CN114058523A - Saccharomyces cerevisiae and fermentation product and application thereof - Google Patents

Saccharomyces cerevisiae and fermentation product and application thereof Download PDF

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CN114058523A
CN114058523A CN202111310132.0A CN202111310132A CN114058523A CN 114058523 A CN114058523 A CN 114058523A CN 202111310132 A CN202111310132 A CN 202111310132A CN 114058523 A CN114058523 A CN 114058523A
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tryptophan
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weight percent
saccharomyces cerevisiae
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曾世超
赵晓岚
郭雅珊
邢晨光
刘刚
林晓慧
刘超霞
蔡秀莲
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Xiamen Oamic Biotechnology Co Ltd
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Abstract

The invention relates to saccharomyces cerevisiae, a fermentation product and application thereof, and belongs to the technical field of fermentation engineering. The saccharomyces cerevisiae or the fermentation product thereof can be used for preparing anthranilic acid by biodegradation of L-tryptophan, and the method has the advantages of high reaction speed, safe process, environmental protection, less byproducts, convenience in downstream extraction and separation and the like, and the obtained anthranilic acid is high in yield and purity.

Description

Saccharomyces cerevisiae and fermentation product and application thereof
Technical Field
The invention relates to the technical field of fermentation engineering, in particular to saccharomyces cerevisiae, a fermentation product and application thereof.
Background
Methyl anthranilate has fruit aroma, flower aroma, powder aroma, mildew aroma and grape aroma, is naturally contained in grapes, strawberries, red wine and white wine, is an important raw material for preparing essences of the types of grapes, citrus sweet berries, strawberries, watermelons and the like, is also an essential raw material for preparing artificial orange oil, and has large market demand.
Methyl anthranilate currently on the market is mainly derived from chemical synthesis. With the progress and development of society, the willingness of human beings to return to nature is stronger and stronger, and the application of natural essence and spice in the food industry is more and more extensive. Compared with a chemically synthesized product, the natural essence and spice obtained by biotransformation by taking a natural substrate as a raw material has higher market acceptance and added value.
In the process of implementing the invention, the inventor finds that at least the following problems exist in the prior art: in the prior art, the production of natural methyl anthranilate mainly relies on the oxidative demethylation of methyl N-methyl anthranilate, which can be obtained from Egyptian bitter orange leaf oil. However, the reaction depends on MnO2The chemical oxidants can not only cause the problem of natural authentication of the produced methyl anthranilate, but also generate a large amount of waste residues which are difficult to treat, thereby causing environmental protection pressure, generating a series of byproducts and being not beneficial to subsequent separation and extraction operations. The preparation of anthranilic acid by means of microbial fermentation is another possible way for preparing natural methyl anthranilate, but the current space-time productivity and product concentration are low, and the method has no industrial application value. For example, V i factor E Balderas-Hern z et al report that the use of genetically engineered E.coli can be used with grapeThe o-aminobenzoic acid is produced by taking sugar as a raw material, and only 14g/L o-aminobenzoic acid can be produced after fermentation for 34 h. Jannis Kuepper et al reported that only 1.54g/L anthranilic acid could be obtained within 70h using glucose as raw material by applying molecularly modified Pseudomonas putida.
Therefore, a method for preparing anthranilic acid and methyl anthranilate, which is safe, green and environment-friendly, has few byproducts, is easy to extract and separate, and has high yield and high purity, is still needed.
Disclosure of Invention
Summary of The Invention
The object of the present document is to provide a saccharomyces cerevisiae and its fermentation product and use, in a first aspect, a saccharomyces cerevisiae; in a second aspect, there is provided a fermentation product of saccharomyces cerevisiae; in a third aspect, a method for producing the fermentation product is provided; in a fourth aspect, a process for preparing anthranilic acid is provided; in a fifth aspect, there is provided a process for the preparation of methyl anthranilate; in a sixth aspect, there is provided a use of the saccharomyces cerevisiae of the first aspect or the fermentation product of the saccharomyces cerevisiae of the second aspect. The provided saccharomyces cerevisiae has good catalytic activity, can be used for degrading L-tryptophan to prepare anthranilic acid, and the obtained anthranilic acid has high purity, high yield, quick reaction, high safety, green and environment-friendly process, few byproducts, convenient downstream extraction and separation and less solvent residue.
