Alpha-L-fucosidase, and related biological material and application thereof

A technology of fucosidase and biomaterials, applied in the field of genetic engineering, can solve the problems of little functional activity of lactose and no research reports on the probiotic activity of 3'-fucosyllactose

Active Publication Date: 2020-03-17
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few reports on the functional activity of 3’-fucosyllactose
[0005] So

Method used

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  • Alpha-L-fucosidase, and related biological material and application thereof
  • Alpha-L-fucosidase, and related biological material and application thereof
  • Alpha-L-fucosidase, and related biological material and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1: Construction of recombinant α-L-fucosidase encoding gene expression plasmid

[0084] 1. Extract the genomic DNA of Pedobacter sp. and use it as a template, artificially synthesized degenerate primers fuDP-F: 5′-ACNACNAARCAYCAYGAYGGNTTY-3′ and fuDP-R: 5′-RTTNACNARCATRTTNCCCNCC-3′ Primers, carry out gradient PCR amplification (annealing temperature is 40 ℃ ~ 60 ℃), the amplification product is detected by 1% agarose gel electrophoresis, and a conserved sequence of about 700bp is obtained. The results are as follows figure 1 .

[0085] Gradient PCR amplification system: 10×LA buffer 5.0μl, dNTP mix (2.5mmol / 1) 4.0μl, fuDP-F / R (10pmol / μl) each 8.0μl, genomic DNA 1.0μl, LA Taq (5.0U / pl) 0.5 μl, ddH 2 O up to 50.0 μl; gradient PCR amplification program: 95°C pre-denaturation for 3 minutes; 95°C denaturation for 30 seconds, 40-60°C annealing for 30 seconds, 72°C extension for 40 seconds, 34 cycles; 72°C for 5 minutes.

[0086] 2. Ligate the amplified conserved s...

Embodiment 2

[0102] Embodiment 2: Expression of recombinant α-L-fucosidase gene

[0103] 1. Construction of recombinant strains and expression of recombinant α-L-fucosidase

[0104] The recombinant plasmid pET-28a(+)-PbFuc was transformed into Escherichia coli BL21(DE3) to obtain a recombinant bacterium, which was named BL21(DE3)-pET-28a(+)-PbFuc. Insert BL21(DE3)-pET-28a(+)-PbFuc into LB liquid medium for seed liquid culture, medium containing kanamycin (50 μg mL -1 ), the seed liquid inoculum size is 1.5% (w / v), and the solid medium is an LB solid plate containing agar. Pick positive transformants from the solid medium plate into liquid medium, culture at 37°C for 12 hours, transfer to 200mL LB medium with 1% inoculum size, culture at 37°C, when the culture medium is OD 600 When it reaches 0.6-0.8, add IPTG (with IPTG as the experimental group, without IPTG as the control group) to a final concentration of 1mmol L -1 , Induced at 20°C for 16h, the cells were collected by centrifugatio...

Embodiment 3

[0113] Example 3: Enzymatic properties of recombinant α-L-fucosidase (PbFuc)

[0114] 1. Definition and determination method of PbFuc enzyme activity

[0115] α-L-fucosidase activity was determined with reference to the method of Janet et al. (Janet et al., α-Fucosidase with different substrate specificities from two species of Fusarium. Appl Microbiol Biotechnol, 2013, 97: 5371-5380.). Add 100 μL 10mM pNP-FUC, 100 μL 0.05M pH 5.0 citric acid-trisodium citrate buffer, 10 μL appropriately diluted enzyme solution, react at 35°C for 20 min, and finally add 200 μL Na 2 CO 3 (1M) Stop the reaction and shake evenly. Take 200 μL and add it to a 96-well plate, and measure the absorbance at 405 nm. The pNP standard was used as a standard curve. Enzyme activity definition: The amount of enzyme required to catalyze pNP-FUC to generate 1 μmol pNP per minute is one enzyme activity unit (U).

[0116] 2. Enzymatic properties of PbFuc

[0117] (1) Optimum reaction pH and pH stability of...

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Abstract

The invention discloses alpha-L-fucosidase and a related biological material and application thereof. The invention discloses a protein which is A1) a protein of which the amino acid sequence is shownin the formula SEQ ID No.4, A2) a protein of which the amino acid sequence is shown in the formula of SEQ ID No.3, A3) a fusion protein obtained by connecting the N end or/and C end of the protein ofA1) or A2) with a protein label, and A4) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in the formula of SEQ ID No.3 or SEQ ID No.4, has 90% or more of identity with the protein shown in A1) or A2) and has the same function as the protein shown in A1) or A2). The invention further discloses a related biological material of the protein and an application of the related biological material. The protein provided by the invention can be used for efficiently synthesizing 3'-fucosyllactose and has a good application prospect in oligosaccharide synthesis.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to α-L-fucosidase and related biological materials and applications thereof. Background technique [0002] Fucosyllactose is composed of one molecule of fucose and one molecule of lactose, and the fucose residue can be connected to the galactose residue of lactose through α(1,2) glycosidic bond, or through α(1,3) Glucose that is glycosidically bonded to the reducing end of lactose. Fucosyllactose exists in a large amount in human milk, and has various functional activities such as regulating intestinal flora, resisting the adhesion of pathogenic bacteria, regulating immunity and promoting brain development (Yvan et al., Human Milk Oligosaccharides: 2'-Fucosyllactose (2'-FL) and Lacto-N-Neotetraose (LNnT) in Infant Formula. Nutrients, 2018, 10: 1161.). At present, chemically synthesized or biosynthesized 2'-fucosyllactose has been approved by the European Union and the...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12P19/14C12P19/00C12P21/00C12N15/70C12N1/20C12R1/01C12R1/19C12R1/245C12R1/225
CPCC12N1/20C12N9/2402C12N15/70C12P19/00C12P19/14C12P21/005C12Y302/01051
Inventor 江正强史然马俊文刘军闫巧娟刘海杰
Owner CHINA AGRI UNIV
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