CN115418323A - Pichia pastoris AtAc3 and application thereof, leavening agent and glycyrrhetinic acid preparation method - Google Patents

Pichia pastoris AtAc3 and application thereof, leavening agent and glycyrrhetinic acid preparation method Download PDF

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CN115418323A
CN115418323A CN202211220324.7A CN202211220324A CN115418323A CN 115418323 A CN115418323 A CN 115418323A CN 202211220324 A CN202211220324 A CN 202211220324A CN 115418323 A CN115418323 A CN 115418323A
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atac3
pichia pastoris
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CN115418323B (en
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吕波
陈林浩
李春
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Beijing Institute of Technology BIT
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Abstract

The invention discloses a Pichia pastoris strain AtAc3 and application thereof, and a preparation method of a leavening agent and glycyrrhetinic acid, and belongs to the technical field of microorganisms. The invention provides a Pichia pastoris (Pichia pastoris) strain AtAc3, the preservation number of which is CGMCC No.25102. The invention also provides application of the strain AtAc3 in hydrolysis of glycyrrhetinic acid monoglucuronide and/or hydrolysis of glycyrrhizic acid, and a starter and a method for preparing glycyrrhetinic acid based on the strain AtAc3. The conversion rate of the strain AtAc3 to glycyrrhizic acid reaches up to 96.58%, and the strain has the advantages of high conversion efficiency, low accumulation of intermediate products, low fermentation viscosity and the like, and lays an important foundation for industrial application of green production of glycyrrhetinic acid.

Description

Pichia pastoris AtAc3 and application thereof, leavening agent and glycyrrhetinic acid preparation method
Technical Field
The invention belongs to the technical field of microorganisms, and relates to a Pichia pastoris (Pichia pastoris) strain AtAc3, application thereof, a leavening agent and a preparation method of glycyrrhetinic acid.
Background
Licorice (Glycyrrhiza uralensis Fisch.) is one of traditional medicinal plants in China, and has the effects of clearing away heat and toxic materials, tonifying spleen and qi, moistening lung and arresting cough, harmonizing the drugs and the like. Research shows that the licorice extract has pharmacological effects of resisting inflammation, resisting bacteria, protecting liver, resisting ulcer, resisting tumor, etc. and has Glycyrrhetinic Acid (GA) as main active component.
Glycyrrhetinic acid can be prepared by hydrolyzing Glycyrrhizic acid (GL) with two molecules of glucuronic acid groups on the structure, is an important pentacyclic triterpene compound, and is widely applied to industries such as medical raw materials, food additives, cosmetics and the like due to wide pharmacological effects such as anti-inflammation, antibiosis, anti-tumor, antioxidation, whitening, skin care, immunity regulation and the like.
The annual market demand of glycyrrhetinic acid exceeds 200 tons, the global demand amount reaches $ 10 billion, the worldwide demand amount is increased at a speed of more than 5 percent per year, and the current global product production capacity is about 150 tons. The glycyrrhetinic acid product belongs to a liquorice extract product with high added value, good bioavailability and high safety, and the main production raw materials are glycyrrhetinic acid ammonium salt and glycyrrhetinic acid powder, so that the production quantity and the scale of the raw materials are large, the output quantity is high, and the supply is relatively stable. But more raw material manufacturers exist and the added value of the product is lower. In addition, although the total number of domestic liquorice product production enterprises exceeds 200, the large-scale production enterprises are small in number and lack high-end product competitiveness.
The production mode of glycyrrhetinic acid mainly focuses on plant extraction and chemical methods. However, because the planting of liquorice is easily affected by the environment and the content of glycyrrhetinic acid is very low (the mass fraction is 0.01% -0.2%), chemical synthesis usually needs to consume a large amount of organic reagents, and the process conditions are harsh, and the like, and the problems are limited to a certain extent. The strain fermentation and transformation has the advantages of mild conditions, environment-friendly process, low cost, capability of preparing a large amount of medicinal precursors and the like, and becomes a glycyrrhetinic acid production mode with great development prospect at present. Therefore, the research and development of the glycyrrhetinic acid product with high added value can not only increase the international competitiveness and the economic benefit of production enterprises, but also contribute to the reasonable utilization of resources and the environmental protection.
