Amino acid dehydrogenase mutant and application thereof

An amino acid and dehydrogenase technology, applied in application, enzyme, genetic engineering and other directions, can solve the problem that amino acid dehydrogenase is not suitable for industrial production, and achieve the effects of improving enzyme specificity, reducing cost, and improving enzyme activity

Active Publication Date: 2018-12-18
天津凯诺医药科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims to provide an amino acid dehydrogenase mutant and its application to solve the technical problem that the wild-type amino acid dehydrogenase in the prior art is not suitable for industrial production

Method used

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  • Amino acid dehydrogenase mutant and application thereof
  • Amino acid dehydrogenase mutant and application thereof
  • Amino acid dehydrogenase mutant and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment approach

[0029] According to a typical embodiment of the present invention, an amino acid dehydrogenase mutant is provided. The amino acid sequence of the amino acid dehydrogenase mutant is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 1, and the mutation includes at least one of the following mutation sites: 64th, 94th, and 133rd , 137th, 148th, 168th, 173rd, 183rd, 191st, 207th, 229th, 248th, 255th and 282nd, and the 64th Lysine at position 94 is mutated to aspartate; aspartate at position 94 is mutated to alanine, glycine, valine, or serine; cysteine ​​133 is mutated to alanine or threonine acid; phenylalanine at position 137 is mutated to alanine; phenylalanine at position 148 is mutated to valine or alanine; asparagine at position 168 is mutated to aspartic acid; 173 Threonine at position 183 is mutated to serine, histidine, tryptophan, phenylalanine, or leucine; arginine at position 183 is mutated to phenylalanine, lysine, cysteine, v...

Embodiment 1

[0048] Add 20mg of substrate 1, 1mL reaction system, 22mg ammonium chloride, 30mg glucose, 4mg glucose dehydrogenase, NAD + 0.4mg, amino acid dehydrogenase 4mg, 0.1M Tris-HCl buffer. React at 30°C for 18 hours, add 900 μL (0.1N HCl:MeOH=1:1) to 100 μL system, centrifuge at 12000 rpm for 3 min, and take the supernatant for detection. Method for detecting ee value: Take 100 μL reaction system, add 100 μL ACN, 100 μL H 2 O, 100 μL 1M NaHCO 3 , centrifuge at 12000rpm for 3min, take out the supernatant, add 200μL of 5mg / mL Na-(2,4-dinitro-5-fluorophenyl)-L-alanamineamide, incubate at 50℃ for 1h, add 500μL of ACN and centrifuge, take the supernatant for liquid phase analysis.

[0049]

[0050]

[0051] Compared with the multiple of the mother parent, the activity is increased by 1-5 times, ++ by 5-10 times, +++ by 10-50 times, and ++++ by more than 50 times.

[0052] ee value between 80-90%*, ee value between 90-98%**, ee value greater than 98%***

Embodiment 2

[0054] Add 20mg of substrate 2, 1mL reaction system, 22mg ammonium chloride, 30mg glucose, 4mg glucose dehydrogenase, NAD + 0.4mg, amino acid dehydrogenase 4mg, 0.1M Tris-HCl buffer. React at 30°C for 18 hours, add 900 μL (0.1N HCl:MeOH=1:1) to 100 μL system, centrifuge at 12000 rpm for 3 min, and take the supernatant for detection. Method for detecting ee value: Take 100 μL reaction system, add 100 μL ACN, 100 μL H 2 O, 100 μL 1M NaHCO 3 , centrifuge at 12000rpm for 3min, take out the supernatant, add 200μL of 5mg / mL Na-(2,4-dinitro-5-fluorophenyl)-L-alanamineamide, incubate at 50℃ for 1h, add 500μL of ACN and centrifuge, take the supernatant for liquid phase analysis.

[0055]

[0056]

[0057] Compared with the multiple of the mother parent, the activity is increased by 1-5 times, ++ by 5-10 times, +++ by 10-50 times, and ++++ by more than 50 times.

[0058] ee value between 80-90%*, ee value between 90-98%**, ee value greater than 98%***

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Abstract

The invention discloses an amino acid dehydrogenase mutant and an application thereof. The amino acid sequence of the amino acid dehydrogenase mutant is an amino acid sequence obtained by mutating anamino acid sequence represented by SEQ ID NO:1, and the mutation includes at least one of the following mutation sites: the 64thmutation site, the 94thmutation site, the 133rd mutation site, the 137thmutation site, the 148th mutation site, the 168th mutation site, the 173rd mutation site, the 183rd mutation site, the 191st mutation site, the 207th mutation site, the 229th mutation site, the 248thmutation site, the 255th mutation site and the 282nd mutation site; or the amino acid sequence of the amino acid dehydrogenase mutant is an amino acid sequence having the mutation sites in the mutated amino acid sequence and having a 80% or more homology with the mutated amino acid sequence. The amino acid dehydrogenase mutant has greatly improved enzyme activity, the enzyme activity is above 50times higher than that of a wild amino acid dehydrogenase, and the enzyme specificity is also correspondingly improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an amino acid dehydrogenase mutant and its application. Background technique [0002] Amino acids are amphoteric compounds with at least one carboxyl group and one amino group. According to the existence of amino acids, they can be divided into two types: natural amino acids and unnatural amino acids. Natural amino acids are amino acids that exist in nature, and unnatural amino acids are artificially synthesized amino acids. Generally, some groups are introduced into the side chains of natural amino acids to optimize their performance. Amino acids and their derivatives are widely used in agriculture, industry, chemical industry, food, medicine, etc. because of their special structural properties. Optically active unnatural amino acids are the chiral synthetic units of some bioactive peptides and important intermediates of many pharmaceuticals and fine chemicals. [0003] With the d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12P13/04
CPCC12N9/0014C12P13/04
Inventor 洪浩詹姆斯·盖吉卢江平张娜焦学成李瑞张克俭张瑜杨益明
Owner 天津凯诺医药科技发展有限公司
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