CN112226395B

CN112226395B - Escherichia coli engineering bacterium and method for producing icariin through whole-cell catalysis of escherichia coli engineering bacterium

Info

Publication number: CN112226395B
Application number: CN202010945305.5A
Authority: CN
Inventors: 杜丽琴; 丁波; 庞浩; 可丛雪; 黄日波; 刘家瑞; 闭海; 韦航; 韦宇拓
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2020-09-10
Filing date: 2020-09-10
Publication date: 2022-07-05
Anticipated expiration: 2040-09-10
Also published as: CN112226395A

Abstract

The invention discloses a recombinant escherichia coli engineering bacterium pQE-SPRHA2-PBGL and a method for producing icariin by using the same through catalyzing icariin in whole cells. The invention constructs a polycistron-containing recombinant escherichia coli strain, and directly realizes the conversion of icariin to icariin by adopting a whole-cell catalysis method, wherein the hydrolysis rate of icariin is more than 98%.

Description

Escherichia coli engineering bacterium and method for producing icariin through whole-cell catalysis of escherichia coli engineering bacterium

Technical Field

The invention relates to the field of bioengineering and biosynthesis of natural compounds, in particular to an escherichia coli engineering bacterium and a method for producing icariin by catalyzing icariin through whole cells thereof.

Background

Icaritin (anhydroicaritin, icaritin) is a polyhydroxy flavonoid monomer component in epimedium herb of the genus epimedium of the family berberidaceae, and has the functions of resisting tumor (Liu Song, Liu super Ming, Yu Li Juan, etc.' pharmacological action research progress [ J ]. Jiangnan institute of Jiangxian institute of medicine, 2017,37(04): 631; 635.), antioxidation, resisting hepatic fibrosis, promoting differentiation, mineralization, resisting osteoporosis (Zheng Z G, Zhang X, Zhou Y P, et al. Anhydroritin, a SREBPs inhibitor, inhibitors RANKL-induced inflammatory and antibodies diabetes mellitus [ J. European Journal of pharmaceutical, 2017,809: 156. 162.), anti-inflammation, promoting angiogenesis and vascular endothelial formation (endothelial cell proliferation, endothelial cell proliferation promotion of Ma Jiangjiang Ujian J.: preliminary metabolism mechanism of Epimeritin and its metabolism, 2015,39(03): 313-. The icaritin is mainly extracted from epimedium plants, but the content of the icaritin in the icaritin plants is extremely low in a natural state. Therefore, with the further development of the medicinal effect of the icariin at home and abroad, how to prepare the icariin in a green and high-efficiency manner becomes a key point.

Epimedium is a traditional Chinese medicinal material, has a long medicinal history in China, and has main active ingredients of total flavonoids of epimedium (Zhang Jia Li, Ding Hui, Song Xin Bo's & ltEpimedium Total flavonoid anti-aging research progress [ J ] research and development, 2018.), wherein the flavonoids compounds take 3-benzopyrone as a mother nucleus, and mainly comprise icariin, icariside I, baohuoside I, epimedin A, epimedin B, epimedin C and the like. Several common epimedium species have similarity in chemical structure, the common aglycone of the common epimedium species is Anhydroicaritin (Anhydroicaritin), and the difference lies in that glycosyl (glucose, rhamnose and xylosyl) with different types and numbers are connected at the C-3 position and the C-7 position of the basic molecular skeleton. The flavone in epimedium extracted from natural environment is mainly in the form of combined glycoside, which accounts for more than 95% of the flavone content in epimedium, wherein the content of epimedium is the most, the content of free icariin is generally less than 5%, but the content of free icariin is obviously higher than that of combined glycoside in the activity action.

