Method for synthesizing L-alanine by catalyzing maleic acid through double-enzyme coupled whole cells

A technology of maleic acid and alanine, applied in the field of bioengineering, can solve the problems of long time consumption, high cost, complex fermentation process, etc., and achieve the effect of simple production method, high production efficiency and high output

Pending Publication Date: 2021-06-11
JIANGNAN UNIV
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  • Application Information

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Problems solved by technology

[0005] However, in the prior art, the conversion of whole cells to synthesize L-alanine has technical problems of high cost and complicated process. For example, it is recorded in the Chinese invention patent application text with the publication number CN109456928A that by constructing recombinant Escherichia coli, a high The enzyme-active L-aspartic acid β-decarboxylase strain, the recombinant strain is fermented in a shake flask, and L-Asp-Na is used as a substrate to carry out a whole-cell catalytic reaction to prepare L-alanine
Although the yield of the final product L-alanine of this method reaches more than 94%, what it adopts is aspartic acid as a substrate, and the price of aspartic acid is relatively expensive, which is not conducive to large-scale industrial production
Although the fermentation method is low in cost, the process operation is complex and time-consuming. For example, the publication number is CN109355242A. It is recorded in the Chinese invention patent application text that the glucose fermentation method is used to prepare L-alanine, and the yield reaches 171.0g after 55 hours of fermentation. / L, the fermentation process requires two stages of fermentation, and the fermentation process is relatively complicated

Method used

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  • Method for synthesizing L-alanine by catalyzing maleic acid through double-enzyme coupled whole cells
  • Method for synthesizing L-alanine by catalyzing maleic acid through double-enzyme coupled whole cells
  • Method for synthesizing L-alanine by catalyzing maleic acid through double-enzyme coupled whole cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Selection of L-aspartate β decarboxylase

[0047] Adjust the L-aspartate β decarboxylase to L-aspartate β decarboxylase from other 3 sources, respectively: ArASD (hereinafter referred to as ASD-1) of GenBank:AP019740.1, nucleic acid sequence such as SEQ ID NO .4 encoding L-aspartate β decarboxylase Pd21192ASD (hereinafter referred to as ASD-2), GenBank: WP_016451742.1 pET28a-Pd19121ASD (hereinafter referred to as ASD-3);

[0048] The recombinant strains E.coli BL21(DE3) / pET28a-ASD-1, E.coli BL21(DE3) / pET28a-ASD-2 and E.coli BL21(DE3) / pET28a-ASD-3 were synthesized by Jinweizhi Company and stored in In the preservation tube, the prepared single colony was inoculated in 5 mL of LB liquid medium with an antibiotic concentration of 50 mg / mL, cultivated at 37 ° C and 200 rpm for 8 h, and prepared a seed liquid; then inoculated with 2% (v / v) Transfer the seed solution to a 250mL shake flask containing 50mL of 2YT medium, the concentration of antibiotic kana is 50mg...

Embodiment 2

[0052] Example 2: Construction of a recombinant vector coupled to express maleate cis-trans isomerase and L-aspartate β decarboxylase

[0053] Specific steps are as follows:

[0054] (1) According to the gene encoding maleate cis-trans isomerase shown in SEQ ID NO.2 and the gene encoding L-aspartic acid β decarboxylase shown in SEQ ID NO.4 according to the nucleic acid sequence and carrier, select the enzyme cutting site, and design primers, as shown in Table 1:

[0055] Table 1: Design of Enzyme Ligation Primers

[0056]

[0057] Note: the underlined mark is the restriction site

[0058] (2) chemically synthesized nucleic acid sequence as shown in SEQ ID NO.2 encoding maleate cis-trans isomerase gene MaiA and nucleic acid sequence as shown in SEQ ID NO.4 encoding L-aspartic acid β decarboxylase The gene ASD, using NdeⅠ, XhoⅠ, EcoRI, NotⅠ, BamHI, HindⅢ enzymes to the enzymes of the above genes and the plasmid vector pRSF Duet-1 Carry out double enzyme digestion for 4 ho...

Embodiment 3

[0061] Example 3: Construction of recombinant strains coupled to express maleate cis-trans isomerase and L-aspartate β decarboxylase and expression of enzymes

[0062] Specific steps are as follows:

[0063] (1) The recombinant plasmid pRSF prepared in Example 1 Duet-l -ASD-MaiA and pRSF Duet-l -MaiA-ASD was transformed into the expression host E.coli BL21(DE3)△fumAC competent cells, and then spread on the LB solid medium resistant to carrier kana, cultured at 37°C for 12h, and prepared E. .coli BL21(DE3)△fumAC / pRSF Duet-l -ASD-MaiA and E.coli BL21(DE3)△fumAC / pRSF Duet-l -MaiA-ASD.

[0064] (2) The single colony prepared in the picking step (1) was inoculated into 5 mL of LB medium with an antibiotic concentration of 50 mg / mL, cultured at 37 °C and 200 rpm for 8 hours, and then inoculated with 2% (v / v) The amount was transferred to a 250mL shake flask containing 50mL of 2YT medium, the concentration of antibiotic kana was 50mg / mL, and cultured to OD at 37°C and 200rpm 60...

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Abstract

The invention discloses a method for synthesizing L-alanine by catalyzing maleic acid through double-enzyme coupled whole cells, and belongs to the field of bioengineering. According to the invention, a recombinant strain for coupling expression of maleic acid cis-trans isomerase and L-aspartic acid beta decarboxylase is established, and host bacterium modification and optimization are carried out on the recombinant strain, so that a recombinant bacterium capable of catalyzing maleic acid to generate L-alanine in a whole-cell high-conversion-rate manner is obtained. The recombinant strain can generate L-alanine by using relatively cheap maleic acid, almost no intermediate products fumaric acid and L-aspartic acid are accumulated, the conversion rate reaches 96% or above, the highest production rate reaches 28.43 g/(L.h), and the highest yield reaches 261.2 g/L.

Description

technical field [0001] The invention relates to a method for synthesizing L-alanine from maleic acid through double-enzyme coupling whole cells, which belongs to the field of bioengineering. Background technique [0002] L-Alanine (L-Alanine, L-Ala) is one of the smallest chiral molecules. It is widely used in food, medicine, chemical industry and other fields and has great market development potential. It is mainly used in biochemical research, tissue culture, liver function test, flavor enhancer, can increase the seasoning effect of condiments, and can also be used as a sour taste corrector to improve the sour taste of organic acids. [0003] At present, the methods for preparing L-alanine in industry are mainly chemical synthesis and biological methods. In chemical synthesis production, propionic acid chlorination is a common way to synthesize L-alanine, but this method has disadvantages such as poor product quality, long synthetic route, low yield, high cost, and serio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/06C12R1/19
CPCC12N9/90C12N9/88C12P13/06C12Y502/01001C12Y401/01012C12Y403/01001
Inventor 周哲敏刘中美崔睿智朱小青周丽崔文璟郭军玲
Owner JIANGNAN UNIV
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