Detailed Description
The present document provides the following technical solutions.
In a first aspect, a saccharomyces cerevisiae is provided.
The saccharomyces cerevisiae has the preservation number of: CCTCC M20211292 (deposited in China center for type culture Collection at 10/18/2021).
The Saccharomyces cerevisiae is named as Saccharomyces cerevisiae OMK-77.
In a second aspect, a fermentation product of Saccharomyces cerevisiae is provided.
A fermentation product of the saccharomyces cerevisiae of the first aspect.
In some embodiments, the fermentation product may be a fermentation broth, a fermentation supernatant, a cell suspension, and/or intracellular material.
In a third aspect, a method of producing the aforementioned fermentation product is provided.
A method of producing a fermentation product of the second aspect, comprising: activating the saccharomyces cerevisiae of the first aspect, performing at least one stage of amplification culture, and collecting a fermentation product.
In some embodiments, the expanded culture comprises a primary expanded culture, a secondary expanded culture, a tertiary expanded culture, or a quaternary expanded culture.
In a fourth aspect, a method of preparing anthranilic acid is provided.
A method for preparing anthranilic acid comprises the following steps:
Figure BDA0003340559300000021
l-tryptophan is fermented with a microorganism or a fermentation product of the microorganism to produce anthranilic acid.
The microorganism includes at least one selected from the group consisting of Corynebacterium (Corynebacterium sp.), Bacillus (Bacillus sp.), Lactobacillus (Lactobacillus sp.), Lactococcus (Lactobacillus sp.), Pseudomonas (Pseudomonas sp.), Clostridium (Clostridium sp.), Rhodococcus (Rhodococcus sp.), Rhodobacterium (Rhodobacter sp.), Streptococcus (Streptococcus sp.), Saccharomyces (Saccharomyces sp.), Aspergillus (Aspergillus sp.), and Streptomyces (Streptomyces sp.).
In some embodiments, the microorganism is selected from the genus saccharomyces; preferably, the microorganism is the saccharomyces cerevisiae of the first aspect.
In some embodiments, the fermentation product of the microorganism is the fermentation product of the second aspect or the fermentation product of the production method of the third aspect.
In some embodiments, a method of making anthranilic acid comprises:
(1) preparing a seed culture solution: carrying out at least one-stage amplification culture on the strains of the microorganisms, or sequentially carrying out solid slant culture and seed culture to obtain a seed culture solution;
(2) inoculating the seed culture solution into a fermentation culture medium, culturing for 6-12h, adding L-tryptophan according to the addition amount of 10-50 g/L, and continuously culturing until all L-tryptophan is converted into anthranilic acid to obtain a biotransformation mixture;
(3) and (3) carrying out aftertreatment 1 on the biotransformation mixture to obtain anthranilic acid.
The culture time for continuing the culture until all L-tryptophan is converted into anthranilic acid can be 40-60 h. In some embodiments, the culturing is continued until all of the L-tryptophan is converted to anthranilic acid for a culture time of 45h to 55 h.
In some embodiments, a method of making anthranilic acid comprises:
(1) preparing a seed culture solution: carrying out at least one-stage amplification culture on the strains of the microorganisms, or sequentially carrying out solid slant culture and seed culture to obtain a seed culture solution;
(2) inoculating the seed culture solution into a fermentation culture medium, culturing for 6-12h, and adding a first part of L-tryptophan to induce the expression of L-tryptophan degradation pathway related enzymes; then adding L-tryptophan in batches every 8-10h and continuing to culture, and continuing to culture for 8-30 h after adding L-tryptophan for the last time to obtain a biotransformation mixture;
(3) and (3) carrying out aftertreatment 1 on the biotransformation mixture to obtain anthranilic acid.
The culture temperature of the strain in the solid slant medium may be about 25 ℃ to about 30 ℃. In some embodiments, the culture temperature of the bacterial species in the solid slant medium is about 28 ℃ to 30 ℃.
The culture time of the strain in the solid slant culture medium may be about 40 hours to about 60 hours. In some embodiments, the culture time of the strain on the solid slant medium is 45h to 55 h.
The seed culture may be grown in seed medium at a temperature of about 28 ℃ to about 30 ℃. In some embodiments, the seed culture is incubated in a seed medium at a temperature of 28 ℃.