Currently, icariin is mainly obtained by hydrolyzing icariin, and the method mainly comprises a method of acid hydrolysis, enzyme hydrolysis and enzyme combination (Yuren, Chen 26163, Bing, and Celluol, etc., the research progress on pharmacological actions of icariin [ J ] in China J.Ouchi J.J., 2018 (17)). The acid hydrolysis is to hydrolyze the glycosidic bond of C-3 and C-7 sites of icariin by dilute acid solution such as low-concentration hydrochloric acid sulfuric acid and the like to prepare anhydroicaritin, for example, CN201110427160.0 hydrolyzes crude extract of epimedium herb by dilute hydrochloric acid solution at high temperature to prepare anhydroicaritin with purity of more than 95 percent, but the method uses acid which is harmful to environment and has over high reaction temperature (90-100 ℃); the acid enzyme binding method is also a more common method, such as ZL201310280568.9 mainly hydrolyzes rhamnosyl on C-3 position of icariin by acid, hydrolyzes glucosyl on C-7 position by beta-glucan or cellulase, and the side products are more and poor in regulation and control in the acid hydrolysis process, and even the mother core structure of the product icariin aglycone can be changed; CN109988137A is subjected to smith degradation, macroporous resin adsorption and purification, alcohol washing and other steps to obtain high-purity icariin, although the problem of structural change caused by acid hydrolysis is successfully solved, the smith degradation process is long in time and complicated in process, and some reaction processes are even as long as 150 hours; the enzyme hydrolysis is usually carried out by breaking off the glycosyl groups at different C positions of icariin by mixed enzyme to generate icariin, for example, CN201410274823.3 utilizes naringinase (commercial pure enzyme) which is a compound enzyme system consisting of alpha-L-rhamnosidase and beta-glucosidase to convert icariin to generate icariin, rhamnosidase can break off the glycosidic bond of icariin C-3 rhamnosyl to generate icariside I, beta-glucosidase can catalyze the glycosidic bond of icariin C-7 glucosyl to generate baohuoside I, and when the rhamnosidase and the beta-glucosidase act on the icariin together, anhydroicariin is generated, but the reaction time is as long as 30 hours, and the efficiency is low as only to prove that the icariin is generated; ZL200910184282.4 uses mixed enzyme system-helicase (commercial pure enzyme) containing more than 20 enzymes such as cellulase, pectinase, amylase, protease, etc. to prepare icariin, the reaction time is as long as 72 hours, and the product yield is low; ZL200710099025.1 can hydrolyze icariin to generate icariin by utilizing commercial beta-glucosidase, but the catalytic reaction process is as long as 24 hours, and the product yield is only 55 percent at most. Therefore, the further improvement of the preparation method of the icariin is particularly important.

The icariin is used as the name of the invention to search a patent search database of the national intellectual property office, 55 search results appear in total, and 55 patents related to the icariin exist. Among them, there are 8 patents (in which there are 2 patents for a two-enzyme combined catalytic method, 1 patent for Smith degradation method, 4 patents for a complex enzyme catalytic method), 2 patents (all are an enzyme binding method) for a method for preparing hydrated icariin (sugar substituents on C-3, C-7 of icariin are hydrolyzed and a molecule of water is added to 8-isopentenyl group of icariin), 2 patents (all are a chemical method) for a method for preparing cyclic icariin (3, 5-dihydroxy-4 ' -methoxy-6 ', 6 ' -dimethyldihydropyrane and <2 ', 3 ': 7, 8> flavone), 17 patents for an application of icariin, 1 patent for an application of cyclic icariin, other search results are related to preparation of a preparation containing icariin or preparation of an icariin derivative and application of the preparation and the derivative. The invention discloses a method for searching a patent search database of the national intellectual property office by taking icaritin as the name of the invention, wherein 138 search results occur in total, and 138 patents related to the icaritin (dehydrated icaritin and icariin) are provided. Among them, there are 22 patents on the preparation method of icaritin (among them, there are 11 items of chemical synthesis methods, 3 items of organic acid hydrolysis methods, 4 items of recrystallization methods after enzymatic hydrolysis, 2 items of mixed enzymatic hydrolysis methods, 2 items of acid enzyme combination methods), 2 items of patents on the preparation method of hydrated icaritin (both are acid enzyme combination methods), 4 items of patents on the preparation method of cyclic icaritin (both are acid hydrolysis methods), 30 items of patents on the application of icaritin, 2 items of patents on the application of hydrated icaritin, 3 items of patents on the application of cyclic icaritin, and other search results are related to preparation or material preparation of preparations containing icaritin or preparation of icaritin derivatives and their applications.

