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Enzyme co-expression system and its application in the synthesis of sialic acid

An expression vector and recombinase technology, applied in the field of biochemistry, can solve the problems that the sialic acid reaction efficiency does not meet the requirements of industrial production, the second-step reaction yield is low, the sialic acid yield is low, etc. The effect of improving catalytic activity, improving yield, and improving balance

Active Publication Date: 2022-07-05
SHENZHEN READLINE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the reaction efficiency of sialic acid in such reports does not meet the requirements of industrial production.
[0004] In addition, from the analysis of enzyme catalytic efficiency, first of all, the first step reaction enzyme (AGE) used in the current literature is severely inhibited by pyruvate
Another key problem is the low yield of the second step reaction
In the second step reaction, the natural N-acetylneuraminic acid aldolase (NAL) has the problem of catalyzing the equilibrium (reversible) reaction, and the low yield of the forward reaction to produce sialic acid also greatly limits the industrialization of this process.

Method used

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  • Enzyme co-expression system and its application in the synthesis of sialic acid
  • Enzyme co-expression system and its application in the synthesis of sialic acid
  • Enzyme co-expression system and its application in the synthesis of sialic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Construction of plasmid vector pRSFDuet-1-age-nal by homologous recombination

[0061] (1) Construction of AGE, NAL target genes

[0062] The target genes AGE and NAL were synthesized by gene synthesis, and the gene sequences are shown in Table 1. The synthesized gene was ligated and inserted into the pET28a expression vector, using two sites of NdeI and XhoI restriction enzymes to form pET28a-age and pET28a-nal original plasmids.

[0063] Table 1 Gene sequence

[0064]

[0065]

[0066]

[0067] Among them, three mutation points, underlined and bold indicate the mutated gene sequence.

[0068] (2) Design primers

[0069] Through PCR amplification, four plasmid fragments required for homologous recombination were obtained. See Table 2 for primer names and sequences:

[0070] Table 2 Primer names and sequences

[0071]

[0072]

[0073] The four PCR reactions were, using pRSFDuet-1 as a template, amplified w...

Embodiment 2

[0076] Example 2 Culture expression of sialic acid synthase

[0077] (1) Plasmid transformation

[0078] The complete plasmid pRSFDuet-1-age-nal obtained in Example 1 was transformed into competent cells large intestine B121(DE3) by heat shock. Plate culture was performed, and finally single clones were picked for improved LB liquid culture. When the cell OD reached about 0.6, about 1 mL of seed was raised, and the remaining cell solution was added with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to induce protein expression at 25 °C for 10 hours, and finally collected by high-speed centrifugation. Cells (6000 rpm, 15 min) to obtain wet cells. Take a small amount of cells and mix them with Tris-HCl buffer (50 mM, pH 8.0), and then use the freeze-thaw method to break the cells. -Determination of protein expression by polyacrylamide gel electrophoresis (SDS-PAGE), please see the protein expression results image 3 .

[0079] (2) Expanded training

[0080] ...

Embodiment 3

[0081] Example 3 Preparation of crude enzyme solution

[0082] Take the Escherichia coli cells after confirming the correct protein expression in Example 2, resuspend them in an appropriate volume of buffer (50 mM Tris-HCl, pH=7.5), use high pressure homogenization to break the cell wall, and centrifuge at high speed (12000 rpm, 10 min) to obtain the supernatant containing the two enzymes, which is the crude enzyme solution.

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Abstract

The present invention relates to the field of biochemistry, in particular to an enzyme co-expression system and its application in synthesizing sialic acid. The present invention provides an N-acetylneuraminic acid aldolase (NAL) mutant having the amino acid sequence shown in SEQ ID NO: 1; or a substitution, deletion, addition and / or substitution 1 on the basis of the above amino acid sequence A sequence of one or more amino acids; or a sequence with more than 90% homology to the above amino acid sequence. The dual-promoter co-expression system adopted in the present invention simultaneously adopts the molecular biological means of increasing expression, realizes the simultaneous overexpression of two enzymes, and realizes the preparation of enzyme liquid by one-time sterilization process, thereby improving the reaction efficiency of the enzyme-catalyzed reaction. . At the same time, for Clostridium Fusarium ( Clostridium scindens ) NAL was modified to improve the synthesis efficiency of the forward reaction to produce sialic acid. The yield of the enzyme-catalyzed process in the present invention meets the requirements of industrial production.

Description

technical field [0001] The present invention relates to the field of biochemistry, in particular to an enzyme co-expression system and its application in synthesizing sialic acid. Background technique [0002] Sialic acid, also known as bird's nest acid, is a class of derivatives of neuraminic acid, usually referring to the compound N-acetylneuraminic acid (N-acetylneuraminic acid) <neu5ac>). Its main food source is breast milk, and sialic acid is also present in milk, eggs and cheese, but in lower amounts. The content of N-acetylneuraminic acid in bird's nest is relatively high, which is also the main indicator of bird's nest grading standard. Sialic acid is widely used in health food, biopharmaceutical, and cosmetic production. [0003] Due to the relatively low natural yield of sialic acid, the development of effective synthetic routes has always been a hot spot in industrial production. Among them, according to the synthetic route of N-acetylneuraminic acid i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21A61K8/60A61Q19/00A61K31/7012A23L33/00C12R1/19
CPCC12N9/88C12N15/70C12Y401/03003A61K8/60A61Q19/00A61K31/7012A23L33/00A61K2800/10A23V2002/00A23V2200/30
Inventor 赵弘秦国富于铁妹潘俊锋刘建
Owner SHENZHEN READLINE BIOTECH CO LTD
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