Formate dehydrogenase mutant and application thereof

A technology for formate dehydrogenase and mutants, which is applied in the field of enzyme engineering, can solve the problems of low market competitiveness, limited application, low synthesis activity, etc., and achieves the effects of market competitiveness and production cost reduction.

Active Publication Date: 2022-07-15
深圳希吉亚生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, NMNH is an atypical reducing coenzyme factor. Except for a very small number of flavin reductases, most of the natural oxidoreductases have extremely ...

Method used

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  • Formate dehydrogenase mutant and application thereof
  • Formate dehydrogenase mutant and application thereof
  • Formate dehydrogenase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Preparation of formate dehydrogenase mutants

[0047] The present embodiment provides a formate dehydrogenase, and its preparation method is as follows:

[0048] 1. Preparation of Recombinant Plasmids and Recombinant Bacteria

[0049] The parent formate dehydrogenase used in this example is from Pandoraea commovens, and its amino acid sequence is shown below (GenBank: VVE30574.1):

[0050] MAKIVCVLYDDPVTGYPKTYARDDLPKIECYPDGQTLPTPRAIDFQPGALLGSVSGELGLRKYLESNGHELVVTSSKDGDNSVLDRELADAEIVISQPFWPAYMTAERIKRAKKLKMIVTAGIGSDHTDLQAAMEHGITVAEVTYCNSNSVAEHVMMTTLALVRNYLPSYQWVLKGGWNIADCVERSYDLEGMHVGTVAAGRIGLRVLRLMKPFGTHLHYLDRHRLPESVEKELNLTHHTSLESLAKVCDVVTLNCPLHPETEHMINADSLKHFKRGAYLINTARGKLCDRDAVAAALESGQLAGYGGDVWFPQPAPADHPWRSMPHHGMTPHISGTSLSAQTRYAAGTREILECYFENRPIRNEYLIVQNGKLAGVGAHSYSAGNATGGSEEAARFKKSA(SEQ ID No.1)。

[0051] The nucleotide sequence encoding this formate dehydrogenase is shown below:

[0052]ATGGCCAAGATTGTTTGTGTACTGTACGACGACCCCGTTACCGGCTACCCGAAGACCTACGCCCGCGACG...

Embodiment 2

[0083] Example 2 Enzymatic activity assay

[0084] A reaction solution with a final concentration of 60 mM NMN, 150 mM formic acid, and 100 mM Tris buffer was prepared, and the pH was adjusted to 8.0. Take 4 reaction solutions (900 μl each), add 100 μl of parental PcFDH with the same protein concentration and the supernatant crude enzyme solution of 3 mutant PcFDHs respectively, react at 30°C for 10 min, and then add 100 μl of 25% trichloroacetic acid to stop the reaction, The NMNH content in the reaction solution was measured by HPLC, and the specific activity of each enzyme was calculated, and the enzymatic activity of converting 1 nmol of NMN to NMNH within 1 minute was defined as 1U. The enzymatic activities of mutant PcFDH were compared, and the results are shown in Table 3.

[0085] Table 3 Relative enzyme activity detection results

[0086]

[0087] It can be seen from the above results that the enzyme activity of the single-site mutant provided in the examples of ...

Embodiment 3

[0088] Example 3 Preparation of NMNH

[0089] A substrate solution containing 100 mM NMN, 200 mM formic acid, and 200 mM Tris-HCl buffer was added to the reactor, and the pH was adjusted to 7.0-8.0. Then, the catalytic enzyme was added in an amount of 20ml / L (crude enzyme solution / substrate solution) of the supernatant crude enzyme solution of the mutant V54N / A121K / E228K, and after stirring evenly, the reaction was carried out in a constant temperature water bath shaker. The rotating speed of the shaker was set at 50 rpm, the reaction temperature was controlled at 30 °C, and the pH was maintained at 7.0-8.0. After 4 hours of reaction, the solution containing the crude product was obtained, and the pre-reaction substrate solution and the post-reaction reaction solution were detected by HPLC ( Figure 1-Figure 2 ), filtered, purified and dried to obtain the final product, hydrogen spectrum ( Figure 3-Figure 5 ), carbon spectrum ( Figure 6-Figure 7 ) test confirmed it to be ...

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Abstract

The invention discloses a formate dehydrogenase mutant and application thereof. The formate dehydrogenase mutant comprises mutation of at least one amino acid in the 54th site, the 121st site and the 228th site of an amino acid sequence as shown in SEQ ID No.1. Compared with conventional wild type formate dehydrogenase, the formate dehydrogenase provided by the invention has higher reductase activity on NMN, the enzyme activity of the formate dehydrogenase reaches about 80-886 times that of the wild type, the formate dehydrogenase can efficiently reduce the NMN into NMNH, and the formate dehydrogenase can be used in the form of crude enzyme without purification or can be used only after partial purification due to the high catalytic activity. Therefore, the production cost can be greatly reduced, and the method is suitable for large-scale industrial production and has better market competitiveness.

Description

technical field [0001] The present application relates to the technical field of enzyme engineering, in particular to formate dehydrogenase mutants and their applications. Background technique [0002] Reduced form of Nicotinamide mononucleotide (NMNH) is one of the precursors of Nicotinamide adenine dinucleotide (NAD+). Decreased intracellular NAD+ levels can lead to cellular aging and aging-related diseases, and additional NMN and NR supplementation can maintain NAD+ levels. Current studies have found that NMNH, as a regulator of nicotinamide mononucleotide acyltransferase (NMNAT), can increase NAD+ levels more effectively than NMN and NR, and effectively control the rate of cell metabolism. Therefore, NMNH becomes a potentially important NAD+ enhancer. [0003] However, NMNH is an atypical reducing coenzyme factor. Except for a very small number of flavin reductases, most of the natural oxidoreductases have extremely low catalytic synthesis activity for NMNH, resulting ...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P19/30C12R1/19
CPCC12N9/0008C12N15/70C12P19/30C12Y102/01002
Inventor 李钊
Owner 深圳希吉亚生物技术有限公司
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