Through literature search, no report of a method for preparing icariin (anhydroicaritin and icaritin) through whole-cell catalysis exists at present.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Disclosure of Invention

Aiming at the problems of low icariin conversion rate and low icariin yield in the prior art, the invention provides an escherichia coli engineering bacterium and a method for producing icariin by using the engineering bacterium to carry out whole-cell catalysis on icariin, so that the conversion from icariin to icariin is directly realized, and the conversion rate is high.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

an escherichia coli engineering bacterium co-expresses a gene coding alpha-L-rhamnosidase SPRHA2 and a gene coding beta-glucosidase PBGL in a polycistronic form; wherein the gene for coding the alpha-L-rhamnosidase SPRHA2 is from Sphingobacterium Novosphingobium sp.GX9, the base sequence is shown as SEQ ID NO.1, and the gene for coding the beta-glucosidase PBGL is from Paenibacillus clarkii cookii GX-4.

The method for producing icariin by catalyzing icariin by using co-expression escherichia coli engineering bacteria pQE-sprha2-pbgl comprises the following operation steps:

(1) constructing co-expression escherichia coli engineering bacteria pQE-sprha2-pbgl, carrying out induction culture on the co-expression escherichia coli engineering bacteria pQE-sprha2-pbgl, carrying out induction for 8h at 30 ℃, collecting thalli after the induction culture is finished, and weighing wet weight of the thalli;

(2) cleaning the thalli for 3 times, and then re-suspending the thalli by using a buffer solution to obtain a coexpression escherichia coli engineering bacterium pQE-sprha2-pbgl whole-cell catalytic solution;

(3) adding herba epimedii plant extract with proper concentration into the coexpression escherichia coli engineering bacterium pQE-sprha2-pbgl whole-cell catalytic solution obtained in the step (4) and reacting for 10 hours;

(4) terminating the reaction after boiling water bath to obtain the product, namely the icariin.

Preferably, the engineered Escherichia coli pQE-sprha2-pbgl in the step (1) is in polycistronic form, and the target gene pbgl is positioned at the downstream of the target gene sprha 2.

Preferably, the induction culture in step (1) is carried out at 30 ℃ for 8h with 0.5mM IPTG.

Preferably, the cells are washed in step (2) by resuspending the cells in a 0.9% NaCl solution.

Preferably, the buffer solution in the step (2) is borax borate buffer solution.

Preferably, the boric acid borax buffer solution contains 0.2M boric acid and 0.05M borax, and the pH value is 8.0.

Preferably, the concentration of the bacterial cells before washing in step (2) is 16.7 g/L.

Preferably, the reaction conditions in step (3) are 55 ℃ and 220 rpm.

Preferably, the epimedium plant extract described in step (3) is a crude icariin extract containing 70% icariin.

Preferably, the epimedium plant extract with a proper concentration described in the step (3) is an icariin crude extract with a concentration of 62.5 g/L.

Preferably, the reaction is terminated after the boiling water bath at 100 ℃ is carried out for 5min in the step (4).

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention provides a novel method for producing icariin by co-expressing whole-cell catalysis of icariin by adopting two glycosidases (alpha-L-rhamnosidase SPRHA2 and beta-glucosidase PBGL) for the first time, a polycistron-containing recombinant escherichia coli strain is constructed, the conversion from icariin to icariin is directly realized by adopting a whole-cell catalysis method, and the hydrolysis rate of icariin is more than 98%;

(2) the two enzymes involved in the method have clear physicochemical properties and can act on icariin in a synergistic manner through co-expression; the whole-cell catalysis method omits the complicated steps of purifying enzyme, has simple operation, and has the advantages of high conversion efficiency, economy, high industrialization potential and the like in the process of catalyzing the hydrolysis of icariin to produce icariin.

