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A kind of α-rhamnosidase and its preparation method and application

A rhamnosidase and glycoside rat technology, which is applied in the fields of genetic engineering technology and biomedicine, can solve the problems of high thermal stability, reduced yield of the target product, low reaction temperature, etc., and achieves excellent thermal stability and optimal reaction. The effect of high temperature and high expression

Active Publication Date: 2017-11-17
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

We analyzed the existing research, found the existing problems and limitations, and made a summary: (1) Most of the α-rhamnosidase produced by fungi is the crude enzyme solution after fermentation, which not only contains α-rhamnoside The enzyme also has a large amount of β-glucosidase and other glycoside hydrolases, which inevitably produce by-products during use and reduce the yield of the target product
(2) Most of the α-rhamnosidases reported so far have low activity and cannot be applied to large-scale production. At the same time, there are many influencing factors in the process of fungal fermentation enzyme production, which is not conducive to production regulation
(3) At present, most of the α-rhamnosidases from different sources are generally mesophilic enzymes, and the reaction temperature is not high. When it is necessary to increase the temperature of the catalytic system to convert some raw materials with low solubility, most of the enzymes cannot be maintained under high temperature conditions. High thermal stability, affecting production efficiency

Method used

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  • A kind of α-rhamnosidase and its preparation method and application
  • A kind of α-rhamnosidase and its preparation method and application
  • A kind of α-rhamnosidase and its preparation method and application

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Effect test

Embodiment 1

[0032] Embodiment 1: the acquisition of α-rhamnosidase gene of the present invention and the construction of recombinant plasmid pET-TPERha

[0033] 1.1 Thermotoga petrophila Culture of DSM 13995

[0034] Thermotoga petrophilaDSM 13995 was purchased from the DSMZ Culture Collection Center (www.dsmz.de) with the number 13995, and its medium formula was: 10 g / L starch, 5 g / L tryptone, 3 g / L yeast extract, 5 g / L L meat extract, 10 g / L 2-morpholineethanesulfonic acid, 10 mg / L ferric sulfate heptahydrate, 1 mg / L resazurin, and adjust the pH to 7.2. Inoculate with a syringe according to the inoculum volume of 0.5 wt.% medium, culture statically at 85°C for 24 h, and collect the cells.

[0035] 1.2 Extraction of genomic DNA

[0036] (1) Statically culture Thermotoga petrophila DSM 13995 for about 24 hours, take 30 mL of bacterial liquid and centrifuge at 4,000 g for 10 min to collect cells.

[0037] (2) Resuspend the cells in 9.5 mL TE buffer, add 0.5 mL 10 wt.% sodium dodecy...

Embodiment 2

[0052] Embodiment 2: the preparation of α-rhamnosidase of the present invention

[0053] The recombinant plasmid pET-TPERha was transformed into Escherichia coli JM109(DE3) host bacteria (purchased from Novagen), and placed on an LB plate 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 6 h, and use a high-speed refrigerated centrifuge to distill the culture solution in Centrifuge at 13,000 rpm for 15 min at 4°C to collect the cells.

[0054] Since the recombinant plasmid pET-TPERha contains a His-tag tag, it was purified by His•Bind Purification Kit (purchased from Novagen) to obtain a purified recombinant enzyme. Specific operation process:

[005...

Embodiment 3

[0071] Embodiment 3: the qualitative determination of α-rhamnosidase of the present invention

[0072] 1. Determination method of enzyme activity

[0073] Reaction system 100 μL, 5 μL 20 mmol / L p-nitrophenyl-α-L-rhamnoside ( p Add 85 μL of 100 mmol / L citric acid-sodium hydrogen phosphate buffer (pH 6.0) to NPR), incubate at 90°C for 3 minutes, then add 10 μL of enzyme solution (diluted to an appropriate multiple) for 10 minutes, and develop color Then 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 assay conditions, produce 1 μmol per minute p - The amount of enzyme required for nitrophenol is 1 enzyme activity unit.

[0074] 2. Determination of the optimum reaction pH

[0075] Under the conditions of different pH (3.5-7.5, 100 mmol / L citric acid-disodium hydrogen phosphate buffer), the enzyme activity was measured at 90°C, and the results were as follows figure 2...

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Abstract

Alpha-L-rhamnosidase and a preparing method and applications thereof are disclosed. The amino acid sequence of the alpha-L-rhamnosidase is shown as SEQ ID NO.1. The recombinase is high in optimum temperature, good in thermal stability, and capable of efficiently expressing target protein under preferred conditions, so that the recombinase has wide applications in the fields of selective hydrolysis of rutoside, hesperidin, naringin, prunin, saikoside and other flavonoid compounds connecting alpha-rhamnoside or rhamnose in terpene fragrance precursor compounds, and the like.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and biomedicine, and specifically relates to an extremely heat-resistant α-rhamnosidase and its preparation method and application. Background technique [0002] α-Rhamnosidase (α-L-Rhamnosidase) is a glycoside hydrolase that can hydrolyze the sugar terminal of various natural compounds and release rhamnose, including hesperidin, rutin, quercetin, naringin And some natural compounds such as terpene glycosides. Because α-rhamnosidase catalyzes a variety of substrates extensively, it is widely used in many fields, such as converting naringin, hesperidin, etc. to reduce the bitterness, thereby improving the taste of fruit juice; converting rutin to produce biological Isoquercetin with higher utilization and stronger biological activity in inhibiting the proliferation of malignant tumors, HIV-1 and HSV-1 / 2; transforming ginsenosides Re and Rg2 to generate rare and more active ginsenoside...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70
Inventor 赵林果解静聪李琦裴建军葛林
Owner NANJING FORESTRY UNIV
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