CN117089465A

CN117089465A - Aspergillus wart and application thereof

Info

Publication number: CN117089465A
Application number: CN202311059294.0A
Authority: CN
Inventors: 薛文娇; 安超; 马赛箭; 刘瑶; 张琪雯; 丁浩; 刘晨; 张婧婧
Original assignee: Microbiology Institute Of Shaanxi
Current assignee: Microbiology Institute Of Shaanxi
Priority date: 2023-08-22
Filing date: 2023-08-22
Publication date: 2023-11-21
Anticipated expiration: 2043-08-22
Also published as: CN117089465B

Abstract

The invention discloses aspergillus wart and application thereof, and relates to the technical field of microorganisms. Aspergillus warts was designated as strain F8889, accession number: CGMCC No.40637. The strain can convert a large amount of flavonoid glycoside (epimedin A, B, C and icariin) in the epimedium extract by a one-pot method to prepare rare high-activity natural product icaritin, and has the advantages of definite target product, strong conversion specificity, simple process and obvious advantages in the process of producing the icaritin by mass fermentation.

Description

Aspergillus wart and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to aspergillus warts and application thereof.

Background

Icaritin is one of active flavonoid components contained in medicinal plant Epimedii Folium, has a molecular formula of C21H20O6 and a relative molecular weight of 368.4, has multiple pharmacological and biological activities including immunoregulation, nerve protection, anti-inflammatory and other biological effects, and has broad-spectrum anti-tumor activity, wherein the icaritin has particularly obvious effects on breast cancer, oral squamous cell carcinoma, prostate cancer, liver cancer and the like, and is a clinically effective, safe and reliable drug candidate molecule for treating various tumors. At present, a new medicine (aclacindine) taking icaritin as a main component passes three-stage clinical tests, and proves the safety and effectiveness of the new medicine, and the new medicine is approved to be marketed in a soft capsule form at 2022 and 1 and 10 days.

However, the flavone glycoside in the medicinal plant icariin exists mainly in the form of epimedin a, B, C and icariin, the content of icaritin is low and is less than 0.1%, and although icaritin can be obtained by a chemical method (including acid hydrolysis or chemical synthesis), the complex process, low yield, environmental pollution problems (waste water generated in the hydrolysis process, organic solvent waste liquid) and the like limit the wider application of icaritin. The green biological production of bioconversion to icaritin provides the possibility and is becoming a future development direction.

According to the structural comparison of epimedin and icaritin, in order to convert epimedin A, B, C and icariin into icaritin, L-rhamnosidase, xylosidase and glucosidase are required to be simultaneously provided, and glycosyl inside and outside the epimedin should be simultaneously hydrolyzed, but only two types of alpha-L-rhamnosidase (Tporha from Thermotoga petrophila DSM 13995 and DthRha from Dictyoglomus thermophilum DSM 3960) which are reported at present can simultaneously cleave the external and internal rhamnoside bonds of epimedin C, and simultaneously cleave the rhamnosidase of epimedin A and B are not reported, and the basic idea for solving the problem is that two enzymes with different functions are obtained, then multiple enzymolysis reactions are carried out or multi-enzyme system fusion is carried out, and the method has the defects of mainly being characterized by multi-step reactions, low product yield, cooperative optimization of enzymatic reaction conditions in multi-enzyme system establishment and the like; the other is to screen a single microorganism capable of producing a multi-enzyme system to carry out one-pot biological transformation to obtain a target product, and the method has the advantages that the target product is obtained by one-pot method, the yield is high, and in addition, the same strain means that in vivo enzymes co-evolve, and enzymatic reaction conditions are relatively consistent. In general, screening and obtaining microbial resources with rich enzyme lines is of great importance for natural product bioconversion.

Therefore, providing a single microorganism capable of producing a multi-enzyme system for performing one-pot bioconversion to icaritin is a problem to be solved by those skilled in the art.

Disclosure of Invention

In view of this, the invention provides a aspergillus wart and application thereof.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

aspergillus warts, named strain F8889, identified as Aspergillus warts Aspergillus protuberus, deposited at China general microbiological culture Collection center with the collection number CGMCC No.40637 at 24 days of 5 months of 2023, and classified as Aspergillus warts Aspergillus protuberus (A. Protuberus) by the national institute of Chinese sciences, national institute of sciences, no. 3, beijing, kogyo, and the collection address.

An application of Aspergillus wart in biotransformation of natural product of high-activity rare high-activity flavonoid glycoside is provided.

Furthermore, the natural product of the high-activity rare high-activity flavonoid glycoside is icaritin.