In the production method for preparing glycyrrhetinic acid by using the existing strain fermentation substrate glycyrrhetinic acid, the problems of high GAMG accumulation amount of an intermediate product, high viscosity of fermentation liquor and the like are easily caused, the inhibition effect on the activity of the strain is possibly generated, and the mass transfer process is greatly influenced, so that the increase of the production efficiency and the energy consumption is obviously reduced.
Therefore, there is a need in the art to develop a novel strain with high catalytic efficiency, low accumulation of intermediate GAMG, and high yield of product. And a foundation is further laid for the industrial production of the glycyrrhetinic acid from the perspective of solving the actual production requirements.
Disclosure of Invention
Based on the objective problems and requirements in the field, the invention provides the Pichia pastoris (Pichia pastoris) strain AtAc3 with high substrate conversion rate, low intermediate product accumulation amount, high target product final concentration and high production efficiency, and the method can be used for industrial scale production of glycyrrhetinic acid.
The technical scheme of the invention is as follows:
a Pichia pastoris (Pichia pastoris) strain AtAc3 is characterized in that the preservation number is CGMCC No.25102.
Use of Pichia pastoris (Pichia pastoris) strain AtAc3 with preservation number of CGMCC No.25102 in hydrolyzing glycyrrhetinic acid monoglucuronide and/or glycyrrhizic acid.
The efficiency of the Pichia pastoris (Pichia pastoris) strain AtAc3 hydrolysis of glycyrrhetinic acid monoglucuronide is higher than that of glycyrrhizic acid.
During the reaction process of hydrolyzing glycyrrhizic acid by using Pichia pastoris (Pichia pastoris) strain AtAc3, the accumulation of glycyrrhetinic acid monoglucuronide is always lower than 4.8%.
A starter culture for preparing glycyrrhetinic acid comprises fermenting active components; characterized in that the fermentation active ingredients comprise: pichia pastoris (Pichia pastoris) strain AtAc3 with the preservation number of CGMCC No.25102.
The starter for preparing glycyrrhetinic acid further comprises: and (5) auxiliary materials.
A method for preparing glycyrrhetinic acid capable of inhibiting the accumulation of glycyrrhetinic acid monoglucuronide is characterized in that a Pichia pastoris (Pichia pastoris) strain AtAc3 fermentation substrate with the preservation number of CGMCC No.25102 is adopted.
The initial concentration of the substrate is 2-40g/L.
The fermentation conditions include: pH4.0-7.0, temperature 30-50 deg.C;
the substrate is glycyrrhizic acid;
preferably, the GL conversion is as high as 96.58% and GA production efficiency is 9.15mM/h.
The invention provides an AtAc3 strain capable of efficiently hydrolyzing glycyrrhizic acid, which is characterized in that the preservation number is as follows: CGMCC No.25102.
The method for industrially producing the glycyrrhetinic acid is characterized by comprising the step of carrying out catalytic reaction on a substrate by adopting a strain AtAc3.
The pH value of the fermentation and catalysis stage is 4.0-7.0, the temperature is 30-50 ℃, and the initial concentration of the substrate for the catalytic reaction is 2-40g/L.
The period of the fermentation stage is 24-60h, the period of the substrate catalysis is 1-48h, and the feeding is carried out for 0-6 times in the catalysis stage.
The invention verifies the effect of the AtAc3 strain on the preparation of glycyrrhetinic acid, and finds that the conversion rate of glycyrrhizic acid and the accumulation amount of an intermediate product GAMG in the reaction process have obvious advantages compared with the prior reported strains. For example, under the same conditions, the amount of intermediate GAMG accumulation in fermentation of glycyrrhizic acid was reduced 5.13-fold in the AtAc3 strain compared to that of AtGUS strain derived from Aspergillus terreus (Aspergillus terreus) strain Li-20. The AtAc3 strain is subjected to fed-batch fermentation, glycyrrhizic acid is used as a conversion substrate, the whole fermentation and catalysis period is 90 hours, the substrate glycyrrhizic acid conversion rate reaches 96.58 percent after 6 times of feeding, the final concentration of glycyrrhetinic acid is 31.53g/L, and the production efficiency is 9.15mM/h. The results lay an important foundation for the industrial production of glycyrrhetinic acid.
The AtAc3 strain of the invention is preserved, and the preservation information is as follows:
the preservation name of the strain is as follows: atAc3
The preservation number is as follows: CGMCC No.25102
And (3) classification and naming: pichia pastoris
Latin name: pichia pastoris
The preservation unit is as follows: china general microbiological culture Collection center
The address of the depository: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: year 2022, 6 months and 16 days.