The culture of the strain in the seed culture medium can be primary culture or secondary culture, wherein the primary culture means that the microorganism is cultured in the seed culture medium only once; the second-stage culture means that the microorganism is cultured in a seed culture medium once to obtain a first-stage seed solution, and then the first-stage seed solution is inoculated in a fresh seed culture medium to be continuously cultured once.
The culture time of the primary culture can be 4-10 h. In some embodiments, the seed culture is incubated for 4h to 6 h.
The culture time of each culture of the secondary culture can be 8-14 h. In some embodiments, the seed culture is incubated for 10h to 12 h.
The stirring speed of the strain during the culture of the seed culture medium can be 200rpm-250 rpm.
When the seed culture solution is inoculated to the fermentation culture medium, the volume ratio of the seed culture solution to the fermentation culture medium can be 6: 100-12: 100.
The pH of the fermentation medium may be 5.0-6.5.
The pH of the seed culture may be 5.0-6.5.
The culture temperature of the culture in the fermentation medium in the step (2) may be 28 ℃ to 30 ℃.
The stirring rotation speed for the culture in the fermentation medium in the step (2) may be 100rpm to 600 rpm. In some embodiments, the stirring speed for culturing in the fermentation medium in the step (2) may be 200rpm to 500 rpm. In some embodiments, the stirring speed for culturing in the fermentation medium in the step (2) may be 250rpm to 400 rpm.
The aeration ratio of the seed culture solution inoculated in the fermentation culture medium in the step (2) during culture can be 1: 0.2-1: 1. In some embodiments, the seed culture solution is inoculated into the fermentation medium in the step (2) and then cultured at an aeration ratio of 1: 0.4 to 1: 0.6. In some embodiments, the aeration ratio of the seed culture solution after being inoculated into the fermentation medium in the step (2) is 1: 0.5.
In some embodiments, a method of making anthranilic acid comprises:
(1) preparing a seed culture solution: coating strains of microorganisms on a solid slant culture medium, performing static culture at 28 ℃ for 48 hours to obtain activated strains, inoculating the activated strains into a seed culture medium, and performing shake culture at 28-30 ℃ and 200-250 rpm to the early logarithmic phase to obtain a seed culture solution;
(2) inoculating the seed culture solution obtained in the step (1) into a fermentation culture medium according to the volume ratio of the seed culture solution to the fermentation culture medium of 6: 100-12: 100, culturing for 6-12h under the conditions of pH 5.0-pH6.5, 28-30 ℃, stirring speed of 100-600 rpm and aeration ratio of 1: 0.2-1: 1, adding a first part of L-tryptophan to induce the expression of the enzyme related to the L-tryptophan degradation pathway, continuously culturing for 8-10h, adding a second part of L-tryptophan and continuously culturing; adding L-tryptophan in batches, continuously culturing, and continuously culturing for 8-30 h after adding L-tryptophan for the last time to obtain a biotransformation mixture;
(3) and (3) carrying out aftertreatment 1 on the biotransformation mixture to obtain anthranilic acid.
In some embodiments, the aeration ratio is 1: 0.4 to 1: 0.6. In some embodiments, the aeration ratio is 1: 0.5.
The first portion of L-tryptophan may be added in an amount of 1g/L to 5 g/L. In some embodiments, the first portion of L-tryptophan is added in an amount of 2g/L to 4g/L or 2g/L to 3 g/L.
The second portion of L-tryptophan or the portion of L-tryptophan added in portions may be added in an amount of 5g/L to 20g/L per time. In some embodiments, the second portion of L-tryptophan or the portion of L-tryptophan added in portions is added in an amount of 10g/L to 15g/L per addition.
The L-tryptophan is added in batches, namely the L-tryptophan is added once every 8 to 15 hours after the second part of L-tryptophan is added according to the addition amount of 5 to 20 g/L. In some embodiments, the addition of L-tryptophan in portions may be performed such that the L-tryptophan is added in an amount of 5g/L to 20g/L every 10h to 15h after the second portion of L-tryptophan is added. In some embodiments, the addition of L-tryptophan in portions may be performed once every 12h to 15h after the addition of the second portion of L-tryptophan, and the addition amount of L-tryptophan is 5g/L to 20 g/L.
The total number of times of addition of the L-tryptophan may be 2 to 8 times. In some embodiments, the total number of additions of L-tryptophan may be 2, 3, 4, 5, 6, 7, or 8.