Drawings

FIG. 1 is a HPLC chart of whole-cell catalysis of icariin to icariin by Escherichia coli engineering bacteria pQE-sprha2-pbgl of the present invention; wherein the a-1 retention time of 7.686min is icariin, the a-2 retention time of 12.49min is icariside I, the a-3 retention time of 13.222min is baohuoside I, and the a-4 retention time of 18.02min is icariin; a is a standard product containing icariin, icariside I, baohuoside I and icariin, b is an HPLC diagram of engineering bacteria pQE-sprha2-pbgl whole cell catalyzing icariin to generate icariin.

FIG. 2 is an HPLC analysis of hydrolyzed icariin at a ratio of SPRHA2 to PBGL plus enzyme of 4: 1; wherein A is a standard sample, 1 is icariin, 2 is icariside I, 3 is baohuoside I, and 4 is anhydroicaritin; b is the result obtained by 5min of reaction, and C is the result obtained by 5h of reaction.

Detailed Description

The following detailed description is to be read in connection with the accompanying drawings, but it is to be understood that the scope of the invention is not limited to the specific embodiments. The raw materials used in the examples were all commercially available unless otherwise specified.

Example 1

1.1 the enzyme adding amount ratio of the purified double-enzyme catalytic icariin is groped

Inducing and culturing pQE-sprha2 at 20 ℃ and 0.5mM IPTG for 20h at 180rpm, and inducing and culturing pSE-pbgl for 8h at 30 ℃ and 0.5mM IPTG for 200 rpm; carrying out cell breaking and purification on the induced pQE30-SPRHA2 and pSE-PBGL thallus to obtain pure enzyme, carrying out protein quantification on the purified pure enzyme to obtain the product with the SPRHA2 of 2.2 mu g/mu L, the PBGL of 1.2 mu g/mu L, the conversion rate of icariin 6h of 0.3% (w/v) of reaction when the enzyme adding amount of SPRHA2 is 50 mu g/mL is known to be above 95%, and the enzyme adding amount of the fixed SPRHA2 is 50 mu g/mL, taking two pure enzymes to dilute by proper times, and then carrying out reaction on 0.3% (w/v) of icariin according to different enzyme adding amount ratios (1:1, 2:1, 3:1, 4:1, 5: 1); in order to observe the reaction speed of the two enzymes in the reaction process, samples are respectively taken after 5 minutes and 5 hours of reaction, and HPLC is used for detecting hydrolysate after 5 minutes of thermal termination; the detection results show that when the enzyme adding ratio of SPRHA2 to PBGL is 4:1, the icariin can completely react for 5h by 0.3% (w/v), and the liquid phase detection results when the enzyme adding ratio of SPRHA2 to PBGL is 4:1 are shown in FIG. 2.

The experimental results from fig. 2 show that: when the double enzymes act for 5min, the intermediate products icariside I and baohuoside I are remained, and the remained amount of baohuoside is more, which is shown in figure 2B; with the reaction time prolonged, icariin is completely hydrolyzed into anhydroicaritin after 5h, as shown in FIG. 2C. The time required for completely converting icariin by the synergy of the two enzymes is shorter than that required for hydrolyzing a substrate by SPRHA2 single enzyme, and the intermediate product generated in the action of the single enzyme is supposed to be quickly converted into the final product icariin, so that the product inhibition effect of the intermediate product on the single enzyme is reduced. The enzyme dosage of alpha-L-rhamnosidase SPRHA2 is more than that of beta-glucosidase PBGL when the anhydroicaritin is generated by reaction, and SPRHA2 is the rate-limiting enzyme of a two-enzyme catalytic system. Therefore, when a polycistron coexpression strain is constructed in an escherichia coli system subsequently, the SPRHA2 is placed in front of the PBGL and is close to the promoter so as to ensure that the constructed polycistron has a good effect of hydrolyzing the icariin.