Drawings
FIG. 1 is a comparison of the GL hydrolyzing ability of four existing Pichia strains (AtGUS, PGUS, atGUSMix, tpGUS) of Experimental example 1 of the present invention with that of AtAc3 strain.
FIG. 2 is a graph showing the effect of temperature in Experimental example 2 of the present invention on the activity of AtAc3 strain. Influence of temperature on the Total conversion Activity, B: temperature effect on conversion of GL.
FIG. 3 is a graph showing the effect of pH on the activity of AtAc3 strain in Experimental example 3 of the present invention. Influence of pH on the Total conversion Activity, B: influence of pH on the conversion of GL.
FIG. 4 is OD of growth stage of AtAc3 strain in Experimental example 4 of the present invention 600 Time-dependent changes, wherein the glucose feeding time was 18h and 42h.
FIG. 5 is a substrate catalysis stage in a 5L fermenter of the AtAc3 strain of Experimental example 4 of the present invention, showing changes in the substrate GL concentration, the intermediate GAMG concentration and the product GA concentration.
Detailed Description
The present invention will be further described below by way of specific examples and experimental examples. It should be understood that these examples and experimental examples are only for explaining and illustrating the present invention and should not be construed as limiting the scope of the present invention. Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the reagents or materials used are commercially available.
Sources and sources of biological materials
(1) AtGUS is derived from Aspergillus terreus strain Li-20 and is described in reference "Effects of a Non-dominant Sequence on the Properties of β -glucuronidase from Aspergillus terreus Li-20", published in 2012, genBank number JF894133.1.
(2) Aspergillus oryzae (Aspergillus oryzae) strain Li-3, the amino acid sequence of which was submitted to the NCBI database in 2007 by the inventor's topic group under GenBank accession number ABU68712.1.
(3) TpGUS is derived from Talaromyces pinophilus strain Li-93 and is described in the invention patent of 'beta-glucuronidase, gene and application thereof', the patent application number is 201610953058.7, and the strain preservation number is CGMCC No.11765.
(4) AtGUSMix is obtained by replacing the structure domain of AtGUS and PGUS and is described in the invention patent 'an engineering GA108/PGAPZ alpha A-Atgusmix for industrially producing glycyrrhetinic acid and a method', the patent application number is 201811564910.7, and the strain preservation number is CGMCC No.16731.
Reagent and consumable
(1) Culture medium
LB medium (low salt): 10g/L tryptone, 5g/L NaCl, 5g/L yeast extract and natural pH; the LB solid medium was supplemented with 20g/L agar powder and sterilized at 121 ℃ for 15min. YPD medium: tryptone 20g/L, dextrose monohydrate 20g/L, yeast extract 10g/L, natural pH; 20g/L agar powder was additionally added to the YPD solid medium, and the mixture was sterilized at 115 ℃ for 15min.
(2) Common solution
Pichia pastoris strain construction and verification
1M sorbitol: 182.17g sorbitol is weighed, 900mL purified water is added, the volume is adjusted to 1L, the pH is natural, and the mixture is sterilized for 20min at 115 ℃. Lysis solution of lysine: 0.372g of EDTA disodium salt and 2g of sodium hydroxide are respectively weighed, 5mL of Triton X-100 is added, and deionized water is used for dissolving and fixing the volume to 1L.4g/L glycyrrhizic acid-YPD verification solution: weighing 4g glycyrrhizic acid, 20g tryptone, 20g dextrose monohydrate, and 10g yeast extract, sterilizing at 115 deg.C for 15min under natural pH.
(3) Reaction buffer
4g/L glycyrrhizic acid in 50mM HAc-NaAc reaction buffer (pH4.0-5.5): weighing 4g monoammonium glycyrrhizinate and 4.1g anhydrous sodium acetate, adding 950mL purified water, and separating with glacial acetic acidAdjusting pH to 4.0, 4.5, 5.0 or 5.5, adding deionized water to reach volume of 1L, sterilizing at 115 deg.C for 15min, and storing at room temperature. 4g/L glycyrrhizic acid 50mM Na 2 HPO 4 -NaH 2 PO 4 Reaction buffer (pH 6.0-6.5): weighing 4g of monoammonium glycyrrhizinate and 17.9g of Na 2 HPO 4 ·12H 2 Adding 950mL of purified water, adjusting the pH to 6.5 or 6.0 by using phosphoric acid, adding deionized water to the volume of 1L, sterilizing at 115 ℃ for 15min, and storing at room temperature. 4g/L glycyrrhizic acid in 50mM Tris-HCl reaction buffer (pH 7.0): weighing 4g of mono-ammonium glycyrrhizinate and 6.1g of Tris, adding 950mL of purified water, adjusting the pH to 7.0 by using concentrated hydrochloric acid, adding deionized water to a constant volume of 1L, sterilizing at 115 ℃ for 15min, and storing at room temperature.
