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Enzyme, encoding gene thereof, application of enzyme and encoding gene, and method for preparing ginseng saponin compound K

A compound and gene technology, applied in the field of genetic engineering, can solve problems such as low conversion rate and unsatisfactory

Active Publication Date: 2016-10-12
KUNMING NOVOGINSENG BIOENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the existing enzymes used for ginsenoside biotransformation are highly specific, that is, they can only act on a certain glycoside structure specifically, but cannot meet the requirements of acting on more than one glycoside structure at the same time, resulting in low conversion rate

Method used

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  • Enzyme, encoding gene thereof, application of enzyme and encoding gene, and method for preparing ginseng saponin compound K
  • Enzyme, encoding gene thereof, application of enzyme and encoding gene, and method for preparing ginseng saponin compound K
  • Enzyme, encoding gene thereof, application of enzyme and encoding gene, and method for preparing ginseng saponin compound K

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] This example is used to illustrate the preparation of the enzyme provided by the present invention.

[0067] (1) Acquisition of genes

[0068] According to the nucleotide sequence shown in SEQ ID NO: 1, the corresponding gene was obtained by artificial chemical synthesis (entrusted to Kunming Shuoqing Biotechnology Co., Ltd.).

[0069] (2) Construction of expression vectors and recombinant strains

[0070] Both the gene obtained in step (1) and the Pichia pastoris expression vector pPICZɑA (purchased from Invitrogen, USA) were digested with EcoR I and Xba I, and then T4 ligase (purchased from Bao Biological Engineering (Dalian) Co., Ltd. ) to connect the above two enzyme-digested products to obtain a recombinant plasmid, and the double-enzyme digestion verification result of the recombinant plasmid is as follows: figure 1 As shown, wherein, 1 is a DNA marker, and 2-5 are recombinant plasmids.

[0071] The obtained recombinant plasmid was transformed into Pichia pasto...

Embodiment 2

[0079] This example is used to illustrate the application of the enzyme provided by the present invention in the preparation of ginsenoside CK.

[0080] Get 2mL by the purified enzyme liquid that embodiment 1 obtains, add ginsenoside Rb 3 Pure product (reported by Yunnan Yunuo Bioengineering Co., Ltd. (Li Haizhou, Zhang Yingjun, Yang Chongren. Further research on the chemical constituents of Panax notoginseng. Natural Product Research and Development, 2006, 18(4): 549-554) Methods Separation and purification from the total saponins of Panax notoginseng; Content: HPLC≥99%; Molecular weight, 1 H and 13 The chemical shift value of C NMR is consistent with that reported in the literature), so that the final concentration is 1.0 mg / mL, and it is hydrolyzed at 45 °C and pH 4.5 for 48 hours, and the treated sample is taken for TLC analysis. The enzyme solution can hydrolyze Ginsenoside Rb 3 For ginsenoside CK (such as Figure 4 Shown in 1, 2 and 4, wherein, 1 is the pure product ...

Embodiment 3

[0083] This example is used to illustrate the application of the enzyme provided by the present invention in the preparation of ginsenoside CK.

[0084] Get 2 mL of the purified enzyme solution obtained in Example 1, add gypenoside IX (Gypenoside IX, Gyp IX) pure product (Yunnan Yunuo Bioengineering Co., Ltd. with reference to literature (Li Haizhou, Zhang Yingjun, Yang Chongren. Notoginseng Further research. Natural product research and development, 2006, 18 (4): 549-554) reported method is separated and purified from Panax notoginseng saponins; Content: HPLC≥99%; Molecular weight, 1 H and 13 The chemical shift value of C NMR is consistent with that reported in the literature), so that the final concentration is 1.0 mg / mL, and it is hydrolyzed at 45 °C and pH 4.5 for 48 hours, and the treated sample is taken for TLC analysis. The enzyme solution can hydrolyze Gypenoside IX is ginsenoside CK (such as Figure 4 Shown in 1, 3 and 5, wherein, 1 is ginsenoside CK pure product co...

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Abstract

The invention relates to the field of biology, and discloses an enzyme, an encoding gene thereof, application of the enzyme and the encoding gene, and a method for preparing a ginseng saponin compound K. The invention particularly provides the enzyme, the encoding gene thereof, a recombinant carrier, expression box, transgenic cell line or recombinant bacterium containing the encoding gene, a composition with the enzyme as the active component, and application of the enzyme to degradation of a glycoside compound, and provides a method for preparing the ginseng saponin compound K. When used for degrading ginsenoside Rb3 and gynostemma saponin IX in notoginseng leaf total saponin, the enzyme probably can also hydrolyze glucose at the C3 site by acting on the xylose glycoside structure at the C20 site in ginsenoside Rb3 and gynostemma saponin IX, and therefore ginseng saponin CK is obtained, and the conversion rate of ginseng saponin CK is 84% or above.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to an enzyme and its coding gene, their application and a method for preparing ginsenoside compound K. Background technique [0002] Ginsenoside compound K (ginsenoside Compound K, ginsenoside CK, structural formula shown in formula I below) is diol type ginsenoside (for example, ginsenoside Rb 3 and Gypenoside IX, whose structural formulas are shown in the following formulas II and III respectively) are the main metabolites in the human intestinal tract, which belong to rare ginsenosides. Studies have found that ginsenoside CK has a good effect on inhibiting the growth and metastasis of tumor cells both in vivo and in vitro. It is a potential anti-tumor drug and has certain curative effects in various aspects such as anti-aging, memory improvement, and anti-inflammation. Therefore, how to obtain a large amount of ginsenoside CK is the focus of current pharmaceutical rese...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/81C12N1/19C12P33/20C12P33/06C12R1/84
CPCC12N9/2402C12P33/005C12P33/06C12P33/20
Inventor 韩秀林李铭刚赵江源黄开心杨胜江马凤灵包崇卯林婷婷张孝龙文孟良艾黎
Owner KUNMING NOVOGINSENG BIOENG
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