Method for preparing monosialotetra-hexosylganglioside (GM1) by applying recombinant sialidase converted gangliosides (GLS)

Abstract

The invention provides a method for preparing monosialotetra-hexosylganglioside (GM1) by applying recombinant sialidase converted gangliosides (GLS). The method comprises the following steps: 1) providing a free recombinant sialidase which is a protein encoded by a base sequence shown in SEQ ID NO: 1; and 2) mixing the recombinant sialidase obtained in the step 1) with GLS so as to convert the GLS into the GM1. By virtue of total synthesis of a full-length gene of the sialidase, soluble expression of the sialidase is successfully realized in escherichia coli. The recombinant sialidase is high in specific activity and low in preparation cost. Moreover, the method can be further used for removing the inhibiting effect of sialic acid by adopting dialysis to in-situ separate a product sialic acid to promote reaction, so that the high concentration GLS is fully converted into the GM1.

Description

technical field [0001] The invention belongs to the field of enzymatic transformation and preparation of monosialotetrahexosyl gangliosides, and more specifically relates to a method for preparing monosialotetrahexosyl gangliosides by using recombinant sialidase to transform polysialogangliosides. Background technique [0002] Gangliosides are glycosphingolipids that contain sialic acid. In 1942, Klenk first found such compounds in ganglion cells, hence the name gangliosides. Gangliosides are composed of ceramides, oligosaccharides and sialic acid. The oligosaccharides contained in gangliosides are specific tetrasaccharides linked by β-glycosidic bonds. Tetrasaccharides are connected sequentially by galactose (Gal), N-acetylgalactosamine (N-GalNAC), galactose and glucose (Glu), and finally connected with ceramide (Cer) through glucose residues. The sialic acid residues of gangliosides are all attached to the galactosyl groups of the oligosaccharides, and the number varies...

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Problems solved by technology

[0010] To sum up, the preparation of GM1 in the prior art has the following defects: 1) GM1 is directly separated from the ganglioside mixture, resulting in waste of polysialogangiosides and high cost; 2) acid conversion requires High temperature and complex transformation products, and the substrate conversion amount is less than 2% (w / v), and the conversion rate is only 50%-70%; 3) the original sialidase-producing bacteria transform polysialogangliosides to produce GM1, and the reaction system The substances in the medium are very miscellaneous and may be pathogenic to the human body; 4) the expression level of sialidase of the original bacteria is low, the unit enzyme activity is low, and the process of purifying the sialidase of the original bacteria is complicated, and the transformation of the sialidase of the original bacteria by free Higher cost of polysialogangliosides; 5) Severe sialic acid inhibition in sialidase-conversion GLS reaction, resulting in low conversion

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