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Chondroitin sulfate synthase and encoding gene and application thereof

A technology of chondroitin sulfate and chondroitin sulfate, applied in the direction of enzymes, enzymes, transferases, etc., can solve the problems of limited types and limited research and development of chondroitin sulfate

Active Publication Date: 2019-07-26
BLOOMAGE BIOTECHNOLOGY CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As far as the current situation is concerned, the types of existing chondroitin sulfate synthases are very limited, which limits the further research and development of chemical enzymatic synthesis of chondroitin sulfate

Method used

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  • Chondroitin sulfate synthase and encoding gene and application thereof
  • Chondroitin sulfate synthase and encoding gene and application thereof
  • Chondroitin sulfate synthase and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1. Preparation of recombinant chondroitin sulfate synthase AuCS

[0053] (1) Construction of expression strains

[0054] The nucleotide sequence of chondroitin sulfate synthase AuCS is shown in SEQ IND NO.1, and the amino acid sequence is shown in SEQ IND NO.2. The nucleotide sequence of chondroitin sulfate synthase AuCS was artificially synthesized, and the above nucleotide sequence was inserted into the plasmid vector pET28a(+) to construct the recombinant plasmid pET28a(+)-His-AuCS. Siri Corporation assisted in the completion. The recombinant plasmid pET28a(+)-His-AuCS constructed by Nanjing GenScript Company was transformed into Escherichia coli BL21(DE3) competent cells (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.), and kanamycin (100 μg / mL) cultured on LB solid medium at 37°C for 12 hours, screened transformants, and used Escherichia coli BL21(DE3) competent cells that had not been transformed with recombinant plasmids as a negati...

Embodiment 2

[0061] Example 2. Verification of the activity of chondroitin sulfate synthase AuCS

[0062] (1) Verification of GalNAc transferase activity of chondroitin sulfate synthase AuCS

[0063] Use commercial GlcA-pNP (final concentration is 0.2mM) as acceptor substrate, UDP-GalNAc (final concentration is 0.3mM) reacts as donor substrate, and reaction system is as shown in table 2; React in a water bath at ℃ for 4 hours, heat in boiling water for 5 minutes to inactivate the enzyme to terminate the reaction, filter the reaction solution through a 0.22 μm filter membrane, and perform liquid phase detection according to the method described in Table 1. There is specific absorption at the detection wavelength, and the flow rate of the mobile phase is 0.5 mL / min.

[0064] Table 2 Reaction system for verification of GalNAc transferase activity of AuCS

[0065]

[0066] Note: AuCS is the chondroitin sulfate synthase AuCS prepared in Example 1.

[0067] The liquid phase test results ar...

Embodiment 3

[0080] Example 3 Properties of Chondroitin Sulfate Synthase AuCS

[0081] (1) Determination of the optimal reaction pH of the in vitro reaction of AuCS

[0082] The reaction system is shown in Table 2 except for the pH of the buffer, and the Tris-HCl buffer was replaced with Tris-HCl / PBS / CH with different pH values. 3 For COONa buffer, set 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.3, 5.9, 6.4, 7.0, 7.5, 8.0, 8.5, a total of 13 pH gradient points, and each gradient has three parallel groups. Each reaction system was reacted in a water bath at 25°C for 4 hours, and heated in boiling water for 5 minutes to inactivate the enzyme to terminate the reaction.

[0083] The result is as Figure 6 As shown, the results indicated that the enzyme was active in a wide pH range (5.0-8.5), and the optimum pH for the reaction was 5.0-6.5.

[0084] (2) Determination of the optimal metal ion for the in vitro reaction of AuCS

[0085] The reaction system is shown in Table 2 except for metal ions, Mn 2+ ...

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Abstract

The invention relates to chondroitin sulfate synthase and a encoding gene and application thereof. The chondroitin sulfate synthase is derived from Actinobacillus ureae and has a nucleotide sequence shown as SEQ ID NO.1 and an amino acid sequence shown as SEQ ID NO.2. The chondroitin sulfate synthase possesses two transferase activities of the acetylgalactosamine transferase GalNAc-T and glucuronosyltransferase GlcA-T except for its native substrate UDP-GalNAc, a unique glycosyl donor UDP-GalNAc can also be used and transferred to a chondroitin sulfate sugar chain with GlcA at a non-reducing end, thereby synthesizing thenon-natural chondroitin sulfate oligosaccharide with an azide group in the sugar chain structure.

Description

technical field [0001] The invention relates to a chondroitin sulfate synthase and its encoding gene and application, in particular to a chondroitin sulfate synthase derived from Actinobacillus Ureae and its encoding gene and application, belonging to the field of biotechnology. Background technique [0002] Glycosaminoglycans (GAGs for short) are straight-chain acidic mucopolysaccharides with multiple physiological functions formed by repeated cross-linking of repeating disaccharide units. Based on the differences in disaccharide types, connection methods, and sulfation sites, glycosaminoglycans can be divided into heparin, hyaluronic acid, chondroitin sulfate (CS for short), skin chondroitin, and keratan sulfate. Glycosaminoglycans have various pharmacological activities such as anticoagulant, antithrombotic and antitumor, and are the main components of articular cartilage and synovial fluid. In addition, it has moisturizing and water-retaining effects in the field of ski...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/70C12N1/21C12P19/26C12P19/18C12R1/19
CPCC12N9/1051C12N15/70C12P19/18C12P19/26
Inventor 生举正王凤山王婷婷曹吉超
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD
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