阅读说明：本技术 一种利用β-葡萄糖苷酶制备宝藿苷I的方法 (Method for preparing baohuoside I by using β -glucosidase ) 是由 傅荣昭 刘立辉 刘滔滔 曹磊 郭杏林 于 2018-04-25 设计创作，主要内容包括：一种制备宝藿苷I的方法,包括利用β-葡萄糖苷酶催化底物水解制备宝藿苷I,所述底物为淫羊藿提取物、淫羊藿苷、朝藿定A、朝藿定C、箭藿苷A和箭藿苷B中的任意一种或多种,所述β-葡萄糖苷酶由来源于极端栖热袍菌Thermotoga petrophila RKU-1的β-葡萄糖苷酶基因编码。(A method for preparing baohuoside I comprises the step of catalyzing substrate hydrolysis by β -glucosidase to prepare baohuoside I, wherein the substrate is any one or more of epimedium extract, icariin, epimedin A, epimedin C, agastachoside A and agastachoside B, and β -glucosidase is encoded by β -glucosidase gene derived from Thermotoga petriphilia RKU-1.)

A method for preparing baohuoside I is characterized in that β -glucosidase is used for catalyzing substrates to hydrolyze to prepare baohuoside I, the substrates are any one or more of epimedium extract, icariin, epimedin A, epimedin B, epimedin C, sagohuoside A and sagohuoside B, β -glucosidase is coded by β -glucosidase gene derived from Thermotoga pegophila RKU-1, β -glucosidase specifically hydrolyzes all icariin, epimedin A, epimedin B, sagohuoside A and sagohuoside B in the epimedium extract to form baohuoside I, and hydrolyzes all the epimedin C in the epimedium extract to form baohuoside I and rhamnosyl icariside II.

The method for preparing baohuoside I according to claim 1, wherein the β -glucosidase has an amino acid sequence shown as SEQ ID NO. 2.

The method for preparing baohuoside I according to claim 1, wherein the β -glucosidase has a nucleotide sequence shown as SEQ ID NO. 1.

The method for preparing baohuoside I according to claim 1, 2 or 3, characterized in that the reaction system for preparing baohuoside I by catalyzing substrate hydrolysis with β -glucosidase also contains buffer solution and cosolvent, the pH value of the buffer solution is 5.0-7.0, the temperature of the reaction system is controlled to be 45-80 ℃, the pH value is 4.5-6.5 during the reaction process, and the reaction time is 2-10 h.

The process for preparing baohuoside i according to claim 4, wherein: the using amount of the buffer solution is 60-90% (v/v) of the total reaction solution, and the using amount of the cosolvent is 5-20% (v/v) of the total reaction solution.

The process for preparing baohuoside i according to claim 4, wherein: the cosolvent is selected from any one of acetone, methanol, ethanol, NMP, Tween 20 and Tween 80.

The method for preparing baohuoside I according to claim 1, 2 or 3, wherein β -glucosidase is added in the form of crude enzyme solution, and the crude enzyme solution is β -glucosidase-containing solution obtained after cell breaking and centrifugation removal of precipitate after induced expression by microorganism strains containing β -glucosidase gene.

The method for preparing baohuoside I according to claim 7, wherein the crude enzyme solution is prepared by constructing a recombinant plasmid containing the β -glucosidase gene, transferring the recombinant plasmid into a microbial strain, culturing the microbial strain, performing induction expression on the β -glucosidase, collecting the microbial strain in a buffer solution, breaking cells, centrifuging, and removing precipitates to obtain the crude enzyme solution.

The process for preparing baohuoside i according to claim 7, wherein: the dosage of the crude enzyme solution is 5-20% (v/v) of the total reaction solution, and the substrate concentration is 5-20% (w/v) of the total reaction solution.

The method for preparing baohuoside I according to claim 4, characterized in that after the reaction for preparing baohuoside I by hydrolyzing the substrate catalyzed by β -glucosidase is finished, the obtained enzyme-catalyzed reaction liquid is subjected to crystallization process treatment, crystals are collected to obtain crude baohuoside I, the crude baohuoside I is subjected to recrystallization process treatment, and the crystals are collected to obtain refined baohuoside I.

The method for preparing baohuoside I according to claim 10, wherein when the cosolvent is acetone, the crystallization process comprises: and (3) distilling the enzyme catalysis reaction liquid in a water bath environment at 45-55 ℃ under reduced pressure to remove acetone, cooling the residual solution to 10-15 ℃, filtering after all crystals are separated out, and washing and drying a filter cake to obtain a baohuoside I crude product.

The method for preparing baohuoside I according to claim 10, wherein the recrystallization process comprises the following steps: dissolving the crude baohuoside I product in absolute ethyl alcohol, adding active carbon, stirring, heating to 55-65 ℃, filtering while hot, washing a filter cake with absolute ethyl alcohol, distilling the filtrate under reduced pressure to remove the ethanol, adding water, maintaining the temperature at 55-65 ℃ in the whole process, cooling to 5-10 ℃, stirring until crystals are completely separated out, filtering, washing the filter cake with an ethanol solution, and drying to obtain the refined baohuoside I product.

β -glucosidase and application of microorganism strains containing β -glucosidase gene in catalyzing any one or more of epimedium extract, icariin, epimedin A, epimedin B, epimedin C, arrow glycosides A and arrow glycosides B to prepare baohuoside I, which is characterized in that β -glucosidase is encoded by β -glucosidase gene derived from Thermotoga pegophila RKU-1, and the β -glucosidase specifically hydrolyzes all icariin, epimedin A, epimedin B, arrow glycosides A and arrow glycosides B in the epimedium extract into baohuoside I and hydrolyzes all the epimedin C in the epimedium extract into baohuoside I and rhamnosyl icariside II.

β -glucosidase and the use of a microorganism strain containing β -glucosidase gene for hydrolysis of xylosidic bonds and rhamnoside bonds, characterized in that the β -glucosidase is encoded by β -glucosidase gene derived from Thermotoga petriphilia RKU-1.