Further, the improved culture medium for culturing the aspergillus wart in the bioconversion comprises the following components: 10g/L glucose, 10g/L rhamnose, 0.5g/L NaNO ₃ ，1g/L KH ₂ PO ₄ ，2g/L Na ₂ HPO ₄ ，0.2g/L FeSO ₄ ，0.1g/L ZnSO ₄ ，0.2g/L CuSO ₄ ，1g/L CaCl ₂ ，0.5g/L MgSO ₄ ·7H ₂ O；

The initial pH of the culture was 6.5.

Because of the co-evolutionary relationship with the host plant, plant endophytes are involved, directly or indirectly, in the production and transformation of natural products of the host plant. In recent years, many plant-derived natural products such as artemisinin have been widely reported to be produced not only by fir plants but also by endophytes of the fir class, host plant endophytes also being involved in bioconversion of plant natural products, most commonly endophytes producing glycosidases involved in hydrolysis of sugar chains of plant natural products such as saponins, flavonoid glycosides.

In order to find out a microbial source capable of bioconverting a large amount of flavonoid glycoside (epimedin A, B, C and icaritin) substances in the epimedium extract into rare icaritin, the invention separates and identifies wild epimedium endophytic fungi, and performs screening of beta-glycosidase and transformation research of the epimedium extract, 7 strains capable of producing beta-glycosidase are obtained through screening, and the strains are identified through molecular biology.

Among them, strain F8889 exhibits the potential of converting a large amount of flavonoid glycoside (epimedin a, B, C and icaritin) substances in an epimedium extract into rare icaritin in an epimedium extract conversion test, and the conversion routes of preparing rare natural products icaritin from a large amount of flavonoid glycoside (epimedin a, B, C and icaritin) are (epimedin a, B, c→icariin→icariside i, icariside ii→icariside), respectively. By constructing a phylogenetic tree for this strain, a. Facilis was identified.

In order to improve the activity of glycosidase, the optimal pH and temperature of the enzymatic reaction are optimized, and the optimal pH and temperature conditions of the enzymatic reaction are determined.

According to structural comparison of epimedin and icaritin, in order to convert epimedin A, B, C and icariin into icaritin, it is necessary to have rhamnosidase, xylosidase and glucosidase simultaneously, wherein rhamnosidase and glucosidase are two major enzymes, and in order to increase the yield of icaritin, the rhamnosidase and glucosidase contents should be increased simultaneously. Experiments show that glucose and rhamnose can induce the strain to simultaneously increase the content of rhamnosidase and glucosidase in the culture medium, and the yield of icaritin is increased.

Compared with the prior art, the invention discloses and provides the aspergillus wart A.protuberant us strain F8889, which can convert a large amount of flavonoid glycoside (epimedin A, B, C and icariin) in the epimedium extract by a one-pot method to prepare rare high-activity natural product icaritin, has definite target product, strong conversion specificity and simple process, and has obvious advantages in the process of producing the icaritin by mass fermentation.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 screening of beta-glucosidase produced by endophytes of wild medicinal plant Epimedii Folium;

FIG. 2 construction of phylogenetic tree of Aspergillus wart A.protuberus F8889 strain;

FIG. 3 HPLC detection chart of a large amount of flavonoid glycoside and conversion products in epimedium extract in fermentation broth before and after fermentation conversion of Aspergillus wart A. Protuberus F8889 strain;

FIG. 4 conversion pathway of a large amount of flavonoid glycoside in Epimedium extract to rare icaritin;

FIG. 5 optimal pH and temperature optimization of strain F8889 glycosidase;

FIG. 6 simultaneously improves the optimization of glucosidase and rhamnosidase in strain F8889.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In order to further understand the present invention, the following details are set forth in the examples, wherein the reagents involved in the examples may be purchased commercially, unless otherwise specified, and the methods not mentioned are conventional experimental methods, and are not described in detail herein.

Example 1: isolation and culture of screening strain of beta glycosidase produced by endophytic fungi of wild medicinal plant Epimedii iFolium

Collecting whole plant of wild medicinal plant Epimedii Folium, taking back to laboratory in shortest time, washing plant with running water, separating root, stem and leaf, shearing partial tissue into small pieces, absorbing water on the surface of tissue with water absorbing paper, sterilizing 1min with 75% ethanol, 2min with 2.5% sodium hypochlorite, sterilizing 1min with 75% ethanol, washing 3-5 times with sterile water, air drying, inoculating the tissue pieces onto PDA culture medium plate added with 100U/mL alcamicin sulfate, and culturing at 28deg.C for 5-7 d until mycelium grows.

Screening and identification of beta glycosidase-producing strain

Inoculating mycelium growing on PDA to R2A culture medium containing esculin, and inversely culturing in a constant temperature incubator at 28 ℃ for 5-7 d, wherein the composition of the esculin-R2A agar culture medium is as follows: 1g/L esculin, 0.5g/L ferric citrate, 15.2g/L R A commercial medium. The culture medium was sterilized at 121℃for 20min.