Group 1 example, the Strain of the invention AtAc3
The group of embodiments provides a Pichia pastoris (Pichia pastoris) strain AtAc3, which is characterized in that the preservation number is CGMCC No.25102.
Any behavior of the Pichia pastoris (Pichia pastoris) strain AtAc3 with the collection number of CGMCC No.25102 is utilized, used, sold, promised, sold, produced, prepared, cultured, propagated and fermented and falls into the protection scope of the invention.
According to the teaching and inspiration of the invention, the skilled in the art selects appropriate auxiliary materials to mix in combination with the common technical means in the field of microbial technology according to the actual production needs, and prepares the Pichia pastoris (Pichia pastoris) AtAc3 with the preservation number of CGMCC No.25102 into various dosage form products, such as powder, tablets and liquid, which meet the production requirements of the technology.
Group 2 examples, use of the Strain AtAc3 of the invention
The group of embodiments provides the application of a Pichia pastoris (Pichia pastoris) strain AtAc3 with the preservation number of CGMCC No.25102 in the hydrolysis of glycyrrhetinic acid monoglucuronide and/or the hydrolysis of glycyrrhizic acid.
In a further embodiment, the efficiency of hydrolyzing glycyrrhetinic acid monoglucuronide is higher than the efficiency of hydrolyzing glycyrrhizic acid.
In a specific embodiment, the GAMG accumulation ratio in the conversion process is always maintained within 4.8% of the total system content.
Group 3 examples of the fermentation Agents of the invention
The group of embodiments provides a starter for preparing glycyrrhetinic acid. All embodiments of this group share the following common features: the leavening agent comprises a leavening active ingredient; the fermentation active ingredients comprise: pichia pastoris (Pichia pastoris) strain AtAc3 with the preservation number of CGMCC No.25102.
In a further embodiment, the fermentation agent for preparing glycyrrhetinic acid further comprises: and (5) auxiliary materials.
In more specific embodiments, the pharmaceutical excipient is selected from the group consisting of: solvents, propellants, solubilizers, solubilizing agents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, and the like.
According to the content of the invention, due to different requirements in practical production and application, by combining with conventional technical means in the fields of preparation, production and processing of microbial inoculum (such as encyclopedia of preparation technology, pharmaceutical preparation technology and the like), a person skilled in the art can select and mix the auxiliary materials, and prepare the Pichia pastoris (Pichia pastoris) strain AtAc3 with the preservation number of CGMCC No.25102 into different dosage forms, such as powder, tablets, injection, oral liquid and the like.
Group 4 example, preparation of Glycyrrhetinic acid of the invention
The present group of embodiments provides a method for preparing glycyrrhetinic acid that can inhibit the accumulation of glycyrrhetinic acid monoglucuronide. All embodiments of this group share the following common features: the substrate is fermented by a Pichia pastoris (Pichia pastoris) strain AtAc3 with the preservation number of CGMCC No.25102.
In specific embodiments, the initial concentration of the substrate is 2-40g/L;
preferably, the catalytic or fermentative conditions comprise: pH4.0-7.0, temperature 30-50 deg.C;
preferably, the substrate is glycyrrhizic acid.
In a more preferred embodiment, the conversion rate of the substrate glycyrrhizic acid reaches 96.58%; the production efficiency of the glycyrrhetinic acid is 9.15mM/h.
Experimental example 1 construction and Activity verification of Strain AtAc3
(1) Obtaining of Strain AtAc3
The experimental example obtains a Pichia pastoris (Pichia pastoris) strain AtAc3, and finds that the Pichia pastoris strain AtAc3 has higher GL hydrolytic activity and higher GAMG hydrolytic activity and can effectively reduce GAMG accumulation in the GA production process. The strain AtAc3 is sent for preservation, and the preservation information is as follows:
the preservation name of the strain is as follows: atAc3
The preservation number is as follows: CGMCC No.25102
And (3) classification and naming: pichia pastoris
Latin name: pichia pastoris
The preservation unit: china general microbiological culture Collection center
The address of the depository: xilu No.1 Hospital No. 3 of Beijing market Chaoyang district
The preservation date is as follows: 6 and 16 months in 2022.
