELGRILEIIMFAARQFFGKVPSFEEETF.

[0065] The sequence structure of said SEQ ID NO.2 is as follows:

[0066] Atggctaaagttctgtacatcaccgctcacccgcacgacgacacccagtcttactctatggctgttggtaaagctttcatcga...

Embodiment 2

[0067] Embodiment 2 has the preparation of the diaphorase mutant of high thermostability

[0068] The preparation method of the diaphorase mutant with high thermostability described in this embodiment comprises the following steps:

[0069] S1. Construction of high thermostability diaphorase mutant genetically engineered strains:

[0070] S101. Carrying out the whole gene synthesis of the diaphorase mutant gene according to the sequence of SEQ ID NO: 2, and amplifying the diaphorase mutant gene through the upstream primer and the downstream primer using the diaphorase mutant gene as a template, wherein, The upstream primer has a sequence as described in SEQ ID NO: 4, and the downstream primer has a sequence as described in SEQ ID NO: 5,

[0071] The sequence structure of said SEQ ID NO: 4 is as follows:

[0072] 5'-GGGAATTCCATATGATGGCTAAACTGCTG-3'.

[0073] The sequence structure of said SEQ ID NO: 5 is as follows:

[0074] 5'-CCGCTCGAGTTAGAAGTTTTTAGC-3'.

[0075] S102. A...

Embodiment 3

[0084] The enzymatic property determination of embodiment 3 high thermostability diaphorase mutants

[0085] Method for assaying activity of diaphorase mutants:

[0086] Step 1. Reaction mixture configuration: 0.50ml of 0.2M KH 2 PO 4 -NaOH solution (pH 7.5), 0.10ml of 0.25% (W / V) NTB (nitro blue tetrazolium) solution, 0.10ml of 1% (W / V) BSA solution, 0.10ml of 10mM NADH solution, 0.20ml distilled water. The reaction mixture should be prepared immediately and placed on ice.

[0087] Step 2. Determination of enzyme activity: Take 1ml of the above reaction mixture, put it in a 1.2ml cuvette, put it in a spectrophotometer at 37°C for 3min, add 100μL of diluted enzyme, react at 37°C for 7 minutes, and continuously measure the absorbance at 550nm . Record the absorbance change ΔA within 1 min.

[0088] Definition of diaphorase mutant enzyme activity: 1 enzyme activity unit is equal to the amount of enzyme required to oxidize 1 μM NADH in 1 min under the above reaction conditi...

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Abstract

The invention provides a diaphorase mutant with high thermal stability, a diaphorase mutant gene with high thermal stability and a preparation method of the diaphorase mutant. The diaphorase mutant isobtained through mutation of the 122nd position amino acid in the amino acid sequence of wild-type diaphorase from glycine to aspartic acid, and the amino acid sequence of the diaphorase mutant is shown in SEQ ID NO: 1. The enzymatic properties of the diaphorase mutant are the same as the enzymatic properties of the wild-type diaphorase, the thermal stability of the diaphorase mutant is significantly higher than that of the wild-type diaphorase, and the stable range of the pH value is wide; an engineering means is adopted by the preparation method of the diaphorase mutant, genes of the diaphorase mutant are cloned into an expression vector, prokaryotic expression is achieved through transformation of host cells, and a large number of highly stable diaphorase mutants can be obtained; and alot of raw materials are provided for development of clinical diagnostic reagents, and the production cost is reduced.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a diaphorase mutant with high thermal stability, a gene and a preparation method thereof. Background technique [0002] Diaphorases (DIAphorases), referred to as DIA, also known as diaphorase, lipoamide dehydrogenase. Diaphorase plays an important role in the electron transfer system in the body. It can catalyze the dehydrogenation of dihydrolipoamide or dihydrolipoic acid to become lipoamide and lipoic acid respectively. It can also catalyze the reduction of artificial electron acceptors. Such as certain dyes, ferricyanate, quinone, etc. The two hydrogens removed can reduce FAD (flavin adenine dinucleotide) to FADH 2 , and then passed to NAD+ (nicotinamide adenine dinucleotide+) to form NADH and H + . In addition, the application of diaphorase in vitro has also been extensively studied, such as in the production of useful materials, energy-related materials, environm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12Q1/32C12R1/19
CPCC12N9/0051C12N15/70C12N2800/22C12Q1/32C12Y108/01004G01N2333/90212
Inventor 岳硕豪郑长龙
Owner 北京达成生物科技有限公司
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