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Protein with sialic acid hydrolase activity and application thereof

A technology of sialic acid hydrolase and protein, which is applied in the direction of hydrolytic enzymes, applications, enzymes, etc., can solve the problems of unsuitable industrial scale applications, degradation of bacterial strains, addition of production processes, etc., to achieve fast biotransformation rate and discharge of three wastes The effect of low and low equipment requirements

Active Publication Date: 2014-08-27
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the sialidohydrolase secreted by bacteria is generally an inducible enzyme, even if GLS is used to induce, the amount of secreted sialidohydrolase is very small, resulting in a very low efficiency of the biotransformation process, and this production process is not suitable for scale-up, not suitable for industrial-scale applications
In addition, bacterial strains are prone to problems such as degradation and variation, adding instability to the production process

Method used

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  • Protein with sialic acid hydrolase activity and application thereof
  • Protein with sialic acid hydrolase activity and application thereof
  • Protein with sialic acid hydrolase activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1, the construction of sialidohydrolase recombinant vector

[0072] The polynucleotide (SEQ ID No.1) of sialidohydrolase from Cellulosimerbium sp. 21 (preservation number: CGMCC No.7587) was obtained by PCR, and cloned into the expression vector pET-28a, A recombinant plasmid capable of expressing sialidohydrolase was obtained. Specific steps include:

[0073] 1) Extraction of Genomic DNA of Cellulosimicrobium sp. 21: Cellulosimicrobium sp. 21 was activated on LB agar medium at 37° C. for 12 hours. The activated strains were put into LB liquid medium in a ring, and cultured with shaking at 37°C and 200rpm for 12h. The bacterial cells were collected by centrifugation at 6000 rpm at 4°C for 10 min, and the genomic DNA of Cellulosimerbium sp.

[0074] 2) Amplification of Cce sia-1 gene:

[0075] The Cce sia-1 gene was amplified by PCR using the genomic DNA of Cellulosimicrobium sp. 21 as a template. The amplification primers are: Primer-F5'-GGAATTCCATATGCAC...

Embodiment 2

[0079] Embodiment 2, the preparation of recombinant sialidohydrolase Cce sia-1

[0080] 1. The recombinant strain pET-28a / Ccesia-1 / E.coli BL21(DE3) of Example 1 was inoculated on LB solid medium containing 50 μg / ml kanamycin, and cultured overnight at 37°C. Pick one ring and inoculate it into 60ml liquid medium containing LB of 50 μg / ml kanamycin and 1% glycerol, and cultivate at 37°C and 200 rpm until the OD600 is 2.0 (with LB containing 50 μg / ml kanamycin and 1% glycerol) Glycerol liquid culture medium is blank control), which is the seed solution. Inoculate the seed liquid into 3L fermentation medium according to the ratio of 1:50 (the solvent of the fermentation medium is water, and its solute and concentration are: 1% (mass percentage concentration) tryptone, 0.5% (mass percentage concentration) yeast extract material, 1% (mass percentage concentration) NaCl, 1% (volume percentage concentration) glycerin, 0.14% (mass percentage concentration) potassium dihydrogen phospha...

Embodiment 3

[0092] Embodiment 3, the biological characteristic of recombinant sialidohydrolase Cce sia-1

[0093] 1. Effect of temperature on activity of recombinant sialidohydrolase

[0094] In a water bath at 20-80°C, using 4-MU-NeuAc as a substrate, the enzymatic activity of the purified recombinant sialidohydrolase Cce sia-1 was measured according to the method of 1) in Step 5 of Example 2. The experimental results showed that the optimal reaction temperature of the recombinant sialidase Cce sia-1 was 45°C ( Image 6 Middle b).

[0095] 2. The effect of pH on the activity of recombinant sialic acid hydrolase Cce sia-1

[0096] Appropriately diluted purified recombinant sialidase Cce sia-1 was reacted with the substrate 4-MU-NeuAc in buffers with different pH values ​​at 37°C for 10 minutes, and the recombinant sialidase Cce sia-1 was measured in Enzyme activity under different pH conditions. The buffers used are: 50mM NaAc-HAc buffer, pH2.0-6.0; 50mM NaAc 2 HPO 4-NaH 2 PO 4 Bu...

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Abstract

The invention discloses a protein with sialic acid hydrolase activity and an application thereof. The protein is a protein of the following (a) or (b) or (c) or (d): (a) a protein with the amino acid sequence of SEQ ID No.2; (b) a protein with the amino acid sequence of SEQ ID No.4; (c) a protein which is obtained by carrying out the substitution and / or deletion and / or addition of one or several amino acid residues on the amino acid sequence as shown in SEQ ID No.2 and has the sialic acid hydrolase activity; and (d) a protein which is obtained by carrying out the substitution and / or deletion and / or addition of one or several amino acid residues on the amino acid sequence as shown in SEQ ID No.4 and has the sialic acid hydrolase activity. An experiment proves that the Cce sia-1 disclosed by the invention achieves obvious high-ratio sialic acid hydrolase activity and can be used for producing the monosialoteterahexosyl ganglioside by catalyzing gangliosides.

Description

technical field [0001] The invention relates to a protein with sialidohydrolase activity and its application in the field of biotechnology. Background technique [0002] Monosialotetrahexosyl ganglioside (GM1) has been proven to have significant nerve repair and regeneration activities, and is a good drug for the treatment of neurodegenerative diseases or nerve damage. It has been used in Alzheimer's disease, In the treatment of diseases such as Parkinson's disease. Due to the constraints of production technology, the price of GM1 is very expensive. In neurological diseases, the drug cycle of GM1 is longer, and a course of treatment takes at least three months. As a result, the cost of treatment has increased significantly, which has limited the general application of GM1. [0003] At present, the production of GM1 is mainly extracted from animal brain tissue, such as pig brain and cow brain. A large amount of organic reagents such as chloroform and methanol were used in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/11C12N5/10C12N1/15C12N1/21C12N1/19C12N15/70C12R1/19C12R1/01
CPCC12N9/2402C12Y302/01018
Inventor 高娟周义发胡彦波勾东霞郭特台桂花
Owner NORTHEAST NORMAL UNIVERSITY