(2) Comparison of hydrolytic Activity of Each Strain
Activity verification and comparison of Pichia pastoris eukaryotic expression are carried out on five strains of AtGUS, PGUS, atGUSMix, tpGUS and AtAc3 to characterize relative activity difference of the strains in GL hydrolysis. The results of examining the GAMG accumulation and GA production in the 5 strains during the reaction by adding 4g/L glycyrrhizic acid to YPD medium are shown in FIG. 1.
In the study of the embodiment, the GAMG concentration of the intermediate product of the new strain AtAc3 is very low, the GAMG accumulation ratio is always lower than 4.8% in the transformation process, the GAMG accumulation of AtGUS, atGUSMix and TpGUS is about 20%, and the GAMG accumulation of PGUS is over 50%.
The results show that the accumulation amount of the intermediate product GAMG of the new strain AtAc3 is respectively reduced by 5.13, 12.60, 4.54 and 4.98 times compared with the intermediate product GAMG of the existing strains AtGUS, PGUS, atGUSMix and TpGUS under the same conditions, which indicates that the accumulation amount of the intermediate product GAMG of the new strain AtAc3 is far lower than that of other strains, namely the extremely low accumulation amount of GAMG in the catalysis process can be ensured. Meanwhile, the new strain AtAc3 has the highest GA conversion rate, so that the overall high conversion efficiency can be ensured, and the new strain AtAc3 can effectively solve the problems that the viscosity of fermentation liquor is too high, the mass transfer process is influenced and the like caused by the GAMG of an intermediate product, and the overall production efficiency is improved. The constructed AtAc3 strain is preserved with the preservation number of CGMCC No.25102.
Experimental example 2 Effect of temperature and pH on the Activity of AtAc3 Strain
Further using YPD culture medium to culture AtAc3 strain, and using 50mM HAc-NaAc reaction buffer solution of 4g/L glycyrrhizic acid as reaction system to make activity comparison so as to obtain comprehensive performance of AtAc3 strain under two investigation modes and provide strain with stronger comprehensive capability for subsequent GA production.
Effect of temperature on the Activity of AtAc3 Strain
Temperature is one of important strain activity influencing factors and directly influences the fermentation reaction rate of the strain and the stability of the strain. And (3) investigating the influence of temperature on the activity of the AtAc3 strain at 30-50 ℃, and comparing the hydrolysis conditions of the AtAc3 strain on GL at different temperatures to obtain the optimal temperature condition for catalytic reaction. The results are shown in FIG. 2. The results in FIG. 2A show that the AtAc3 strain exhibits optimal GL hydrolytic activity at 45 ℃ with the highest overall conversion. The results in FIG. 2B show that at 30-50 deg.C, GAMG is very low in the process of catalyzing GL to generate GA by AtAc3 strain, and GAMG can be effectively inhibited. Namely, the comprehensive effect of AtAc3 is better, high total conversion rate can be kept under the two investigation modes, and extremely low-level GAMG accumulation of the whole reaction system is kept in the investigation temperature range. Therefore, atAc3 was determined to be the optimum strain, and 45 ℃ was the optimum temperature condition.
Experimental example 3 Effect of pH on the Activity of AtAc3 Strain
This experimental example was conducted by examining the optimum pH of the AtAc3 strain in the pH range of 4.0-7.0. Using 4g/L GL as reaction substrate, respectively preparing 50mM reaction systems with different pH values, namely HAc-NaAc buffer solution (pH 4.0-5.5) and Na 2 HPO4-NaH 2 PO 4 Buffer solution (pH6.0-6.5), tris-HCl buffer solution (pH7.0), the optimum reaction temperature was 45 ℃. The results are shown in FIG. 3. Among them, the results of fig. 3A show that at pH5.5, the total conversion of the AtAc3 strain is highest, showing the highest GL hydrolysis activity. The results in FIG. 3B show that more GAMG was accumulated in the intermediate product at pH4.0-5.0, and that the highest GAMG accumulation rate was observed at pH 4.5. Almost no GAMG was accumulated at pH 6-7.0. When the pH was 5.5, the GA productivity was the highest and the GAMG accumulation in the reaction system was low. Therefore, it was confirmed that pH5.5 was the optimum pH condition.
