Technology for biotransformation preparation of panoxadiol saponins by recombinant glucoside hydrolase

A technology of ginsenoside and ginseng diol, which is applied in the direction of biochemical equipment and methods, hydrolytic enzymes, enzymes, etc., and can solve problems such as degradation, inapplicability to industrial-scale applications, and easy mutation of microorganisms

Active Publication Date: 2015-07-08
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the small amount of glycoside hydrolase secreted by bacteria or fungi, and the microorganisms are easy to mutate and degrade, the efficiency of this biotransf...

Method used

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  • Technology for biotransformation preparation of panoxadiol saponins by recombinant glucoside hydrolase
  • Technology for biotransformation preparation of panoxadiol saponins by recombinant glucoside hydrolase
  • Technology for biotransformation preparation of panoxadiol saponins by recombinant glucoside hydrolase

Examples

Experimental program
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Effect test

Embodiment 1

[0076] Embodiment 1, the construction of ginsenoside hydrolase recombinant vector

[0077] The polynucleotide (SEQ ID No.1) of ginsenoside hydrolase from Cellulosimerbium sp. 21 (preservation number: CGMCC No.7587) was obtained by PCR, and cloned into the expression vector pET-28a, A recombinant plasmid capable of expressing ginsenoside hydrolase was obtained. Specific steps include:

[0078] 1) Extraction of Genomic DNA of Cellulosimerbium sp. 21:

[0079] Cellulosimicrobium sp. 21 was activated on LB agar medium at 37° C. for 12 hours. The activated strains were put into LB liquid medium in a ring, and cultured with shaking at 37°C and 200rpm for 12h. Bacterial cells were collected by centrifugation at 5000 rpm and 4°C for 10 min, and Genomic DNA of Cellulosimerbium sp.

[0080] 2) Amplification of CcBgl1A gene:

[0081]The CcBgl1A gene was amplified by PCR using the genomic DNA of Cellulosimicrobium sp. 21 as a template. The amplification primers were: Primer-F 5′-GGA...

Embodiment 2

[0085] Embodiment 2, the preparation of recombinant ginsenoside hydrolase CcBgl1A

[0086] 1. The recombinant strain pET-28a / CcBgl1A / E.coli BL21(DE3) of Example 1 was inoculated on LB solid medium containing 50 μg / ml kanamycin, and cultured overnight at 37°C. Pick one ring and inoculate it into 5ml LB liquid medium containing 50μg / ml kanamycin, and culture it at 37°C and 200rpm for 12h (the LB liquid medium containing 50μg / ml kanamycin is used as blank control), which is the seed liquid. The seed solution was inoculated into 200ml LB liquid medium containing 50μg / ml kanamycin at a ratio of 1:50, and cultured in a 1L shake flask at 37°C and 200rpm. When OD600 reached 0.5 (fermentation medium was used as blank control), the temperature was lowered to 25°C, and 0.4mM IPTG was added for induction for 20h. After the induction, collect the bacteria by centrifugation at 5000rpm for 10min, wash the bacteria once with PBS, resuspend in 50ml of PBS buffer, break the cells by ultrasoni...

Embodiment 3

[0098] Embodiment 3, the biological characteristic of recombinant β-glucosidase CcBgl1A

[0099] 1. The effect of pH on the activity of recombinant β-glucosidase CcBgl1A

[0100] Appropriately diluted purified recombinant β-glucosidase CcBgl1A was reacted with substrate pNPG in buffers with different pH values ​​at 37°C for 10 min, and the enzyme activity of recombinant β-glucosidase CcBgl1A at different pH conditions was determined. The buffers used are: pH 2.0-6.0, 50mM NaAc-HAc buffer; pH 6.0-8.0, 50mM Na 2 HPO 4 -NaH 2 PO 4Buffer; pH 8.0-11.0, 50 mM Glycine-NaOH buffer. The experimental results showed that the optimal pH value of recombinant β-glucosidase CcBgl1A was 5.5 ( Figure 6 Middle a).

[0101] 2. Effect of pH on the stability of recombinant β-glucosidase CcBgl1A

[0102] Mix 10 μl of suitably diluted purified recombinant β-glucosidase CcBgl1A enzyme solution with 50 μl of 100 mM buffer solution with various pH values ​​(4.0-10.0) (the buffer solutions used ...

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Abstract

The invention discloses a method for preparing ginsenosides of Gyp XVII, compound O, Mb and F2. The method comprises the following steps that panoxadiol saponins as substrates and a protein undergo a catalytic reaction to produce reactants containing ginsenosides of Gyp XVII, compound O, Mb and F2, wherein the protein is a protein (a) or (b), the protein (a) has an amino acid sequence shown in the formula of SEQ ID No.2 or shown in the formula of SEQ ID No.4, and the protein (b) is derived from the amino acid sequence of the protein shown in the formula of SEQ ID No.2 or shown in the formula of SEQ ID No.4 by replacement and/or deletion and/or addition of one or more amino acid residues and has ginsenoside hydrolase activity. An experiment proves that the method for preparing ginsenosides of Gyp XVII, compound O, Mb and F2 has good practicality.

Description

technical field [0001] The invention relates to a method for preparing ginsenosides in the field of biotechnology. Background technique [0002] Panaxadiol-type saponins Gyp XVII, compound O, Mb and F2 are reported to have good pharmacological activities, such as anti-tumor activity, anti-inflammatory activity, etc., and have certain application potential. However, the content of these ginsenosides in natural ginseng is very small or even does not exist at all, and it is very difficult to extract and separate them from natural ginseng, which greatly limits their application. [0003] Among the total ginsenosides, the content of diol type ginsenosides Rb1, Rb2, Rc and Rd exceeds 60% of the total ginsenoside content. Comparing the structures of ginsenoside Rb1 and Gyp XVII, Rb2 and compound O, Rc and Mb, Rd and F2, it can be seen that their sapogenin structures are the same, but the number of sugar groups attached to the C-3 position is different. Therefore, ginsenosides Gyp...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12P33/20
CPCC12N9/2402C12P33/20
Inventor 周义发高娟原野胡彦波孙成新陈红磊胡晨星
Owner NORTHEAST NORMAL UNIVERSITY
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