In some embodiments, the incubation is continued for 8h to 30h after the last addition of L-tryptophan. In some embodiments, the incubation is continued for 10h to 30h after the last addition of L-tryptophan. In some embodiments, the incubation is continued for 10h to 25h after the last addition of L-tryptophan. In some embodiments, the incubation is continued for 15h to 25h after the last addition of L-tryptophan. In some embodiments, the incubation is continued for 18h to 25h after the last addition of L-tryptophan.
The post-treatment 1 may include: filtering to remove thalli, adjusting pH with sulfuric acid to separate out anthranilic acid, filtering and collecting filter residue to obtain crude anthranilic acid, and recrystallizing the obtained crude anthranilic acid with water as a solvent.
The adjusting the pH with sulfuric acid may include adjusting the pH to 2-3 with sulfuric acid.
The seed medium may comprise or consist of, based on the total mass of the seed medium: 1.5 to 5 weight percent of peptone, 0.5 to 3 weight percent of yeast extract, 2 to 5 weight percent of glucose and a solvent, wherein the solvent is water. In some embodiments, the seed medium comprises, based on the total mass of the seed medium: peptone 2.0 wt%, yeast extract 1.0 wt%, glucose 2.0 wt%, and water in balance.
The solid slant medium may comprise or consist of, based on the total mass of the solid slant medium: peptone 1.5 wt% -5 wt%, yeast extract 0.5 wt% -3 wt%, glucose 2 wt% -5 wt%, agar 1 wt% -5 wt%, and solvent, wherein the solvent is water. In some embodiments, the solid slant medium comprises, based on the total mass of the solid slant medium: peptone 2.0 wt%, yeast extract 1.0 wt%, glucose 2.0 wt%, agar 2.0 wt%, and water in balance.
The fermentation medium may comprise or consist of, based on the total mass of the fermentation medium: 3.0 to 5.0 weight percent of glucose, 0.5 to 1.0 weight percent of ammonium dihydrogen phosphate, 0.2 to 0.5 weight percent of monopotassium phosphate, 0.1 to 0.2 weight percent of magnesium sulfate, 0.05 to 0.1 weight percent of calcium sulfate, 0.2 to 1.0 weight percent of yeast extract powder and a solvent; the solvent is water. In some embodiments, the fermentation medium comprises, based on the total mass of the fermentation medium: 4 wt% of glucose, 1 wt% of ammonium dihydrogen phosphate, 0.5 wt% of potassium dihydrogen phosphate, 0.2 wt% of magnesium sulfate, 0.1 wt% of calcium sulfate, 0.5 wt% of yeast extract powder and the balance of water.
In a fifth aspect, a process for preparing methyl anthranilate is provided.
A process for the preparation of methyl anthranilate which comprises esterifying anthranilate obtained by the process of the second aspect to obtain methyl anthranilate.
The esterification may comprise any of the methods mentioned in the prior art.
In some embodiments, a method for preparing methyl anthranilate comprises dissolving anthranilic acid obtained from the method of the second aspect in methyl tert-butyl ether, adding natural methanol, Novozyme 435, reacting at 30 ℃ to 50 ℃ for 16h to 20h, and post-treating 2 to obtain methyl anthranilate.
The post-treatment 2 may include: filtering to recover Novozyme 435, distilling the filtrate to recover methyl tert-butyl ether to obtain a pot bottom; and rectifying the obtained pan bottom to obtain methyl anthranilate.
In a sixth aspect, there is provided a use of the saccharomyces cerevisiae of the first aspect or the fermentation product of the second aspect.
Use of a saccharomyces cerevisiae strain according to the first aspect or a fermentation product according to the second aspect for the biodegradation of L-tryptophan to anthranilic acid.
Advantageous effects
Compared with the prior art, one of the technical solutions has one of the following beneficial technical effects:
(1) the Saccharomyces cerevisiae OMK-77 prepared by the technical scheme is used for fermenting and degrading L-tryptophan to prepare anthranilic acid, the space-time yield of the anthranilic acid can reach 0.72g/(L h), the product concentration can reach 30.2g/L, the production cost is greatly reduced compared with the existing production process, the production of methyl anthranilate can be realized by simple enzyme-catalyzed esterification reaction subsequently, and the industrial application prospect is good.