1.2 construction of polycistronic Co-expression Strain of Escherichia coli

The construction method of the engineering bacterium of escherichia coli comprises the following operation steps:

(a) designing a primer by using pQE-sprha2 recombinant plasmid which is automatically constructed in the laboratory, and cloning to obtain linearized pQE-sprha 2:

pQE30-sprha 2-F: 5'-aattagctgagcttggactcctgttgatagatcca-3' is shown as SEQ ID NO. 2;

pQE30-sprha 2-F: 5'-aagctttcagcgggtcgtgcccagcgtgaccgggc-3' is shown as SEQ ID NO. 3;

(b) designing a primer by taking pSE-pbgl recombinant plasmid which is automatically constructed in the laboratory as a template, and cloning to obtain a pbgl gene sequence with pQE30 carrier Ribosome Binding Site (RBS) before the gene sequence:

Pbgl-F：

5'-ggcacgacccgctgaaagcttaggagaaattaactatgagaaaccatacttcagacacg-3' is shown as SEQ ID NO. 4;

Pbgl-R：

5'-aacaggagtccaagctcagctaatttcagcttctacggtatttcttggtt-3' is shown as SEQ ID NO. 5;

(c) the linearized pQE-sprha2 and a pbgl gene sequence with a pQE30 vector Ribosome Binding Site (RBS) In front of the gene sequence are connected by using In-fusion enzyme (purchased from Takara company), a recombinant plasmid pQE-sprha2-pbgl is obtained and is transformed into Escherichia coli JM109, a recombinant co-expression Escherichia coli engineering bacterium pQE-sprha2-pbgl is obtained, the obtained Escherichia coli engineering bacterium pQE-sprha2-pbgl is In a polycistronic form, and the target gene pbgl is positioned at the downstream of the target gene sprha 2.

Example 2

The method for catalyzing icariin to generate icariin by using the co-expression escherichia coli engineering bacteria pQE-sprha2-pbgl constructed in the example 1 comprises the following operation steps:

(1) carrying out induction culture on the co-expression escherichia coli engineering bacteria pQE-sprha2-pbgl 8h obtained in the example 1 at the temperature of 30 ℃ by using 0.5mM IPTG, collecting thalli after fermentation is finished, and weighing wet weight of the thalli;

(2) washing 23.8mg wet-weight thallus with 0.9% NaCl solution for 3 times, and re-suspending the thallus with boric acid borax buffer solution containing 0.2M boric acid and 0.05M borax and having a pH value of 8 to obtain 4.76mg/ml co-expression escherichia coli engineering bacteria pQE-sprha2-pbgl whole-cell catalytic solution;

(3) adding 47.62g/L epimedium plant extract into the coexpression escherichia coli engineering bacterium pQE-sprha2-pbgl whole-cell catalytic solution obtained in the step (2), and reacting at 55 ℃ and 220rpm for 10 hours; the herba Epimedii plant extract is icariin crude extract containing 70% icariin;

(4) terminating the reaction after boiling water bath at 100 ℃ for 5min to obtain a product, namely icariin, and carrying out High Performance Liquid Chromatography (HPLC) analysis on the obtained product icariin.

Example 3

(2) washing 55.5mg wet-weight thallus with 0.9% NaCl solution for 3 times, and re-suspending the thallus with boric acid borax buffer solution containing 0.2M boric acid and 0.05M borax and having a pH value of 8 to obtain 11.1mg/ml co-expression escherichia coli engineering bacteria pQE-sprha2-pbgl whole-cell catalytic solution;

(3) adding epimedium herb plant extract with the concentration of 44.4g/L into the coexpression escherichia coli engineering bacterium pQE-sprha2-pbgl whole-cell catalytic solution obtained in the step (2), and reacting for 10 hours at the temperature of 55 ℃ and the rpm of 220; the herba Epimedii plant extract is icariin crude extract containing 70% icariin;

Example 4

(1) carrying out induction culture on the co-expression escherichia coli engineering bacteria pQE-sprha2-pbgl 8h obtained in the example 1 at 30 ℃ by using 0.5mM IPTG, collecting bacteria after fermentation is finished, and weighing wet weight of the bacteria;

(2) cleaning 83.5mg wet-weight thalli for 3 times by using 0.9% NaCl solution, and re-suspending the thalli by using boric acid borax buffer solution containing 0.2M boric acid and 0.05M borax and having the pH value of 8 to obtain 16.7mg/ml co-expression escherichia coli engineering bacteria pQE-sprha2-pbgl whole-cell catalytic solution;