Experimental example 4 validation of AtAc3 Strain in 5L fermenter
The optimum fermentation conditions obtained in the previous trial were combined and verified in a 5L fermentor. The AtAc3 strain plated out was inoculated into a test tube containing YPD liquid medium and activated at 30 ℃ and 200rpm for 36 hours to serve as a primary seed liquid. The primary seed solution was transferred to a 250mL shake flask containing 100mL YPD liquid medium at OD =0.1 and activated at 30 ℃ and 200rpm for 24 hours to serve as a secondary seed solution. The secondary seed liquid was transferred to a 5L fermentor containing 3L YPD liquid medium. The fermentation tank parameters are set to pH5.5, the fermentation temperature is 30 ℃, and the rotation speed is 200rpm. The fermentation process lasts for 60 hours, and glucose is supplemented twice. OD for real-time detection of thallus growth in fermentation process 600 The results are shown in FIG. 4.
In the substrate catalysis stage, the catalytic effect of the AtAc3 strain is quantified and verified by measuring the residual concentration of the substrate GL, the accumulation concentration of the intermediate product GAMG and the generation concentration of the product GA in the reaction system. The results are shown in FIG. 5.
The results show that the period of the whole substrate catalysis phase is 30h, 6 times of substrate GL feeding are carried out totally, and the concentration of each feeding GL is 20g/L. At 24h, the GA concentration was 31.45g/L, the GL conversion rate reached 96.33%, at 30h, the GA final concentration reached 31.53g/L, the GL conversion rate reached 96.58%, and the GA production efficiency was 9.15mM/h. In addition, in the 5L fermentation tank scale-up test, GAMG still meets the above characteristics, and can maintain the low GAMG concentration of the intermediate product in the catalytic reaction system, the GAMG concentration at 30h is less than 1.67g/L, and the GAMG content in the system is only 4.78%. In conclusion, the AtAc3 strain has the advantages of mild reaction conditions, high catalytic efficiency, low accumulation of intermediate GAMG, high product yield and the like, and further lays a solid foundation for industrial production of glycyrrhetinic acid.

Claims (10)

1. A Pichia pastoris (Pichia pastoris) strain AtAc3 is characterized in that the preservation number is CGMCC No.25102.
2. Use of Pichia pastoris (Pichia pastoris) strain AtAc3 with preservation number of CGMCC No.25102 in hydrolyzing glycyrrhetinic acid monoglucuronide and/or glycyrrhizic acid.
3. The use according to claim 2, characterized in that the efficiency of the Pichia pastoris (Pichia pastoris) strain AtAc3 for hydrolysis of glycyrrhetinic acid monoglucuronide is higher than the efficiency for hydrolysis of glycyrrhizic acid.
4. The use of claim 3, wherein the accumulated amount of glycyrrhetinic acid monoglucuronide is always less than 4.8% during the hydrolysis reaction of glycyrrhizic acid by the Pichia pastoris (Pichia pastoris) strain AtAc3.
5. A starter culture for preparing glycyrrhetinic acid comprises fermenting active components; characterized in that the fermentation active ingredients comprise: pichia pastoris (Pichia pastoris) strain AtAc3 with the preservation number of CGMCC No.25102.
6. The fermentation agent for preparing glycyrrhetinic acid according to claim 5, further comprising: and (5) auxiliary materials.
7. A method for preparing glycyrrhetinic acid capable of inhibiting the accumulation of glycyrrhetinic acid monoglucuronide is characterized in that a Pichia pastoris (Pichia pastoris) strain AtAc3 fermentation substrate with the preservation number of CGMCC No.25102 is adopted.
8. The method for preparing glycyrrhetinic acid capable of inhibiting the accumulation of glycyrrhetinic acid monoglucuronide according to claim 7, wherein the initial concentration of the substrate is 2-40g/L.
9. The method for preparing glycyrrhetinic acid according to claim 7, wherein the fermentation conditions include: pH4.0-7.0, and temperature 30-50 deg.C.
10. The method for producing glycyrrhetinic acid capable of inhibiting the accumulation of glycyrrhetinic acid monoglucuronide according to claim 7 or 8, wherein the substrate is glycyrrhizic acid;
and/or, the GL conversion rate is up to 96.58%, and the GA production efficiency is 9.15mM/h.
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