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Novel glycosphingolipid endoglycosidase, and gene engineering preparation method and application thereof

An endoglycosidase and genetic engineering technology, which is applied in the application field of glycosphingolipid endoglycosidase and its genetic engineering preparation, analysis and synthesis of glycosphingolipids, can solve the problem of limited application range, narrow substrate spectrum, and activity low level problem

Active Publication Date: 2018-07-06
SHANGHAI JIAO TONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the EGCase II glycoside synthase has problems such as low activity and narrow substrate spectrum (Journal of the American Chemical Society, 2006, 128(19): 6300-6301), which limit the scope of application of the enzyme

Method used

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  • Novel glycosphingolipid endoglycosidase, and gene engineering preparation method and application thereof
  • Novel glycosphingolipid endoglycosidase, and gene engineering preparation method and application thereof
  • Novel glycosphingolipid endoglycosidase, and gene engineering preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1, the cloning of wild-type glycosphingolipid endoglycosidase 103S_EGCase I gene

[0070] Firstly, the polynucleotide sequence of 103S_EGCase I was cloned from the Rhodococcus equi 103S genome. Through codon optimization, it was unexpectedly found that the optimized sequence (SEQ ID NO: 1) can express a large amount of soluble glycosphingose ​​in E. coli host Endolipid glycosidase I 103S_EGCase I (SEQ ID NO: 2) overcomes the disadvantage that the glycosphingolipid endoglycosidase EGCase I derived from Rhodococcus sp. M-750 cannot be solublely expressed, which limits its application. The specific cloning method is as follows:

[0071] Use upstream primer 5'-AAACGCGGATCCGCCCCGCCGGCGACCCCGATTAC-3'

[0072] (underlined base is restriction endonuclease BamH I recognition site, SEQ ID NO:3)

[0073] and downstream primer 5'-AAACCCAAGCTTTCAGGACGAACCGCTAC-3'

[0074] (the underlined base is the restriction endonuclease Hind III recognition site, SEQ ID NO: 4)

[...

Embodiment 2

[0076] Example 2 Expression, purification and activity determination of 103S_EGCase I

[0077] The engineered bacteria in the glycerol tube were inoculated into a 4 mL LB medium test tube containing 100 ug / mL kanamycin at a volume ratio of 1%, and cultured at 37° C. at 220 rpm for 12 hours. Transfer the 4mL bacterial liquid to a 1L LB medium shake flask containing 50ug / mL kanamycin, culture at 220rpm at 37°C for about 2.5h, make the OD600 reach about 0.9, add 0.1mM IPTG inducer, and induce at 25°C at 200rpm Cultivate for 12-16h. The Escherichia coli cell suspension harvested after fermentation was sonicated, and after one-step Ni-NTA affinity chromatography treatment, the target protein with a purity of more than 95% could be obtained ( image 3 ).

[0078] Using monosialotetrahexosyl ganglioside (GM1) as the substrate to test the hydrolysis activity of the recombinant enzyme, 10nmol substrate and an appropriate amount of enzyme solution in 20 μL of 50mM sodium acetate buffe...

Embodiment 3

[0079] Example 3 Design, Construction, Expression, Purification and Characterization of Synthetic Active Mutants of 103S_EGCase I

[0080] Site-directed mutation of the nucleophilic catalytic residue of glycoside hydrolase to an amino acid that does not have nucleophilic function, resulting in the loss of the original hydrolysis activity of the enzyme. Synthesis of glycosidic bonds, thus becoming glycoside synthase. Using sequence alignment, we determined that the nucleophilic catalytic residue of 103S_EGCase I was glutamic acid at position 339, and mutated it into a series of non-nucleophilic amino acids, including alanine, serine and methionine. Using the recombinant plasmid pET28a-103S_EGCase I as a template, and using a pair of complementary oligonucleotides with mutation sites as primers, use PrimeSTAR Mix high-fidelity enzyme (TakaRa Company) to perform PCR amplification of the whole plasmid to obtain specific mutation sites. Spot the recombinant plasmid. The primer se...

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Abstract

The invention relates to a method for realizing the engineering expression of glycosyl endoglycosidase EGCase I. The method can realize the effective soluble expression of the EGCase I and maintain excellent hydrolysis and transglycosylation activity; a novel EGCase I mutant enzyme developed on the basis of the method has a broad substrate spectrum, and also has the activity of glycosidase synthetase; and the obtained glycosphingolipid endoglycosidase is very suitable for being applied to the analysis and the synthesis of industrial glycosphingolipids.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, the invention relates to a glycosphingolipid endoglycosidase and its genetic engineering preparation method, as well as its application in the analysis and synthesis of glycosphingolipids. Background technique [0002] Glycosphingolipid (Glycosphingolipid, GSL) is a component of eukaryotic cell membrane, and it is a kind of amphipathic molecule formed by glycosidic linkage of ceramide and oligosaccharide chain. Studies have shown that glycosphingolipids are involved in a variety of physiological processes, including signal transduction, cellular immunity, and brain development. In addition, glycosphingolipids are also associated with some pathological processes, including pathogen invasion, cancer, and insulin resistance. [0003] Due to the complex structure of glycosphingolipids, its source mainly depends on natural extraction. However, a large amount of organic solvents such as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12P19/44
CPCC12N9/2402C12P7/6436C12P19/44C12Y302/01123
Inventor 杨广宇韩云宾冯雁李卓刘桂祯陈柳青谭玉萌
Owner SHANGHAI JIAO TONG UNIV
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