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α-l-arabinofuranosidase and its application in the preparation of ginsenoside rd

The technology of furanosidase and ginsenoside is applied in the application field of α-L-arabinofuranosidase, enzymatically converting multi-component ginsenoside Rc to prepare ginsenoside Rd, and achieves the effects of high tolerance and strong transformation ability.

Active Publication Date: 2018-02-09
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the lack of hydrolytic enzymes capable of efficiently converting ginsenosides Rb2 or Rc has become one of the technical bottlenecks for enzymatic conversion and preparation of rare components of ginsenosides. Saponin rare component Rd provides new technology

Method used

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  • α-l-arabinofuranosidase and its application in the preparation of ginsenoside rd
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  • α-l-arabinofuranosidase and its application in the preparation of ginsenoside rd

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Experimental program
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Embodiment 1

[0039] Embodiment 1: The acquisition of the α-L-arabinofuranosidase gene of the present invention and the construction of the recombinant plasmid pET-TthArf

[0040] 1.1 Thermotoga thermarum Cultivation of DSM 5069

[0041] Thermotoga thermarum DSM 5069 was purchased from the DSMZ Culture Collection Center (www.dsmz.de) with the number 5069, and its medium formula was: 10 g / L starch, 5 g / L tryptone, 3 g / L yeast extract, 5 g / L L meat extract, 10g / L 2-morpholineethanesulfonic acid, 10 mg / L ferric sulfate heptahydrate, 1 mg / L resazurin, adjust the pH to 7.2. Inoculate with a syringe according to the inoculum volume of 0.5%, culture statically at 85°C for 24 h, and collect the cells.

[0042] 1.2 Extraction of genomic DNA

[0043] (1) static culture Thermotoga thermarum DSM 5069 for about 24 hours, take 30 mL of the bacterial liquid and centrifuge at 4,000 g for 10 min to collect the cells.

[0044] (2) Resuspend the cells in 9.5 mL TE buffer, add 0.5 mL 10% sodium dod...

Embodiment 2

[0059] Embodiment 2: Preparation of α-L-arabinofuranosidase TthArf of the present invention

[0060] The recombinant plasmid pET-TthArf was transformed into Escherichia coli JM109(DE3) host bacteria (purchased from Novagen), on LB plates containing kanamycin (50 μg / mL) (LB medium: tryptone 10 g / L, yeast Extract 5 g / L, NaCl 5 g / L, agar 15 g / L) after culturing overnight at 37°C, pick the transformant into 200 mL of LB medium (50 μg / mL kanamycin) at 37°C, 200 Shake the culture at rpm until the OD600 is 0.6, add a final concentration of 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) inducer, culture at 30 ° C for 8 h, and use a high-speed refrigerated centrifuge to distill the culture solution Centrifuge at 13,000 rpm for 15 min at 4°C to collect the cells.

[0061] Since the recombinant plasmid pET-TthArf contains a His-tag tag, it was purified by His•Bind Purification Kit (purchased from Novagen) to obtain a purified recombinant enzyme. The specific operation process:

[0...

Embodiment 3

[0078] Embodiment 3: Qualitative determination of α-L-arabinofuranosidase TthArf of the present invention

[0079] 1. Determination method of enzyme activity

[0080] Reaction system 100 μL, 5 μL 20 mmol / L p-nitrophenyl α-D-arabinofuranoside ( p NPArf) was added to 85 μL 100 mmol / L citric acid-disodium hydrogen phosphate buffer (pH 6.0), first at 90 o C for 3 min, then add 10 μL of enzyme solution (diluted to an appropriate concentration) and react for 10 min, after color development, add 600 μL of 1 mol / L sodium carbonate solution to terminate the reaction. Absorbance was measured at 405 nm. Enzyme activity unit (U) is defined as: under the assay conditions, the amount of enzyme required to produce 1 μmol p-nitrophenol per minute is 1 enzyme activity unit.

[0081] 2. Determination of the optimum reaction pH

[0082] Under the conditions of different pH (3.0-7.5, 100 mmol / L citric acid-disodium hydrogen phosphate buffer solution), the enzyme activity was measured at 90°C,...

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Abstract

The invention discloses alpha-L-arabinofuranosidase and application thereof in preparing ginsenoside Rd, and belongs to the field of gene engineering techniques and biological medicines. The amino acid sequence of alpha-L-arabinofuranosidase is as shown in SEQ ID NO.1. The alpha-L-arabinofuranosidase disclosed by the invention is high in conversion property for ginsenoside Rd, and after alpha-L-arabinofuranosidase and ginsenoside Rd are cultured for a certain time, the detection shows that ginsenoside Rc is nearly completely converted into ginsenoside Rd. Alpha-L-arabinofuranosidase is relatively high in resistance to arabinose and is not inhibited by glucose feedback.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and biomedicine, and specifically relates to an α-L-arabinofuranosidase and its application, especially the application of enzymatic conversion of multi-component ginsenoside Rc to prepare ginsenoside Rd. Background technique [0002] Ginseng (Panax ginseng C.A. Meyer) is a perennial herb that likes shade. The fleshy root of wild ginseng is a traditional precious Chinese herbal medicine in my country. It has the functions of regulating blood pressure, protecting the heart, resisting neurasthenia, and improving symptoms of diabetes. Modern medical research has proved that ginsenoside is one of the main effective components of ginseng. Ginsenosides have significant anti-cancer effects, making in-depth research on ginsenosides a hot topic. [0003] According to the existing reports, more than 180 monomers of ginsenosides have been isolated and identified, but there are large differences ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70C12P33/20
CPCC12N9/2402C12P33/005C12P33/20C12Y302/01055
Inventor 赵林果解静聪赵东霞萧伟丁岗王振中
Owner NANJING FORESTRY UNIV
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