(3) adding 62.5g/L epimedium herb plant extract into the coexpression escherichia coli engineering bacterium pQE-sprha2-pbgl whole-cell catalytic solution obtained in the step (2), and reacting at 55 ℃ and 220rpm for 10 hours; the herba Epimedii plant extract is icariin crude extract containing 70% icariin;

Example 5

The method for catalyzing icariin pure substances (the purity is more than or equal to 98%) to generate icariin by using the co-expression escherichia coli engineering bacteria pQE-sprha2-pbgl constructed in the example 1 comprises the following operation steps:

(2) washing 15mg of wet-weight thallus with 0.9% NaCl solution for 3 times, and re-suspending the thallus with boric acid borax buffer solution containing 0.2M boric acid and 0.05M borax and having the pH value of 8 to obtain 5mg/ml co-expression escherichia coli engineering bacteria pQE-sprha2-pbgl whole-cell catalytic solution;

(3) adding 2g/L icariin standard substance into the coexpression escherichia coli engineering bacteria pQE-sprha2-pbgl whole-cell catalytic solution obtained in the step (2), and reacting at 55 ℃ and 220rpm for 1 h; the icariin standard product contains icariin with purity more than or equal to 98%;

(4) terminating the reaction after boiling water bath at 100 ℃ for 5min to obtain a product, namely icariin, and carrying out nuclear magnetic resonance NMR analysis on the product icariin.

Detection and analysis:

firstly, the high performance liquid chromatography HPLC analysis conditions for catalyzing icariin to generate icariin by using the co-expression escherichia coli engineering bacteria pQE-sprha2-pbgl constructed in the embodiment 1 are as follows:

the instrument equipment comprises: agilent G1311C 1260Quat Pump VL quaternary Pump, Agilent G1329B 1260ALS autosampler, CO-1000column oven, Agilent G1314F 1260 VWD ultraviolet detector.

A chromatographic column: c18 column (Alltima, 250X 4.6mm, 5 μm)

Detection conditions are as follows: the detection wavelength is 270nm, the column temperature is 30 ℃, and the flow rate is 1 mL/min.

Mobile phase A: 100% acetonitrile; mobile phase B: ultrapure water, gradient elution procedure was as follows:

32％A	0-5min
		32-80％A	5-12min
80％A	12-17min
		80-32％A	17-20min
32％A	20-25min

HPLC purification conditions for catalyzing icariin pure substances to generate icariin by using the co-expression escherichia coli engineering bacteria pQE-sprha2-pbgl constructed in the example 1 are as follows: the instrument equipment comprises: waters ACQUITY UPLC^TM(Waters, Milford, MA, USA), Waters 2535 Quaternary gradient Module, 2707 Autosampler, 2489UV/Visible Detector, reverse C18 column (SunAire TM, 250X 10mm, 5 μm) and fraction collector III (Waters Corporation, Milford, USA).

The nuclear magnetic resonance analysis conditions for generating the icariin are as follows: instrument equipment Bruker Avance 600NMR spectrometer (Swiss Avance), deuterated reagent: dimethyl sulfoxide (DMSO-d)₆) 600MHz analysis at 25 ℃¹H, 150MHz analysis¹³C。

Calculation of conversion and retention:

percent conversion of icariin (initial amount of icariin-remaining amount of icariin)/initial amount of icariin × 100%;

the amount of baohuoside I produced is the final amount of baohuoside I-the initial amount of baohuoside I;

the generation amount of icariside I is equal to the final yield of icariside I-the initial amount of icariside I;

the amount of icaritin produced is the final yield of icaritin-the initial amount of icaritin.

Second, result in

The hydrolysate obtained in the above example was analyzed by HPLC, and E.coli engineering bacteria pQE-sprha2-pbgl were used to convert icariin into icariside or baohuoside I by whole-cell catalysis, and then converted into icaritin or baohuoside I. The icariin conversion rates in examples 2 to 4 were analyzed and counted, and are shown in Table 1:

TABLE 1 Total cell catalytic herba Epimedii plant extract of Escherichia coli engineering bacteria

Examples	Cell concentration (mg/mL)	Substrate concentration (mg/mL)	Icariin hydrolysis ratio (%)
				2	4.76	47.62	72.4
3	11.1	44.4	98.2
				4	16.7	62.5	99.4

As can be seen from Table 1, when the substrate concentration is about four times of the cell concentration, the hydrolysis rate of icariin is more than 98% in the method for preparing icariin by co-expressing the engineering bacteria pQE-sprha2-pbgl of Escherichia coli 3 and 4 in the embodiment of the invention.

