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Aspartate ammonia lyase mutant and application thereof

A technology of ammonia aspartate and lyase, applied in the field of enzyme catalysis, can solve the problems of low enzyme activity and low conversion rate, and achieve the effect of improving enzyme activity

Active Publication Date: 2020-02-14
ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] We have studied the synthesis of β-alanine catalyzed by aspartate ammonia lyase AspB derived from Bacillus sp.YM55-1, and found that the enzyme can catalyze the reaction of β-alanine with acrylic acid or acrylonitrile as substrate Amino acid, but the conversion rate is low, which is judged to be caused by the low activity of AspB enzyme, so it is necessary to optimize this technology

Method used

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  • Aspartate ammonia lyase mutant and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] The construction of embodiment 1 initial aspartic acid ammonia lyase gene recombinant Escherichia coli

[0058] 1.1 For the aspartate ammonia lyase derived from Bacillus sp.YM55-1, according to the amino acid sequence published in the literature (RuifengLi.et al, 2018), that is, SEQ ID NO: 1, codon optimization was performed on this basis , the whole gene synthesis gene sequence SEQ ID NO: 2, and restriction endonuclease sites Nde I and XhoI were designed at both ends of the gene, subcloned into the corresponding sites of the vector pET24a (Novagen), and the recombinant plasmid pET24a-AspB1 was obtained, The plasmid map constructed see figure 1 .

[0059] 1.2 The recombinant plasmid pET24a-AspB1 was transformed into the expression host Escherichia coli BL21 (DE3) (Invitrogen Company) by electrotransformation to obtain the recombinant Escherichia coli AspB1 expressing the initial aspartate ammonia lyase.

Embodiment 2

[0060] Example 2 Construction of random mutation point library and screening by error-prone PCR method

[0061] 2.1 Error-prone PCR method to construct random mutation point library

[0062] Using the original enzyme gene SEQ ID NO: 2 as a template, an error-prone PCR technique was used to construct a random mutant library. The forward primer AspB1-F is 5’-ATGAACACCGACGTTCGTATC-3’, the reverse primer AspB1-R is 5’-TTATTTACGACCAGCGATACCCGG-3’

[0063]50μL error-prone PCR reaction system includes: 10ng plasmid template pET24a-AspB1, 50pmol pair of primers AspB1-F and AspB1-R, 1×Taq buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 7mM MgCl 2 , (0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM) MnCl 2 , 2.5 units of Taq enzyme (fermentas company). The PCR reaction conditions were: 95°C for 5 minutes; 94°C for 30s, 55°C for 30s, 72°C for 2min / kbp; 30 cycles; 72°C for 10min. The 1.4kbp random mutation fragment was recovered from the gel as a large primer (Axygen DNA Gel Extraction Kit AP-GX...

Embodiment 3

[0073] Embodiment 3 Fermentation and transformation of mutant strain AspB-11-D4

[0074] 3.1 Shake flask fermentation

[0075] Pick a single colony from the LB culture plate of AspB-11-D4, inoculate it into 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate, and culture overnight at 37°C and 250 rpm. Inoculate 2mL of the overnight culture into 200mL TB medium, culture at 37°C, 250rpm for 2-3h, until OD 600 At 0.6-0.8, add 0.1 mM IPTG, and culture overnight at 28°C and 200 rpm. Then at 4° C., 10000 rpm, centrifuge for 10 min, and collect the bacterial cells.

[0076] 3.2 Fermentation of initial enzyme expression strain

[0077] According to the method in step 3.1, perform shake flask fermentation on the initial enzyme expression strain AspB1, and collect the cells.

[0078] 3.3 Determination of bacterial specific activity

[0079] Add the bacterial cells AspB-11-D4 obtained in the above step 3.1 and the bacterial cells AspB1 obtained in the step 3.2 to 500 μL ...

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Abstract

The amino acid sequence of an aspartate ammonia lyase mutant, an amino acid sequence is SEQ ID NO: 3, and compared with an initial aspartate ammonia lyase AspB, the enzyme activity of catalyzing acrylic acid to generate beta-alanine is remarkably improved. When the whole cell expressing the aspartate ammonia lyase mutant is used for catalyzing a 120g / L acrylic acid substrate to react to generate beta-alanine, the conversion rate exceeds 95%, and the aspartate ammonia lyase mutant has an industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of enzyme catalysis, and in particular relates to an aspartate ammonia lyase mutant and its use for enzymatically catalyzing acrylic acid to produce β-alanine. Background technique [0002] β-alanine has aroma and sweet taste, is easily soluble in water and slightly soluble in ethanol, and is a natural β-type amino acid that exists in nature. β-alanine is a natural non-protein amino acid that does not directly participate in the synthesis of proteins in living organisms, but participates in the synthesis of various functional substances such as carnosine and vitamin B5. It is a functional amino acid and is widely used In the pharmaceutical, food, chemical and other industries. β-alanine is mainly used in the synthesis of pantothenic acid, calcium pantothenate, carnosine, sodium pamidronate, octalazine, etc. It can also be used as a dietary supplement to provide energy for muscles. [0003] There are three ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/06C12R1/19
CPCC12N9/88C12N15/70C12P13/06C12Y403/01001
Inventor 范文超王金刚梁岩高书良
Owner ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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