If the strain is capable of producing beta-glucosidase and hydrolyzing esculin, a red halo will be produced around the growth of the strain (shown in FIG. 1). Seven strains capable of producing beta-glycosidase are obtained through co-screening, namely F8443, F8438, F8442, F8446, F8475, F8509 and F8889, ITS sequences are obtained through sequencing, NCBI databases are aligned, and the strains are isolated and have high similarity with Dactylonectria alcacerensis, collectotrium godetiae, collectotrium fioriniae, fusarium perseae, plectosphaerella niemeijorarum, aspergillus ochraceu and Aspergillus protuberus. In particular, for the strain F8889 sequence, F8889 was further determined to have a high similarity with penicillium Aspergillus protuberusNRRL3505 using construction and analysis of MEGA 5.05 phylogenetic tree (see fig. 2).

Example 2: performance study of a large amount of flavonoid glycosides (epimedin A, B, C and icariin) in conversion of Epimedium extract by endogenous strain with beta glycosidase activity

The epimedium extract (water extraction) is a commercial commodity, and is sold by a merchant: chengdu Biotechnology Co., ltd., production lot number: DST230127-850.

Seven strains with beta-glycosidase activity were initially screened and inoculated into a liquid fermentation medium consisting of: 20g/L glucose, 10g/L yeast powder, 0.5g/LNaNO ₃ ，1g/L KH ₂ PO ₄ ，2g/LNa ₂ HPO ₄ ，0.2g/L FeSO ₄ ，0.1g/L ZnSO ₄ ，0.2g/L CuSO ₄ ，1g/L CaCl ₂ ，0.5g/L MgSO ₄ ·7H ₂ O, initial pH is 6.5, after culturing for 72 hours at the culture temperature of 28 ℃ and the rotating speed of 180rpm, adding the water solution of the epimedium extract which is filtered and sterilized, so that the final concentration of the epimedium extract is 1g/L, continuously culturing for 12 hours, taking 2mL of supernatant liquid, adding 2 times of n-butanol for extraction, then removing the n-butanol phase of the upper layer to a new culture dish, and placing the culture dish in a fume hood for volatilizing until the weight is constant, thus obtaining a dry matter.

The dried material was dissolved in methanol and then subjected to liquid phase HPLC detection under the following chromatographic conditions: the chromatographic column adopts YMC-Pack ODS-AQ with the specification of 250mm multiplied by 4.6mm and 5 μm; the mobile phase adopts A phase-B phase, the A phase is water, the B phase is 23wt% acetonitrile, and the gradient elution mode is as follows: 0 to 28min, 72 weight percent of A is adopted; 25-30 min, 72-30wt% of A is adopted; 30-42 min, 30wt% of A is adopted; 42-45 min, 30-72wt% of A is adopted. The volume flow rate in the gradient elution process is 1mL/min, the column temperature is 30 ℃, and the sample injection amount is 10 mu L. All samples were subjected to absorbance detection at 270 nm.

HPLC detection is carried out on the strain fermented icariin extract, the strain fermented icariin extract is compared with HPLC spectrograms and data of standard substance series icariin (see figure 3), a large amount of flavonoid glycoside such as epimedin A, B, C, icariin and the like in the conversion product are hydrolyzed by beta-glycosidase to generate icariside I, icariside II and icaritin, and only the conversion product of strain F8889 in the strain contains icaritin, so that the strain contains more abundant hydrolytic enzyme systems, and a large amount of flavonoid glycoside (epimedin A, B, C and icaritin) in the icaritin extract can be converted by a one-pot method to prepare rare high-activity natural product icaritin. The transformation route is shown in figure 4, and is presumed to be epimedin A, B, C, icariin, icariside I, icariside II and icaritin.

Example 3: optimum reaction temperature optimization for crude glycosidase production reaction condition optimization of aspergillus wart A.protuberus F8889 strain

Culturing Aspergillus wart A.protuberus F8889 in shake flask with the above liquid culture medium for one week, taking out 1mL of bacterial liquid in an ultra-clean workbench under aseptic operation, centrifuging for 5min at 9000r/min, taking out supernatant, adding pNPG substrate in equal volume, and mixing well. Enzyme activity was determined using the pNPG method at 30, 40, 50, 60, 70, 80 ℃ temperature gradients, 3 replicates per group. The buffer used was a pH5.0, 50mM citric acid/phosphate buffer. As can be seen from FIG. 5A, the enzyme has the maximum relative enzyme activity at a reaction temperature of 50℃and thus the optimum temperature of the enzyme is 50 ℃.

