Method for producing N-acetylneuraminic acid by spore surface display system

A surface display system, acetylneuraminic acid technology, applied in the field of N-acetylneuraminic acid production, can solve the problems of whole-cell catalytic conversion rate of toxic substances, difficulty in large-scale production, and large-scale preparation of free enzymes, etc.

Inactive Publication Date: 2010-12-08
SHANDONG UNIV
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Problems solved by technology

[0027] In view of the above-mentioned existing methods for catalytically synthesizing N-acetylneuraminic acid, there are too many protection and deprotection processes in chemical methods, free enzymes are not easy to be prepared on a large scale, and toxic substances will be introduced during the preparation process, and the catalytic conversion rate of whole cells is relatively low. low, difficult to large-scale production, the problem to be solved in the present invention is to provide a spore surface display system, that is, a spore surface display system containing a surface display carrier, and a method for N-acetylneuraminic acid production thereof

Method used

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  • Method for producing N-acetylneuraminic acid by spore surface display system

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Embodiment 1

[0065] Embodiment 1: Construction of high copy shuttle vector pHPGD

[0066] 1. Cloning of pGDV1 replicon

[0067] The plasmid pGDV1 of Bacillus subtilis 168 was extracted by a conventional method. For this process, reference may be made to the method for small-scale preparation of bacterial plasmids in the "Guidelines for Molecular Biology" published by Science Press. Using the synthesized primers, a high-copy replicon gene was amplified by PCR from the extracted plasmid pGDV1.

[0068] Among them, pGDV1 was purchased from Bacillus Genetic Stock Center (BGSC), Germany, and the query number was 1E60; the above-mentioned Bacillus subtilis was used as the source of the replicon gene, and primers were designed according to the known sequence of pGDV1: upstream primer pGDV1F: 5 '-ATCGGTCTCACGCCCGAGACCATGTATAAAAAACAATCATG-3', carrying a BasI restriction site; downstream primer pGDV1R: 5'-CCAAGGTCCCTTACTTCCAAAATCTAAA-3', carrying an EcoO109I restriction site.

[0069] 2. Construct...

Embodiment 2

[0074] Example 2: Construction of high-efficiency spore surface display system Bacillus subtilis (Bacillus subtilis) WB600 (PHPGD-cotG-nanA)

[0075] 1. Cloning of spore coat protein CotG gene (cotG)

[0076] Genomic DNA of Bacillus subtilis 168 was prepared by a conventional method. For this process, reference may be made to the method for small-scale preparation of bacterial genomes in "Guidelines for Molecular Biology" published by Science Press. The cotG gene was amplified by PCR from the genomic DNA of Bacillus subtilis 168 using synthetic primers cotGF and cotGR.

[0077] Wherein, the above-mentioned Bacillus subtilis 168 was purchased from Bacillus Genetic Stock Center (BGSC), Germany, and the query number is 1A1.

[0078] Primers were designed according to the reported genome sequence of Bacillus subtilis 168 and the reported cotG gene sequence.

[0079] Upstream primer cotGF:

[0080] 5'-GCCTTTGAATTCAGTGTCCCTAGCTCCGAGA-3', carrying an EcoRI restriction site;

[00...

Embodiment 3

[0107] Embodiment 3: The method that spore surface display system is used for the production of N-acetylneuraminic acid

[0108] (1) Plate culture: Streak the above-mentioned Bacillus subtilis (Bacillus subtilis) WB600 (PHPGD-cotG-nanA) strain onto a chloramphenicol LB plate containing 1.5% agar by mass volume ratio and containing 40 μg / ml, at 37° C. Incubate for 12 hours.

[0109] (2) first-class seed: under sterile conditions, pick a single colony on the flat plate of step (1) with a sterile toothpick, then inoculate into 5ml of liquid medium containing 40 μg / ml chloramphenicol, Incubate on a shaker at 37°C for 12 hours.

[0110] (3) Shake flask culture: under aseptic conditions, take the culture solution cultivated in step (2) with a volume ratio of 5% inoculum, and inoculate it into 1L of GYS medium containing 40 μg / ml of chloramphenicol, Incubate on a shaker at 37°C for 24 hours.

[0111] Wherein, the formula of the LB medium in the above (2) is: add 5 g of yeast powde...

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Abstract

The invention discloses a method for producing N-acetylneuraminic acid by a spore surface display system. The method comprises the following step of: after recombining with a spore coating protein gene and an N-acetylneuraminic acid aldolase gene by utilizing a high-copy shuttle vector, constructing a surface display expression carrier; and converting the surface display expression carrier into bacillus subtilis to obtain a recombined strain, wherein a spore of the recombined strain can catalyze the synthesis of the N-acetylneuraminic acid. Compared with other methods in the field, the invention can finish the expression, purification and immobilization of enzyme at one step and has concise and efficient operating process. In addition, by utilizing the characteristics of stable spore, easy separation and stress resistance of the bacillus subtilis, the following separating process is simplified, the stability of N-acetylneuraminic acid aldolase is improved, and the safety of the whole catalyzing process is improved.

Description

technical field [0001] The invention relates to a method for producing N-acetylneuraminic acid, in particular to a method for displaying N-acetylneuraminic acid aldolase by a spore surface display system for the production of N-acetylneuraminic acid. Background technique [0002] Spore surface display technology, as a kind of microbial surface display, has attracted the attention of researchers because of its unique advantages such as the expression of heterologous proteins without transmembrane process and the stress resistance of spores. In 2001, Pozzi established the first spore surface display system with the spore coat protein CotB as the anchor protein. Since then, the spore surface display technology has entered a stage of rapid development, not only widely used in vaccine production, but also in the field of biocatalysis. Emerging [Isticato et al, 2001; Kim et al, 2005; Kwon et al, 2007]. Due to the special formation mechanism of spores, when spores are used as carr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/02C12N15/75C12N1/21C12R1/125
Inventor 许平马翠卿徐小曼
Owner SHANDONG UNIV
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