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A technology of rhamnosidase and icariin, which is applied in the fields of genetic engineering and enzyme engineering, and can solve problems such as ineffective degradation
Pending Publication Date: 2018-08-31
NANJING FORESTRY UNIV
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[0009] Technical problem to be solved: Aiming at the fact that the α-L-rhamnosidase reported so far cannot effectively degrade the α-1,2 glycosidic bond linked by the natural product rhamn
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Embodiment 1
[0029] 1. Cloning of α-L-rhamnosidase BtRha gene and construction of expression vector
[0030] Using the Bacteroidesthetaiotaomicron VPI-5482 genome purchased from DSMZ (https: / / www.dsmz.de / ) as a template, degenerate primers P1 and P2 were designed according to the amino acid sequence of bacterial rhamnosidase on NCBI , for the upstream and downstream primers to amplify the BtRha gene fragment, use Ex Taq polymerase (purchased from Takara Company) to prepare 50 μL reaction solution at the recommended ratio for fragment amplification, the PCR reaction conditions are 95 ° C, 5 min; 35 cycles (95 ° C, 30s; 52°C, 30s; 72°C, 2min 20s); 72°C, 10min; the reaction was stopped and kept at 4°C. PCR amplification products were purified by gel recovery kit. To obtain the Bacteroides thetaiotaomicron VPI-5482 α-L-rhamnosidase BtRha gene, the resulting PCR amplified product and pET28a were digested with Nco I and Xhol I respectively, and the fragments recovered were combined with pET-28a...
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Abstract
The invention relates to alpha-L-rhamnosidase and application thereof. The amino acid sequence is shown as SEQ ID NO.2. The alpha-L-rhamnosidase BtRha capable of specifically degrading alpha-1,2 glycosidic bonds is cloned and recombinantly expressed for the first time, and a blank of the alpha-L-rhamnosidase for hydrolyzing the alpha-1,2 glycosidic bonds between rhamnose is filled up. The alpha-L-rhamnosidase can efficiently and specifically degrade epmedin C and convert the epmedin C into icariin, and the molar conversion rate of 1 g/L of the epmedin C hydrolyzed by 1.5 U/mL of the alpha-L-rhamnosidase is as high as 90.5% or above under the conditions that the pH is 5.5 and the temperature is 55 DEG C.
Description
technical field [0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to the expression, purification, qualitative and enzymatic preparation of icariin from α-L-rhamnosidase BtRha gene derived from Bacteroidesthetaiotaomicron VPI-5482 . Background technique [0002] α-L-rhamnosidase (α-L-rhamnosidase, EC 3.2.1.40) widely exists in animals, plants and microorganisms, and can specifically hydrolyze various glycosidic bonds, such as α-1, α-1,2 , α-1,3, α-1,4, α-1,6 glycosidic bonds, these glycosidic bonds are contained in many natural plant extract components, such as rutin, hesperidin, naringin, chaohu Glycosides such as Ding C and icariin have α-L-rhamnose groups at the ends, and the rhamnosyl groups at these ends can be hydrolyzed to improve their bioavailability, which can be used in food, medicine and other fields. The main functions and functions include: [0003] (1) Debittering effect: flavanone gl...
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