FIG. 1 is a high performance liquid chromatography HPLC analysis chart of the conversion product of icariin in example 4, wherein a is a standard containing icariin, icariside I, baohuoside I and icariin aglycone, a-1 is icariin, a-2 is icariside I, a-3 is baohuoside I, a-4 is icariin aglycone; b is a reaction product analysis diagram of engineering bacteria pQE-sprha2-pbgl whole-cell catalysis icariin.

The results of nuclear magnetic resonance NMR analysis of icariin, a conversion product of icariin in example 5, are shown in Table 2:

TABLE 2 NMR analysis table of E.coli engineering bacterium pQE-sprha2-pbgl whole-cell catalysis icariin hydrolysate

In the table 2, a is an icariin standard substance, b is an engineering bacteria whole-cell catalysis icariin hydrolysate, the result of the conversion rate of the hydrolysate of the example 5 being more than 95 percent is subjected to preparative liquid-phase purification, the product with the standard purity is subjected to NMR analysis, and the icariin standard product is obtained from the results in the table 2¹H-NMR、¹³The nuclear magnetic resonance spectrum data of C-NMR and DEPT135 can obtain the engineered Escherichia coli pQE-sprha2-pbThe substance generated by catalyzing icariin by gl is icariin.

In conclusion, the invention provides a colibacillus coexpression engineering bacterium for the first time, a gene coding alpha-L-rhamnosidase and a gene coding beta-glucosidase are coexpressed by polycistrons to prepare icariin under the synergistic action of icariin, and the colibacillus coexpression engineering bacterium has the advantages of high efficiency, high conversion rate, economy, high industrialization potential and the like.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

SEQUENCE LISTING

<110> Guangxi university

<120> escherichia coli engineering bacterium and method for producing icariin through whole-cell catalysis

<130> JC

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 3498

<212> DNA

<213> Novosphingobium sp.

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ttcgacttgc gccacttccg cctgcacggg gagccgcgca tcgaccggtt cgaggccaag 1200

