L-aspartic acid-alpha-decarboxylase mutant and application thereof

An aspartic acid and mutant technology, applied in the field of genetic engineering, can solve the problems of complicated side reactions, polluted environment, influence of later purification, etc., and achieve the effect of high enzyme activity and catalytic stability

Active Publication Date: 2019-05-10
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the chemical method has high reaction efficiency and fast production rate, it requires strong acid and strong alkali as well as high temperatu...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Construction of L-aspartic acid-α-decarboxylase mutant library by random mutation

[0023] The error-prone PCR kit (the kit was purchased from Beijing Tianenze Gene Technology Co., Ltd.) was used in vitro to express the L-aspartic acid-α-decarboxylase gene PanD (the nucleotide sequence is shown in SEQ ID NO.2, The amino acid sequence is shown in SEQ ID NO.1) to introduce nucleotide mutations.

[0024] Error-prone PCR reaction conditions and primers (30μL system):

[0025] 1 μL of L-aspartic acid-α-decarboxylase gene (SEQ ID NO.2) DNA template with a concentration of 1ng / μL, 1 μL of primer 1 and primer 2 with a concentration of 10 μM, 10× error-prone PCR Mix 3 μL, 10× 3 μL of dNTP for error-prone PCR and MnCl for error-prone PCR 2 4 μL, 2 μL of dGTP for error-prone PCR, 0.5 μL of 5 U / μL Taq DNA polymerase for error-prone PCR, and 14.5 μL of ultrapure water.

[0026] Primer 1: TAAGAAGGAGATATA CCATGG GCATGCCCGCTACCG

[0027] Primer 2: GTGGTGGTGGTGGTG CTC...

Embodiment 2

[0031] Example 2: Mutant library screening

[0032] The transformant that embodiment 1 obtains is carried out as follows:

[0033] 1. Establishment of mutation library in 96-well plate: use sterilized pipette tips to select 3000 transformants on the plate and inoculate them into 96 deep-well plate with 500 μL of LB medium containing 100 μg / mL kanamycin, each well corresponds to a transformant. At the same time, EcoliBL21(DE3) / pET28a and EcoliBL21(DE3) / pET28a-PanD were used as controls and inoculated in 96 deep-well plates. Culture at 37°C with shaking at 200r / min for 24h.

[0034] 2. Induced expression of the library: under sterile conditions, take 50 μL of the seed solution from the 96 deep-well plate in step 1, and transfer it to 500 μL containing 100 μg / mL kanamycin + 0.2 mM isopropylthiogalacto Glycosides were expressed in another 96-deep-well plate in LB medium at 30°C and shaken at 200r / min for 24h. Centrifuge at 4000r / min for 10min, discard the supernatant, and collec...

Embodiment 3

[0043] Example 3: Mutant enzyme PanD R98H-K305E 、PanD R98H-K305E-I451V Efficient expression

[0044] The mutant strain Ecoli BL21(DE3) / pET28a-PanD that embodiment 2 obtains respectively R98H-K305E , EcoliBL21(DE3) / pET28a-PanD R98H-K305E-I451V Perform the following operations with the control strain Ecoli BL21(DE3) / pET28a-PanD without mutated enzyme:

[0045] 1. The mutant strain Ecoli BL21(DE3) / pET28a-PanD R98H-K305E , Ecoli BL21(DE3) / pET28a-PanD R98H-K305E , Ecoli BL21(DE3) / pET28a-PanD R98H-K305E-I451V Inoculate them with the unmutated strain EcoliBL21(DE3) / pET28a-PanD in 50ml of LB medium containing 100μg / mL kanamycin, culture at 37°C and shake at 200r / min for 12h.

[0046] 2. Take the seed solution obtained in step 1, transfer it to 50ml LB medium containing 100μg / mL kanamycin with 2% inoculum size (v / v), and cultivate it to OD at 37°C and 200r / min 600 At 0.6-0.8, a final concentration of 0.2 mM isopropylthiogalactopyranoside was added, and then expression was induce...

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PUM

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Abstract

The invention discloses an L-aspartic acid-alpha-decarboxylase mutant and application to converting L-aspartic acid to produce beta-alanine. The mutant is obtained in a manner that the 98th site, the305th site, the 451th site, the 111th site, the 139th site, the 403th site, the 469th site, the 372th site and the 325th site of the amino acid sequence shown in SEQ ID NO.1 are subjected to single mutation or multi-mutation. According to the L-aspartic acid-alpha-decarboxylase mutant, the catalytic capacities of mutant enzymes PanDR98H-K305E and PanDR98H-K305E-I451V and non-mutated enzymes underthe partial neutrality condition are compared, the result discovers that under the condition of pH7.0, the conversion rates obtained when the mutant enzymes PanDR98H-K305E and PanDR98H-K305E-I451V catalyze the L-aspartic acid to generate the beta-alanine are 3.33 times and 2.76 times that of the non-mutated enzymes respectively.

Description

(1) Technical field [0001] The invention relates to an L-aspartic acid-α-decarboxylase mutant obtained by machine mutation and application thereof, belonging to the technical field of genetic engineering. (2) Background technology [0002] β-alanine is the only β-type non-protein amino acid naturally occurring in nature, also known as β-alanine or 3-alanine. β-amino acid is a potential functional amino acid. In industry, β-amino acid, as a very important precursor substance, is widely used in medicine, health products, chemical products, food and feed, etc. In medicine, β-alanine is widely used in the synthesis of pantothenic acid and calcium pantothenate, and is also the main precursor for the synthesis of carnosine. β-alanine can also be used to synthesize pamidronate disodium, which can be used to relieve tumor bone pain and treat hypercalcemia, and to synthesize balsalazide to treat colitis and proctitis. In terms of chemical products, in terms of food and feed, β-ala...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/06C12R1/19
Inventor 于欣君陈虹汪钊
Owner ZHEJIANG UNIV OF TECH
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