Alpha-L-arabinfuranosidease and coding gene and application thereof

A furanosidase and gene technology, applied in the fields of genetic engineering technology and biomedicine, can solve the problems of poor sugar tolerance, low Rd yield and the like

Active Publication Date: 2019-08-23
KUNMING NOVOGINSENG BIOENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provid...

Method used

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  • Alpha-L-arabinfuranosidease and coding gene and application thereof
  • Alpha-L-arabinfuranosidease and coding gene and application thereof
  • Alpha-L-arabinfuranosidease and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] (1) Acquisition of genes

[0064] According to the nucleotide sequence shown in SEQ ID NO: 2, the corresponding gene was obtained by artificial chemical synthesis (entrusted to Kunming Shuoqing Biotechnology Co., Ltd.).

[0065] (2) Construction of expression vectors and recombinant strains

[0066] Both the gene obtained in step (1) and the expression vector pET28a (+) were subjected to EcoRI / HindIII double enzyme digestion, and after the digestion product was purified by nucleic acid, DNA ligase was used to connect the above two digested products (16 ℃ overnight reaction) to obtain a recombinant plasmid, the sequence of which is shown in SEQ ID NO: 2 after restriction digestion and sequencing verification.

[0067] The obtained recombinant plasmid was transformed into Escherichia coli BL21 to obtain a recombinant strain (BL21-pET28a-CaAraf51).

Embodiment 2

[0069]The recombinant strain (BL21-pET28a-CaAraf51) constructed in Example 1 was inoculated in 10 ml of LB liquid medium, and cultivated overnight at 180 rpm at 37°C for about 13 hours; the next day, it was inoculated into 100 ml of fresh In LB medium, until OD 600 When it was 0.8, IPTG with a final concentration of 0.5 mM was added, and cultured at 180 rpm and 25° C. for 15 h.

[0070] Preparation of crude protein solution: collect the cells by centrifugation at 9000rpm, 4°C for 5 minutes, pour off the supernatant; wash the cells twice with PB buffer, and finally resuspend the cells in 20ml equilibration buffer; ultrasonically break the cells (working 4s, Pause for 4s, amplitude 40%) for 30min; 12000rpm, centrifuge at 4°C for 20min to collect the supernatant (crude protein solution) for SDS-PAGE analysis; the supernatant contains the protein expressed by the target gene, ie crude protein.

[0071] Protein purification: filter the crude protein solution with a 0.45mm micropor...

Embodiment 3

[0074] Qualitative determination of α-L-arabinofuranosidase (CaAraf51) of the present invention

[0075] 1. Determination method of enzyme activity

[0076] Add the substrate (p-nitrophenyl-α-L-arabinofuranoside) to 100 μl reaction system to make the final concentration 5 mM, then add 5 μl purified enzyme solution, pH7.5 (phosphate buffer), 37 ° C React under the reaction conditions for 10min, then immediately add 100μl, 1M Na 2 CO 3 , Measure the absorbance of the reaction product p-nitrophenol (pNP) at 405 nm.

[0077] 2. Determination of optimum pH and pH stability

[0078] Determination of pH optimum and pH stability was performed using the following buffers: 50 mM glycine-HCl (pH 2 and 3), 50 mM acetic acid-sodium acetate (pH 4 and 5), 50 mM Na 2 HPO 4 -NaH 2 PO 4 (pH 6-8), 50 mM Glycine-NaOH (pH 9 and 10).

[0079] Add the substrate to 100 μl of buffer solution with different pH to make the final concentration 5mM, then add 5 μl of purified enzyme solution, incub...

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Abstract

The invention relates to the fields of genetic engineering technology and biomedicine, discloses alpha-L-arabinfuranosidease and a coding gene and application thereof, and specifically provides the alpha-L-arabinfuranosidease and the coding gene thereof, a recombinant vector, an expression cassette and a transgenic cell line or a recombinant strain which contain the coding gene and application ofthe alpha-L-arabinfuranosidease in preparing ginsenoside Rd. The alpha-L-arabinfuranosidease can more efficiently catalyze the reaction of ginsenoside Rc to prepare the ginsenoside Rd, and the enzymeactivity is higher; the higher activity can be exhibited both in a wider range of pH and temperature, and the stability is strong; the alpha-L-arabinfuranosidease has higher tolerance against arabinose and glucose, and facilitates the synergistic effect with other ginsenoside hydrolases.

Description

technical field [0001] The invention relates to the fields of genetic engineering technology and biomedicine, in particular to α-L-arabinofuranosidase and its coding gene and application, especially the application in the preparation of ginsenoside Rd by enzymatic conversion of ginsenoside Rc. Background technique [0002] Ginseng (Panax ginseng C.A. Meyer) is the root of the Araliaceae plant Panax genus. Panax in Chinese means longevity and cures all diseases. It has been used as a panacea in China, Korea, Japan and other Asian countries for more than 2,000 years. year history. The earliest Chinese pharmacy monograph "Shen Nong's Materia Medica" records that ginseng can strengthen the body and improve intelligence, soothe the nerves, relieve palpitations, and prolong life after long-term use. Modern pharmacology has proved that active ingredients in ginseng include saponins, polysaccharides, polypeptides, fatty acids, etc., among which saponins are one of the most importan...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N1/21C12R1/19
CPCC12N9/2402C12N15/70C12Y302/01055
Inventor 韩秀林李铭刚赵江源蒋明星包崇卯李芮刘兴艾黎文孟良
Owner KUNMING NOVOGINSENG BIOENG
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