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Hyperthermophilic glycosidase mutant and application thereof in preparation of ginsenoside CK

A thermophilic glycosidase and mutant technology, applied in the field of biotechnology engineering, can solve the problems of difficulty in artificial cultivation of thermophilic bacteria, low glycosidase content, unfavorable production of thermostable glycosidase, etc.

Active Publication Date: 2015-04-01
CHANGCHUN UNIV OF CHINESE MEDICINE
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Problems solved by technology

[0007] The invention provides a hyperthermophilic glycosidase mutant and its application in the preparation of ginsenoside CK to solve the difficulty in artificial cultivation of thermophilic bacteria, the growth cycle is long, and the content of glycosidase is very low, so it is not conducive to the direct utilization of ginsenoside CK. Problems of thermostable glycosidase produced by thermobacteria

Method used

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  • Hyperthermophilic glycosidase mutant and application thereof in preparation of ginsenoside CK
  • Hyperthermophilic glycosidase mutant and application thereof in preparation of ginsenoside CK
  • Hyperthermophilic glycosidase mutant and application thereof in preparation of ginsenoside CK

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030]The construction of embodiment 1 thermophilic bacterium glycosidase engineering bacteria and its enzyme expression

[0031] (1) Cultivation of thermophilic bacteria Fervidobacterium pennivorans DSM 9078 and extraction of chromosomal DNA

[0032] Thermophilic bacteria Fervidobacterium pennivorans DSM 9078 was purchased from DSMZ (Germany). The composition is as follows: NH 4 C1, 0.5g; MgSO 4 ×7H 2 O, 0.16g; KH 2 PO 4 , 1.6g; Na 2 HPO 4 ×H 2 O, 1.0g; CaCl 2 ×2H 2 O, 0.06g; trace mineral solution, 10ml; Vitamin solution, 10ml; yeast extract, 2g; trypticase, 2g; resazurin, 0.5mg; glucose, 3g; Cysteine-HCl×H 2 O, 0.3g; Na 2 S×9H 2 O, 0.3 g. The composition of each liter of trace mineral solution is as follows: nitrilotriacetic acid, 1.5g; MgSO 4 ×7H 2 O, 3g; MnSO 4 ·H 2 O, 0.5g; NaCl, 1.0g; FeSO 4 ·7H 2 O, 0.1g; CoSO4 7H 2 O, 0.18g; CaCl 2 2H 2 O, 0.1g; ZnSO 4 ×7H 2 O, 0.18g; CuSO 4 ×5H 2 O, 0.01g; KAl(SO 4 ) 2 ×12H 2 O, 0.02g; H 3 BO 3 , 0.01g;...

Embodiment 2

[0054] The characteristics of embodiment 2 hyperthermophilic recombinant glycosidase mutants

[0055] (1) Substrate specificity of wild-type and mutant glycosidases

[0056] Various p-nitrophenol glycoside substrates were weighed, and the enzyme activity was measured referring to the glycosidase measurement conditions described in (1).

[0057] It can be seen from Table 1 that thermophilic glucosidase and its mutants showed different hydrolysis activities to a series of p-nitrophenol glycoside substrates, among which the hydrolysis activity of p-nitrophenyl-β-D-glucoside was the highest, and the mutants hydrolyzed The activity of p-nitrophenyl-β-D-glucoside is about 2.5 times that of the wild type.

[0058] Table 1 Substrate selectivity of wild-type hyperthermophilic glycosidase and its mutants

[0059]

[0060] (2) Comparison of conversion rates of wild-type and hyperthermophilic glycosidase mutant glycosidases for substrate-specific catalysis of ginsenoside conversion ...

Embodiment 3

[0071] Example 3 Transformation of rare ginsenoside CK by thermophilic glycosidase Fpglu1a

[0072] Add 1mL of 1mg / ml solution of ginsenosides Rb1, Rb2 and Rc, and then add an equal volume of the purified enzyme solution obtained above, and react at 70°C for 12 hours. After the reaction, add an equal volume of water-saturated n-butyl Alcohol terminated the reaction, vortexed and mixed, centrifuged at 8000rpm for 2min, and after standing for 5min, took the upper n-butanol layer solution, evaporated to dryness in a water bath at 60°C, collected solid samples, and used chromatographic grade methanol to dilute to 1ml to obtain the total saponins of ginseng root. The converted samples were filtered with a 0.22 μm organic membrane and used for HPLC detection. The detection condition of high performance liquid chromatography is, C 18 Reverse-phase chromatographic column (5 cm×3.0 mm, 2.7 μm; Supelco. USA). Mobile phase is water (A) and acetonitrile (B), gradient elution conditions:...

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Abstract

The invention relates to a hyperthermophilic glycosidase mutant and an application thereof in preparation of ginsenoside CK, and belongs to the technical field of biotechnology engineering. An amino acid sequence of the hyperthermophilic glycosidase mutant is as shown in SEQ ID NO:2; and the hyperthermophilic glycosidase mutant is applied to preparation of rare ginsenoside CK. The hyperthermophilic glycosidase mutant has high heat resistance and stability; relatively high catalytic activity can still be kept after heat preservation at 70-80 DEG C for over 300 hours, and thus hyperthermophilic glycosidase mutant has the advantages of low storage and transportation cost, and low requirement standards on a cooling system of a reactor when applied to production; the kinetic reaction is accelerated; meanwhile, the hyperthermophilic glycosidase mutant has beta-glucosaccharase activity; glucosides of main protopanaxadiol type saponins C3 site and C20 site can be hydrolyzed on the basis that the saponin structure is not destroyed; and the hyperthermophilic glycosidase mutant is efficient, specific, few in byproducts, high in yield and the like, so that the Fpglula can be applied to industrial production of a rare ginsenoside Compound K.

Description

technical field [0001] The invention belongs to the technical field of biotechnology engineering, and in particular relates to a hyperthermophilic glycosidase mutant gene derived from bacteria of the genus Fervidobacterium, a recombinant vector, a genetically engineered bacterium for producing a mutant, and the hyperthermophilic glycosidase mutation Application of body in preparation of rare ginsenoside CK. Background technique [0002] Ginseng (Panax ginseng) is a traditional Chinese herbal medicine with a long history. Chinese traditional medicine believes that ginseng can "replenish the five internal organs, soothe the spirit, soothe the soul, stop palpitations, eliminate evil spirits, improve eyesight, improve intelligence, make light of body and prolong life after long-term use". Ginsenoside is the main active substance in ginseng, and more than 100 kinds of natural ginsenoside products have been found in ginseng, and more than 40 kinds have been isolated. The content...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/70C12N1/21C12N9/24C12P33/20
Inventor 于珊珊刘淑莹李晶郑飞许春春
Owner CHANGCHUN UNIV OF CHINESE MEDICINE
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