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Pmst1 mutants for chemoenzymatic synthesis of sialyl lewis x compounds

a technology of sialyl lewis and x compounds, which is applied in the direction of enzymology, biochemistry apparatus and processes, and transferases, can solve the problems of limited application of glycosyltransferases in the synthesis of carbohydrate-containing structures, limited application of glycosyltransferases in the preparation and large-scale synthesis of slesup>x, and poor yield (20%), so as to reduce the 2-3

Inactive Publication Date: 2017-07-20
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes an isolated glycosyltransferase that has specific amino acids at certain positions. This glycosyltransferase has lower activity towards sialidase or donor substrate hydrolysis compared to control glycosyltransferases. It can also be a member of the glycosyltransferase family 80. The patent also includes a recombinant nucleic acid and a cell containing this glycosyltransferase. Additionally, a method of preparing oligosaccharides using this glycosyltransferase is also described.

Problems solved by technology

Nevertheless, despite the discovery of many bacterial glycosyltransferases which have promiscuities for both donor and acceptor substrates, the application of glycosyltransferases in the synthesis of carbohydrate-containing structures is limited by the availability and the substrate specificity of wild-type enzymes.
Nevertheless, the low expression level of the enzyme in E. coli (−1 culture) limits its application in preparative and large-scale synthesis of SLex.
It can use Lex as an acceptor for the synthesis of SLex but the yields are poor (<20%) in spite of different conditions tested.

Method used

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  • Pmst1 mutants for chemoenzymatic synthesis of sialyl lewis x compounds
  • Pmst1 mutants for chemoenzymatic synthesis of sialyl lewis x compounds
  • Pmst1 mutants for chemoenzymatic synthesis of sialyl lewis x compounds

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Effect test

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on of Oligosaccharides

[0122]One-pot three-enzyme synthesis of SLexβProN3 with different sialic acid forms. LexβProN3 (20-25 mg),9 a sialic acid precursor (mannose, ManNAc, ManNGc or their derivatives, 1.5 equiv.), sodium pyruvate (5 equiv.), and CTP (1.5 equiv.) were dissolved in Tris-HCl buffer (10 mL, 100 mM, pH 7.5-8.5) containing MgCl2 (20 mM) and appropriate amounts of Pm aldolase (0.5 mg), NmCSS (0.3-0.5 mg), and PmST1 mutant M144D (0.5-0.9 mg). The reactions were carried out by incubating the reaction mixture in an incubator shaker at 37° C. for 4-6 h. The product formation was monitored by TLC developed with EtOAc:MeOH:H2O:HOAc=4:2:1:0.2 (by volume) and stained withp-anisaldehyde sugar stain. When an optimal yield was achieved, the reaction was stopped by adding the same volume (10 mL) of cold EtOH and incubation at 4° C. for 30 min. The mixture was then centrifuged and the precipitates were removed. The supernatant was concentrated, passed through a BioGel P-2 gel filtratio...

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Abstract

The present invention provides mutants of PmST1 for the preparation of sialyl-Lewisx oligosaccharides, and other sialosides with decreased sialidase activity.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS[0001]This application is a continuation application of the U.S. application Ser. No. 14 / 237,334 filed Jun. 18, 2014, now U.S. Pat. No. 9,255,257, the National Stage Entry of PCT Application No. PCT / US2012 / 049748, filed Aug. 6, 2012, which claims priority to U.S. Provisional Application No. 61 / 515,702, filed Aug. 5, 2011 which is incorporated in its entirety herein for all purposes.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT[0002]This invention was made with Government support under Grant Nos. R01HD065122 awarded by National Institutes of Health. The Government has certain rights in this invention.BACKGROUND OF THE INVENTION[0003]Glycosyltransferase-catalyzed reactions have gained increasing attention and application for the synthesis of complex carbohydrates and glycoconjugates. Most mammalian glycosyltransferases suffer from no or low expression in E. coli systems and more restricted substrate...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/10C12P19/04C12P19/18
CPCC12N9/1081C12P19/04C12P19/18C12Y204/99C12N9/1077
Inventor CHEN, XISUGIARTO, GOLAU, KAM
Owner RGT UNIV OF CALIFORNIA
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