(2) The Saccharomyces cerevisiae OMK-77 prepared by adopting the technical scheme is used for fermenting L-tryptophan to prepare anthranilic acid, the purity of the obtained anthranilic acid can reach more than 99 percent, the yield is as high as 85 percent, the purity of the further prepared methyl anthranilate can reach more than 99 percent, and the yield is as high as 78 percent.
(3) The preparation method adopting the technical scheme is adopted to prepare the anthranilic acid and methyl anthranilate, organic solvents are not used or the use of the organic solvents is greatly reduced, and MnO is not needed2The preparation method has the advantages of fast reaction, high safety, green and environment-friendly process, less by-products, convenient downstream extraction and separation, less solvent residue and the like.
Definition of terms:
unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. The term "aeration ratio" refers to the ratio of aeration per minute to the actual feed volume of the tank. If the aeration ratio is 1: 04, the ratio of aeration per minute to the actual feed liquid volume of the tank body is 1: 0.4.
The term "weight percent" or "percent by weight" or "wt%" is defined as the weight of an individual component in a composition divided by the total weight of all components of the composition and multiplied by 100.
The term "g/L" denotes the ratio of the weight of the substance described to the volume of the mixture containing the substance.
The term "rpm" means the rotational speed "rpm".
The term "room temperature" refers to the ambient room temperature, which may range from 15 ℃ to 30 ℃, or from 20 ℃ to 30 ℃, or from 25 ℃ to 30 ℃, or about 25 ℃.
All numbers disclosed herein are approximate, whether or not the word "about" or "approximately" is used herein. Based on the numbers disclosed, the numerical values of each number may vary by less than + -10% or reasonably as recognized by one of ordinary skill in the art, such as by + -1%, + -2%, + -3%, + -4%, or + -5%.
The term "natural methanol" is methanol obtained by fermentation, for example from sucrose as a starting material.
The term "space-time yield" means the mass of anthranilic acid obtained per unit volume of culture broth (including seed culture broth and fermentation medium) per unit time under given reaction conditions.
The description of the terms "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of this document. Throughout this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Drawings
FIG. 1 is a HPLC chart of the fermentation degradation of L-tryptophan by Saccharomyces cerevisiae OMK-77 of example 2 to produce natural anthranilic acid.
Detailed Description
In order to make the technical solutions of the present document better understood by those skilled in the art, some non-limiting examples are further disclosed below to further explain the present document in detail.
The reagents used herein are either commercially available or can be prepared by the methods described herein. Example 1: production of anthranilic acid (triangular shake flask fermentation)
(1) Strain acquisition
A strain capable of degrading L-tryptophan into anthranilic acid is separated from the surface of the apple peel, and is identified as Saccharomyces cerevisiae through 18S rDNA and ITS sequencing, and the Saccharomyces cerevisiae OMK-77 is named as Saccharomyces cerevisiae. The strain is preserved in China center for type culture Collection (China, Wuhan university) at 18 months 10 in 2021, with the preservation number: CCTCCM 20211292.
(2) Activating strains: coating Saccharomyces cerevisiae OMK-77 on solid slant culture medium from-80 deg.C glycerin pipe, and standing at 28 deg.C for 48 hr to obtain activated strain.
The formula of the solid slant culture medium comprises the following components in percentage by mass: peptone 2.0 wt%, yeast extract 1.0 wt%, glucose 2.0 wt%, agar 2.0 wt%, and water in balance.
(3) Preparing a seed culture solution: inoculating the obtained activated strain into 500mL triangular shake flask containing 30mL seed culture medium, and culturing at 28 deg.C and 250rpm for 4-6h to obtain seed culture solution.
The formula of the seed culture medium comprises the following components in percentage by mass: peptone 2.0 wt%, yeast extract 1.0 wt%, glucose 2.0 wt%, and water in balance, initially pH6.0, sterilized at 115 deg.C for 30 min.
(4) Fermentation and transformation: inoculating 5mL of seed culture solution into a 500mL triangular shake flask containing 50mL of fermentation medium, fermenting at 28 deg.C and 250rpm for 10h, adding L-tryptophan at a ratio of 20g/L, and fermenting at 28 deg.C and 250rpm for 48 h.
