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A kind of metal ion tolerance, heat stability and acid stability sialidase and its application

A sialidase and amino acid technology, applied in the field of sialidase, can solve the problems of difficult availability, weak substrate specificity, and complicated enzyme purification process, and achieve the effects of inhibiting migration and strong tolerance to divalent metal ions.

Active Publication Date: 2017-10-03
桐庐纳泰医学检验实验室有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the strains used in the above reports are mostly pathogenic bacteria or uncommon strains obtained by screening, the genetic information is not clear and difficult to obtain, difficult to cultivate, and even harmful. In addition, the enzyme purification process in most reports is cumbersome and substrate-specific. Not strong, which brings great restrictions to the application and promotion of this type of enzyme

Method used

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  • A kind of metal ion tolerance, heat stability and acid stability sialidase and its application
  • A kind of metal ion tolerance, heat stability and acid stability sialidase and its application
  • A kind of metal ion tolerance, heat stability and acid stability sialidase and its application

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Embodiment 1

[0029] Embodiment 1: the acquisition of sialidase

[0030] 1. Cloning and expression

[0031] The sequence information of the neuA3 gene was obtained through the website http: / / avermitilis.ls.kitasato-u.ac.jp / , and the signal peptide of the enzyme was predicted through the SignalP4.1Server website, and the results showed that the enzyme did not contain a signal peptide A small sialidase of approximately 38 kDa in size. Primers were designed in the coding region of the neuA3 gene, and the following primers were synthesized by the company: neuA3-S: GGAATTCCATATGGCGATGACAGAAGCCAGTACAC (the restriction site of EcoRI is underlined) and neuA3-AS: CCCAAGCTTCAGTAGAGGTCATGGGGTGA (the restriction site of HindIII is underlined), Using the Streptomyces avermitilis genome extracted above as a template for PCR amplification, the amplification conditions were as follows: 95°C for 10 min, 96°C for 1 min, 65°C for 45 s, 72°C for 1 min and 30 s for 34 cycles, and 72°C for 10 min. The PCR ampl...

Embodiment 2

[0042] The enzymatic property research of embodiment 2 novel sialidases

[0043]Dilute the enzyme solution to 0.1 mg / mL, use the derivative of Neu5Ac 4-MU-Neu5Ac as the substrate, add 10 μL of the enzyme solution of this concentration, 10 μL of 1 mM substrate 4-MU-Neu5Ac, and 80 μL of reaction buffer After reacting for a certain period of time, add 100 μL of stop buffer (glycine 0.133M, NaCl 0.06M, NaCl 2 CO 3 0.083M, pH10.7) to terminate the enzymatic hydrolysis reaction to obtain free 4-MU, because free 4-MU can produce 450nm fluorescence at an excitation wavelength of 365nm, while non-free 4-MU-Neu5Ac can only produce 450nm fluorescence at this excitation wavelength A weak fluorescent signal is generated, so the fluorescent signal of free 4-MU can be collected by a fluorescent microplate reader, and the enzyme activity can be calculated according to the standard curve of the relationship between the fluorescent signal and the concentration of 4-MU. One enzyme activity uni...

Embodiment 3

[0048] Example 3 Application research of sialidase biotransformation of gangliosides

[0049] Mix a certain concentration of enzyme and total gangliosides extracted from pig brain in the reaction buffer, and after the reaction is terminated, dry the reacted system with a nitrogen blower and then use chloroform / methanol (V / V =2 / 1) solution was redissolved, the total gangliosides before the reaction was used as a control, and the ganglioside standard was used as a Marker to carry out HPTLC, and the silica gel plate after the expansion layer was used for color reaction with orcinol. At the same time, the silica gel plate after spreading can also be used for immunoblotting experiments. Soak the silica gel plate in 1% BSA / PBS (W / V), seal at room temperature for 30 minutes, and add biotin markers specifically recognized by corresponding gangliosides in a certain proportion. After incubation at room temperature for 2 hours, wash with TBS-T three times, each time for 5 minutes. Add h...

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Abstract

The invention discloses sialidase with metal ion tolerance, heat stability and acid stability. The amino acid sequence of the sialidase is as shown in SEQ ID No. 1 or is an amino acid sequence obtained through deletion and mutation of SEQ ID No. 1 and maintaining unchanged sialidase functions; and the amino acid sequence is purified after expression by Escherichia coli. The sialidase provided by the invention has strong tolerance to divalent metal ions, heat stability and acid stability, can hydrolyze substrates in the connection forms of alpha-2,3 and alpha-2,6 and realizes biotransformation of total gangliosides extracted from pig brains for preparation of high-purity GM1; meanwhile, it is found out for the first time that the sialidase can inhibit migration of bladder cancer cells by transforming ganglioside on the surface of the cancer cells into GM1, so the sialidase has important application prospects.

Description

technical field [0001] The invention relates to a sialidase, in particular to an exo-sialidase and its application in inhibiting tumor migration. Background technique [0002] Sialic acid (SA) is a widely distributed negatively charged nine-carbon sugar, which exists at the end of the sugar chain of glycoconjugates such as glycoproteins and glycolipids through different linkages. In eukaryotes, sialylation of glycoconjugates plays a vital role in stabilizing cell membrane structure, promoting neural development, and signal recognition. Mammalian cell sialylation of sugar chains is closely related to the occurrence and metastasis of cancer. Correlation. [0003] Bacterial exosialidases can hydrolyze sialic acid at the end of glycoconjugates. It has been reported that some bacterial sialidases have application value in the biotransformation of gangliosides to prepare drug GM1 and the treatment of spinal cord injuries. However, the strains used in the above reports are mostly...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N5/10C07K16/40A61K38/47A61P35/00C12R1/19
CPCC12N9/2402C12N15/70C12Y302/01018
Inventor 关锋郭佳王宜成杨刚龙宋波
Owner 桐庐纳泰医学检验实验室有限公司
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