Application of haloferula sp.β-N-acetylhexosaminidase in the synthesis of human milk oligosaccharides

A technology of human milk oligosaccharides and amino acids, which is applied in the application field of synthesizing human milk oligosaccharides, can solve the problems that there is no report on the production of β-N-acetylhexosaminidase, and achieve excellent hydrolysis properties, high transglycoside activity, The effect of excellent transglycosidic activity

Active Publication Date: 2022-04-08
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Haloferula sp. is a branch of the Verrucomicrobia phylum (Bibi et al. Haloferula luteola sp nov., an endophytic bacterium isolated from the root of ahalophyte, Rosa rugosa, and emended description of the genus Haloferula. Int. J. Syst. Evol. Microbiol .,2011,61,1837-1841), there are few international reports on this genus, and there is no report of Haloferula sp. producing β-N-acetylhexosaminidase

Method used

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  • Application of haloferula sp.β-N-acetylhexosaminidase in the synthesis of human milk oligosaccharides
  • Application of haloferula sp.β-N-acetylhexosaminidase in the synthesis of human milk oligosaccharides
  • Application of haloferula sp.β-N-acetylhexosaminidase in the synthesis of human milk oligosaccharides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1, excavation of β-N-acetylhexosaminidase gene and construction of engineering bacteria

[0078] 1. Determination of β-N-acetylhexosaminidase gene

[0079] The protein sequence of the reported β-N-acetylhexosaminidase was searched through the CAZy database, and the sequences were compared and analyzed by homologous sequences, and the β-N-acetylamino group of a glycoside hydrolase 20 family derived from Haloferula sp. Hexosidase gene (designated HaHex74). The nucleotide sequence of the HaHex74 gene is "ATG+No. 280-2238 of SEQ ID No.3", encoding the amino acid sequence shown in "Met+No. 94-745 of SEQ ID No.2". HaHex74 gene had the highest homology of 61% with a soil metagenome β-N-acetylhexosaminidase (AKC34129). Then send it to Shanghai Sangon Biological Co., Ltd. to synthesize the gene.

[0080] 2. Construction of engineering bacteria expressing β-N-acetylhexosaminidase

[0081] 2.1. Design primers according to the sequence of the glyceraldehyde triphosph...

Embodiment 2

[0090] Embodiment 2, preparation of β-N-acetylhexosaminidase and its enzymatic properties

[0091] 1. High copy screening of β-N-acetylhexosaminidase

[0092] MD plate (1.34%YNB, 4 × 10 -5 % biotin, 2% glucose), His obtained from MD plate + After the transformants were scraped with sterilized water, 100 μL was spread on YPD-G418 plates with different concentrations (1% yeast extract, 2% tryptone, 2% glucose, G418 concentrations were 2, 3, 4, 6 mg / mL ). After culturing at 30°C for 3-5 days, pick the transformant and culture it in a BMGY medium shake flask for 16-18 hours, centrifuge at 3000rpm for 5 minutes, collect the bacteria, and resuspend the bacteria in BMGY medium to OD 600 is about 1.0, add glycerol in batches to express the target protein. The above culture conditions are 100mL Erlenmeyer flask with 20mL culture medium, temperature 30°C, rotation speed 200rpm. Glycerol was added every 24 hours to a final concentration of 1% for 3 days, the enzyme activity of the f...

Embodiment 3

[0133] Embodiment 3, the optimization of synthetic LNT2 condition

[0134] The amount of synthesized LNT2 was determined by HPLC. The HPLC determination conditions are: Waters XBridge BEH Amide 5 μm chromatographic column (250×4.6mm), 72% acetonitrile, column temperature 45°C, flow rate 0.5mL / min, 45min, differential (RID) detector.

[0135] Transglycoside product yield (%) = concentration of synthetic product (mM) / initial concentration of glycoside donor (mM) × 100

[0136] 1. Determination of optimum pH

[0137] The HaHex74 pure enzyme solution prepared in Example 2 was used as the enzyme solution to be tested, which were respectively placed in different buffer systems to determine the LNT2 content. The various buffers are as follows:

[0138] 1) MES buffer (pH 5.5-6.5)

[0139] 2) Phosphate buffer (pH 6.0-8.0)

[0140] 3) Tris-HCl buffer solution (pH 7.0-9.0)

[0141] see results Figure 4 Middle A: The optimum pH for the synthesis of LNT2 by HaHex74 is 7.5.

[01...

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Abstract

The invention discloses the application of Haloferula sp.β-N-acetylhexosaminidase in synthesizing human milk oligosaccharides. The present invention provides the application of HaHex74 protein or its related biological materials in any of the following: (a1) synthesis of human milk oligosaccharides; (a2) synthesis of lactose-N-triose II and / or lactose-N-neotetrasaccharide; The HaHex74 protein is derived from Haloferula sp., and its amino acid sequence is shown in SEQ ID No.1. The β-N-acetylhexosaminidase of the present invention has high enzyme production level, excellent hydrolysis characteristics and high transglycoside transfection activity, and has important application value in enzymatically synthesizing human milk oligosaccharides.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the application of Haloferula sp.β-N-acetylhexosaminidase in the synthesis of human milk oligosaccharides. Background technique [0002] β-N-acetylhexosaminidase (EC 3.2.1.52) is a glycoside hydrolase (GH) that catalyzes the cleavage of the β bond in N-acetylamino-β-D-hexosamine to produce N-acetylamino -β-D-glucosamine (GlcNAc) or N-acetylamino-β-D-galactosamine (GalNAc) (Chen et al.Efficient and regioselective synthesis ofβ-GalNAc / GlcNAc-Lactose by a bifunctional transglycosylatingβ-N-Acetylhexosaminidase from Bifidobacterium bifidum. Appl. Environ. Microbiol., 2016, 82, 5642-5652). According to the homology of amino acid sequence, the existing β-N-acetylhexosaminidase belongs to GH3, 20, 84 and 116 families. In recent years, GH20 family β-N-acetylhexosaminidases have been used for the synthesis of functional oligosaccharides due to their high transglycosidic acti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/81C12P19/00C12P19/04
CPCC12N9/2402C12Y302/01052C12P19/00C12P19/04C12N15/815C12P21/02C12Y302/01014C12Y302/01023
Inventor 江正强刘翊昊闫巧娟马俊文杨绍青李婷
Owner CHINA AGRI UNIV
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