Optimum reaction pH condition optimization

The mixed enzyme solution is prepared by the method, the prepared PBS buffer solution and the citric acid/phosphoric acid buffer solution are mixed into a series of gradient buffer solutions with the pH value of 2.00-9.00 according to different proportions, the enzyme solution with the pH value of 2.00-8.00 is prepared by the buffer solution, and the hydrolysis capacities of beta-glucosidase pNPG with different pH values are respectively measured at 50 ℃ and repeated for 3 times in each group. As can be seen from FIG. 5B, there is a maximum relative enzyme activity at pH5.0, and therefore, the optimum pH of the enzyme is 5.0.

Example 4: optimization of glycosidase production conditions of Aspergillus wart A.protuberus F8889 strain

The liquid culture medium is improved.

Basal medium CK:20g/L glucose, 10g/L yeast powder, 0.5g/L NaNO ₃ ，1g/L KH ₂ PO ₄ ，2g/L Na ₂ HPO ₄ ，0.2g/L FeSO ₄ ，0.1g/L ZnSO ₄ ，0.2g/L CuSO ₄ ，1g/L CaCl ₂ ，0.5g/L MgSO ₄ ·7H ₂ O, initial pH6.5.

The improvement of the culture medium mainly optimizes the carbon source in the culture medium.

Improvement medium 1:10g/L glucose, 10g/L rhamnose, 0.5g/L NaNO ₃ ，1g/L KH ₂ PO ₄ ，2g/L Na ₂ HPO ₄ ，0.2g/L FeSO ₄ ，0.1g/L ZnSO ₄ ，0.2g/L CuSO ₄ ，1g/L CaCl ₂ ，0.5g/L MgSO ₄ ·7H ₂ O, initial pH6.5.

Improvement medium 2:20g/L rhamnose, 10g/L yeast powder, 0.5g/L NaNO ₃ ，1g/L KH ₂ PO ₄ ，2g/L Na ₂ HPO ₄ ，0.2g/L FeSO ₄ ，0.1g/L ZnSO ₄ ，0.2g/L CuSO ₄ ，1g/L CaCl ₂ ，0.5g/L MgSO ₄ ·7H ₂ O, initial pH6.5.

After culturing for 120h at the culture temperature of 28 ℃ and the rotation speed of 180rpm, taking out 1mL of bacterial liquid in an ultra-clean workbench in a sterile operation mode, centrifuging for 5min at 9000r/min, taking out the supernatant, respectively adding an equal volume of pNPG or pNPL substrate, and uniformly mixing. The enzyme activities of glucosidase and rhamnosidase were determined at 50℃and pH5.0 (see FIG. 6).

In CK medium, the enzyme activity of the glucosidase is 0.157U/mL, but the enzyme activity of the rhamnosidase is only 0.014U/mL;

in the modified medium 2, however, the enzyme activity of the glucosidase was reduced to 0.023U/mL and the enzyme activity of the rhamnosidase was increased to 0.148U/mL because glucose was replaced with rhamnose;

in the modified medium 1, because glucose and rhamnose are simultaneously contained in the medium, the enzyme activity of the glucosidase is 0.12U/mL, and the enzyme activity of the rhamnose is 0.128U/mL, which are at a higher level, the use of glucose and rhamnose in the medium can induce the strain to simultaneously increase the content of the rhamnose and the glucosidase.

The crude enzyme solution obtained using the above culture medium was subjected to conversion of icariin extract, then, the supernatant was extracted by adding 2 volumes of n-butanol, then, the upper n-butanol phase was removed to a new culture dish, the culture dish was placed in a fume hood and volatilized to constant weight to obtain a dried product, the dried product was dissolved with methanol, then, liquid phase HPLC detection was performed, determination of icaritin content was performed according to peak area, yield was calculated according to the amount of material charged, in the initial medium, the icaritin yield was 5%, in the modified 2 medium, the icaritin yield was 6%, and in the modified 1 medium, the icaritin yield was 18%. The method proves that the glucose and the rhamnose can induce the strain to simultaneously increase the content of rhamnosidase and glucosidase in the culture medium, and the yield of icaritin is increased.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An aspergillus wart characterized in that the aspergillus wart is named as a strain F8889 and has a preservation number of: CGMCC No.40637.

2. The use of a. Wart as claimed in claim 1 for the bioconversion of highly active rare highly active flavonoid glycoside natural products.

3. The use according to claim 2, wherein the highly active rare highly active flavonoid glycoside natural product is icaritin.

4. The use according to claim 2, wherein the modified medium for culturing the aspergillus wart in the bioconversion consists of: 10g/L glucose, 10g/L rhamnose, 0.5g/L NaNO ₃ ，1g/L KH ₂ PO ₄ ，2g/L Na ₂ HPO ₄ ，0.2g/L FeSO ₄ ，0.1g/L ZnSO ₄ ，0.2g/L CuSO ₄ ，1g/L CaCl ₂ ，0.5g/L MgSO ₄ ·7H ₂ O；

The initial pH of the culture was 6.5.