Beta-glycosidase mutant and coding gene thereof, and application thereof in producing ginsenoside CK

A glycosidase and gene technology, applied to β-glycosidase mutant and its encoding gene and its application in the production of ginsenoside CK, can solve the problems of inability to meet large-scale production, limited conversion efficiency and the like, and achieve good industrial application Prospect, vitality-enhancing effect

Active Publication Date: 2014-05-21
香河县天一合益生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] β-Glycosidase (LacS, β-Glycosidase) from Sulfolobus solfataricus has been reported to have the ability to synthesize ginsenoside CK, but the conversion efficiency is limited and cannot meet the needs of large-scale production

Method used

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  • Beta-glycosidase mutant and coding gene thereof, and application thereof in producing ginsenoside CK
  • Beta-glycosidase mutant and coding gene thereof, and application thereof in producing ginsenoside CK
  • Beta-glycosidase mutant and coding gene thereof, and application thereof in producing ginsenoside CK

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Experimental program
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Embodiment 1

[0029] Embodiment 1, the acquisition of beta-glucosidase mutant and its coding gene

[0030] 1. Acquisition of β-glucosidase and its coding gene

[0031] Genomic DNA of Sulfolobus solfataricus (ATCC 35091, DSM 1616) was extracted and used as a template, and 5'-atggctagcatgtactcatttccaaatagc-3'(forward) and 5'-gtgctcgagttagtgccttaatggctttac-3'(reverse) were used as primers Perform PCR amplification. The conditions for PCR amplification were as follows: pre-denaturation at 95°C for 4 minutes, followed by 30 cycles of 45s at 95°C, 30s at 55°C, and 1min30s at 72°C; and extension at 72°C for 10 minutes.

[0032] The above PCR reaction product was recovered and detected by agarose gel electrophoresis. As a result, a fragment with a size of about 1470 bp was obtained.

[0033] The PCR product obtained above was double-digested with NheI and XhoI, and the digested product was ligated with the same digested pET28a vector (Novagen, No. 69864-3) with T4 ligase (purchased from Treasure ...

Embodiment 2

[0044] Example 2, lacS-mut has β-glucosidase activity

[0045] 1. Obtaining of β-glucosidase mutants

[0046] 1. Induced expression

[0047] Inoculate the single colony of BL21(DE3) / pET28a-lacS-mut obtained above into LB liquid medium containing kanamycin (final concentration: 50 μg / ml), culture at 37°C for 12 hours, collect the fermentation broth, and ferment The solution was transferred to 100ml fresh LB liquid medium according to the inoculum amount of 1% (volume percentage content), and cultivated to OD at 37°C 600 When it reached 0.6, sterile IPTG was added to the culture medium, so that the final concentration of IPTG in the culture medium was 0.4mM, and the induction fermentation was carried out at 30°C. After 16 hours, the fermentation was finished, and the fermentation liquid was centrifuged at 4000 g for 10 minutes to collect the bacteria.

[0048] Resuspend the bacteria in 50ml of 50mM binding buffer (Tris-hydrochloric acid, pH 8, containing 300mM NaCl and 10mM i...

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Abstract

The invention discloses a beta-glycosidase mutant and a coding gene thereof, and an application thereof in producing ginsenoside CK. The protein disclosed by the invention is named as lacS-mut, and is one of defined as the following items: 1) the protein is composed of amino acid residue sequence of a sequence 4 in the sequence table; 2) the protein is derived from the protein as defined in the item 1) by substituting and / or deleting and / or adding of one or several amino acid residues in the amino acid residue sequence of the sequence 4 in the sequence table, and has beta-glycosidase activity. Experiments prove that the beta-glycosidase mutant is obtained by mutating a wild type beta-glycosidase gene; compared with the wild type, the beta-glycosidase mutant has the advantage that the activity of the beta-glycosidase mutant in synthesizing the ginsenoside CK is obviously improved, and therefore the beta-glycosidase mutant has better industrial application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a beta-glucosidase mutant and its coding gene and its application in the production of ginsenoside CK. Background technique [0002] Ginseng is a traditional Chinese medicine. According to modern pharmacological research reports, ginseng has the effects of enhancing human immunity, improving nutrition, anti-aging and relieving fatigue. As one of the main effective active ingredients of ginseng, ginsenoside CK is more easily absorbed by the human body, and has medical effects such as promoting the death of malignant tumor cells and curbing the spread or deterioration of tumors, so it has extremely high production value and application prospects. However, ginsenoside CK does not exist in natural ginseng extracts, but is composed of other ginsenoside components (such as ginsenoside Rb 1 ,Rb 2 ,Rd,Re and Rg 1 etc.) are produced through the metabolic transformation of intestinal bacte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N5/10C12N1/21C12P33/20C12R1/01
CPCC12N9/2445C12P33/20C12Y302/01021
Inventor 梁朝宁唐双焱
Owner 香河县天一合益生物科技有限公司
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