(5) Precipitation and recrystallization: after the fermentation is finished, heating to 60 ℃ for inactivation, and centrifuging to remove thalli. Adjusting the pH of the fermentation supernatant to 2.0 by using sulfuric acid, and centrifuging and collecting precipitates. Dissolving the precipitate in a proper amount of hot water of 90 ℃, and then cooling the system to room temperature to separate out anthranilic acid crystals. The crystal is dried to remove water, thus obtaining 0.628g of pure anthranilic acid product with purity more than 99 percent and yield of 85 percent.
The formula of the fermentation medium comprises the following components in percentage by mass: glucose 4 wt%, ammonium dihydrogen phosphate 1 wt%, potassium dihydrogen phosphate 0.5 wt%, magnesium sulfate 0.2 wt%, calcium sulfate 0.1 wt%, yeast extract powder 0.5 wt%, and water in balance, the initial pH is 6.0, and the mixture is sterilized at 115 ℃ for 30 min.
Example 2: production of methyl anthranilate (fermentation tank fermentation)
The steps (1) to (2) are the same as in example 1;
(3) preparing a seed culture solution: inoculating the obtained activated strain into a 500mL triangular shake flask filled with 50mL seed culture medium, and culturing at 28 ℃ and 250rpm for 10-12h to obtain a primary seed solution; completely inoculating 50mL of the primary seed solution into a 10L seed tank filled with 6L of seed culture medium, and culturing at 28 deg.C and stirring speed of 250rpm for 10-12h with aeration ratio of 1: 0.5 to obtain secondary seed solution;
the formulation of the seed medium was the same as in example 1.
(4) Fermentation and transformation: 30L of fermentation tank is filled with 15.0L of fermentation medium, the fermentation medium is sterilized for 30min at the temperature of 115 ℃, the cultured secondary seed liquid is inoculated into a 30L fermentation tank for culture according to the inoculum concentration of 10 percent, and the culture conditions are as follows: the temperature is 28 ℃, the stirring speed is 400rpm, the aeration ratio is 1: 0.5, L-tryptophan is added according to the addition of 2g/L after fermentation culture is carried out for 8h, the fermentation is continued, and then the L-tryptophan is added once according to the addition of 15g/L after fermentation is carried out for 16h, 28h and 40h respectively (the fermentation time is calculated from the start of the secondary seed solution being inoculated into a 30L fermentation tank), wherein the total fermentation time is 58 h. (58h reaction progress as shown in FIG. 1, L-tryptophan has been converted essentially completely to anthranilic acid, the space-time yield is 0.72g/(L x h), the product concentration is 30.2 g/L).
(5) Precipitation and recrystallization: after the fermentation is finished, heating to 60 ℃ for inactivation, and filtering to remove thalli. Adjusting pH of the fermentation supernatant to 2.0 with sulfuric acid, filtering and collecting precipitate. Dissolving the precipitate in 90 deg.C hot water as little as possible, and cooling the system to room temperature to separate out anthranilic acid crystal. Drying and dewatering the crystal to obtain 458.3g of pure anthranilic acid product with purity higher than 99% and yield of 88%.
(6) Esterification: dissolving 458.3g of anthranilic acid crystals obtained in the step (5) in 7L of methyl tert-butyl ether, adding 350g of Novozyme 435 and 1kg of activated molecular sieve 3A, starting stirring, controlling the reaction temperature to be 45 ℃, supplementing 172ml of natural methanol in the reaction for 0h, 4h, 9h and 15h respectively, and reacting for 22h in total to obtain a biotransformation mixture.
(7) Recovering and rectifying a catalyst and a solvent: filtering the biotransformation mixture obtained in the step (6) to recover Novozyme 435 and molecular sieve 3A, transferring clear liquid to a distillation flask, and recovering methyl tert-butyl ether at normal pressure to obtain a residue; and (3) transferring the obtained residue to a rectifying tower for rectification, controlling the rectification temperature, and collecting fractions at the corresponding temperature to obtain 447.8g of methyl anthranilate with the purity of more than 99% and the total yield of 78%.
The formulation of the fermentation medium was the same as that of example 1.
While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods and applications described herein, as well as other variations and combinations of the techniques described herein, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention.

Claims (10)

1. The saccharomyces cerevisiae has the preservation number of: CCTCC M20211292.
2. A fermentation product of the Saccharomyces cerevisiae of claim 1.
3. A method of producing the fermentation product of claim 2, comprising: activating the Saccharomyces cerevisiae of claim 1, and performing at least one stage of scale-up culture to collect the fermentation product.