gccggtttct ccatcgagca ggactattac gccctgggca tgcccgatgc cggcgcggcc 1260

gggccggatg ccgtcaaggt ggtgaacctg acaggcaagc tgcgccccga cggcacgctg 1320

gactggacac cgccggcggg cagcgcctgg cgcatcctgc gctttggcac cagcctgctg 1380

ggcactacca accaccccgc agccccggaa gcgaccgggc tggaagtgga caagttcgat 1440

ggcgcggcgg tgcggcgcta cctgacgcac tacttgggga tgtatcgcga cgcgaccggg 1500

ccggaactga tgggccagca tggcgtgaaa gccctgctga cggattcgat cgaagtgggc 1560

gcggccaact ggacgcccaa gatgatcgac cagttcaagc gcttgcgcgg ctatgatccc 1620

accccctggc tgccgacgct ggccggcgtg ctgatcggca gccgcgccga tgccgaccgt 1680

ttcctctatg actggcgccg cacgctcgcc gatctgctcg cctcggagca ttatggcatg 1740

gtggcgcaag tggcgcacga gaacggcctc aaggtctacg gcgaggcgct ggaggacaac 1800

cgcccgatgc tgggcgatga catggccatg cgcatgcgcg ccgatgtgcc gatggcggcg 1860

ctgtggacct ttggccgcga gacggggccg aaccccagct atctcgccga catgaaaggc 1920

gccgcctcgg tcgcgcacgt ctatgggcag aacctggttg cggctgagtc catgacgtct 1980

gccctggcgc cctgggccta cacgcccaag gacttgcgcc ggatcatcga cctggaattc 2040

gtcagcggga tcaaccgccc ggtggtgcac acctcggtcc accagccggt ggacgacaag 2100

gtgccgggcc tgtcgctgat gatctttggc cagttcttca accgccacga aagctgggcc 2160

gagatggcgc ggccctgggt ggattacatg gcgcgcaatt cgctgctgtt gcagcaaggg 2220

cgcaacgtgg ccgatgtcgc ttatttctat ggcgaggaag ccccactgac cgggctttac 2280

ggcaaggcac cggtggccga tgcgcccaag gccaacgcct atgacttcgt caatgccgac 2340

gcgctgggtg gcgtgctgct caaccaaggc aacgaactcg tcagccaggg cggggcacgc 2400

tatcgcgcgg tctacctcgg cggttcgtcg cggatgatga cgctcaagac cctgcgccgc 2460

ctcgccgaac tggtggatgg tggcgccacg gtaattggcc tggcgccgca gggttcgccc 2520

gcgctcgacg atgcgcaagc ggccaaggcg gcggagtggc agcaactggt ggcgcggctc 2580

tggccaggca gcggcgacgc cacggtgggc aagggccgcg tgattgcgtt ggccgatgtc 2640

gacgccgggc tggcgcggct gggcgtggcg cccgatttcc ggctggtggg cgcaagcgat 2700

gcgcaagtgc cgttcgtgca ccgccaactc gccgatggcg acgcctggtt cctggtcaac 2760

cgccgcaacc gcgatgaaac gttcgaggcg catttccgcg tcaccggcaa gcagcccgag 2820

ctgtggcacg ccgacagcgg cagggtggag ccggtatcgt ggcgcagcga gaacggcgaa 2880

accatcgtgc cgctcagcct gccggcggaa agttcggtct tcgtggtgtt ccgcaagccg 2940

gccacgacca cccatggcga agtgcgggcg gtgcatgaca tgccgctcgg cactctcggc 3000

gcggcgaccg gatcgacccg cagcaagtcg cgcgatgccg gctggatggt cgcgttccag 3060

gccggccggg gtgcgcccgc ttcgctggcc atgcctacct tggcgcggct ggaccagaat 3120

gccgcgcccg gcgtgcgcta tttctccggc atcgcgacct atgcccgcag cttccgcctg 3180

cccaagggct ggaagaaagg ccagccgctg tggctcgacc tgggtgaagt gcacgatatt 3240

gcgcaagtca ctgtcaacgg ccaggatctg ggcacgcagt ggcacgcgcc ctatcgcttc 3300

gatgtcggca gcgcggtgca caatggcacc aacgcgatcc aggtgcgcgt ggccaatagc 3360

tgggtcaacc gcctgatcgg cgatgcgcag cccggcgcca ccaaggtgac ctggaccgcg 3420

attcccacct acaatgccga tgccccgctg cgaccttcgg gcctggttgg cccggtcacg 3480

ctgggcacga cccgctga 3498

<210> 2

<211> 35

<212> DNA

<213> Artificial sequence

<400> 2

aattagctga gcttggactc ctgttgatag atcca 35

<210> 3

<211> 35

<212> DNA

<213> Artificial sequence

<400> 3

aagctttcag cgggtcgtgc ccagcgtgac cgggc 35

<210> 4

<211> 59

<212> DNA

<213> Artificial sequence

<400> 4

ggcacgaccc gctgaaagct taggagaaat taactatgag aaaccatact tcagacacg 59

<210> 5

<211> 50

<212> DNA

<213> Artificial sequence

<400> 5

aacaggagtc caagctcagc taatttcagc ttctacggta tttcttggtt 50

Claims

1. The application of the escherichia coli engineering bacteria in the whole-cell catalysis of icariin to the production of icariin is characterized in that: the escherichia coli engineering bacteria co-express a gene for coding alpha-L-rhamnosidase SPRHA2 and a gene for coding beta-glucosidase PBGL in a polycistronic form, the base sequence of the gene for coding alpha-L-rhamnosidase SPRHA2 is shown in SEQ ID NO.1, and the gene for coding beta-glucosidase PBGL is from paenibacillus klebsiella (Bacillus clarkii)Paenibacillus cookii) GX-4。