4. A method for preparing anthranilic acid is characterized in that: the method comprises the following steps: l-tryptophan is fermented with a microorganism or a fermentation product of the microorganism to produce anthranilic acid.
5. The method according to claim 4, wherein the microorganism comprises at least one selected from the group consisting of Corynebacterium, Bacillus, Lactobacillus, lactococcus, Pseudomonas, Clostridium, Rhodococcus, Streptococcus, Saccharomyces, Aspergillus, and Streptomyces; preferably, the microorganism is the Saccharomyces cerevisiae as claimed in claim 1.
6. The method according to any one of claims 4-5, comprising:
(1) preparing a seed culture solution: carrying out at least one-stage amplification culture on the strains of the microorganisms, or sequentially carrying out solid slant culture and seed culture to obtain a seed culture solution;
(2) inoculating the seed culture solution into a fermentation culture medium, culturing for 6-12h, adding L-tryptophan according to the addition amount of 10-50 g/L, and continuously culturing until all L-tryptophan is converted into anthranilic acid to obtain a biotransformation mixture; or
Inoculating the seed culture solution into a fermentation culture medium, culturing for 6-12h, and adding a first part of L-tryptophan to induce the expression of L-tryptophan degradation pathway related enzymes; adding L-tryptophan in batches every 8-10h, continuously culturing, and continuously culturing for 8-30 h after adding L-tryptophan for the last time to obtain a biotransformation mixture;
(3) and (3) carrying out aftertreatment 1 on the biotransformation mixture to obtain anthranilic acid.
7. The method according to any one of claims 4 to 6, wherein the seed medium comprises, based on the total mass of the seed medium: 1.5-5 wt% of peptone, 0.5-3 wt% of yeast extract, 2-5 wt% of glucose and a solvent, wherein the solvent is water; and/or
The solid slant culture medium comprises the following components in percentage by mass: 1.5 to 5 weight percent of peptone, 0.5 to 3 weight percent of yeast extract, 2 to 5 weight percent of glucose, 1 to 5 weight percent of agar and a solvent, wherein the solvent is water; and/or
Based on the total mass of the fermentation medium, the fermentation medium comprises: 3.0 to 5.0 weight percent of glucose, 0.5 to 1.0 weight percent of ammonium dihydrogen phosphate, 0.2 to 0.5 weight percent of monopotassium phosphate, 0.1 to 0.2 weight percent of magnesium sulfate, 0.05 to 0.1 weight percent of calcium sulfate, 0.2 to 1.0 weight percent of yeast extract powder and a solvent; the solvent is water.
8. A process for the preparation of methyl anthranilate which comprises esterifying anthranilate obtained by the process of any one of claims 4 to 7.
9. The method according to claim 8, which comprises dissolving anthranilic acid obtained by the method of claim 6 in methyl tert-butyl ether, adding natural methanol, Novozyme 435, reacting at 30-50 ℃ for 16-20 h, and performing post-treatment 2 to obtain methyl anthranilate.
10. Use of the saccharomyces cerevisiae according to claim 1 or the fermentation product of saccharomyces cerevisiae according to claim 2 for the preparation of anthranilic acid by biodegradation of L-tryptophan.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
EP0549421A1 (en) * 1991-12-20 1993-06-30 Pernod-Ricard Process for the production of anthranilic acid and methylanthranilate by using saccharomyces cerivisiae mutants
CN102311927A (en) * 2011-08-19 2012-01-11 华南理工大学 Medium and method for high density fermentation of saccharomyces cerevisiae
US20200270587A1 (en) * 2019-02-26 2020-08-27 Korea Advanced Institute Of Science And Technology Recombinant microorganism capable of producing methyl anthranilate and method of producing methyl anthranilate using the same

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Publication number Priority date Publication date Assignee Title
EP0549421A1 (en) * 1991-12-20 1993-06-30 Pernod-Ricard Process for the production of anthranilic acid and methylanthranilate by using saccharomyces cerivisiae mutants
CN102311927A (en) * 2011-08-19 2012-01-11 华南理工大学 Medium and method for high density fermentation of saccharomyces cerevisiae
US20200270587A1 (en) * 2019-02-26 2020-08-27 Korea Advanced Institute Of Science And Technology Recombinant microorganism capable of producing methyl anthranilate and method of producing methyl anthranilate using the same

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