CN102154166B - A kind of Pseudomonas alcaligenes and its method and application for preparing L-menthol - Google Patents

Abstract

The invention discloses pseudomonas alcaligenes, a method for preparing L-menthol by the pseudomonas alcaligenes and application of the pseudomonas alcaligenes. The method for preparing the L-menthol by the pseudomonas alcaligenes comprises the following steps of: (1) adding the pseudomonas alcaligenes into a culture medium to carry out fermentation culture; (2) processing fermentation liquor and performing a conversion reaction on a substrate to obtain reaction solution, wherein the substrate is a mixture of menthol isomer esterified esters and the mixture of the menthol isomer esterified esters contains L-menthol esterified ester and one or more of L-isomenthol esterified ester, L-neoisomenthol esterified ester, L-neomenthol esterified ester, d-menthol esterified ester, d-isomenthol esterified ester, d-neoisomenthol esterified ester and d-neomenthol esterified ester; and (3) carrying out reduced pressure evaporation on the reaction solution, separating the reaction substrate from a product and extracting to obtain 1-menthol.

Description

A kind of Pseudomonas alcaligenes and its method and application for preparing L-menthol

technical field

The present invention relates to a kind of Pseudomonas alcaligenes (Pseudomonas alcaligenes) and preparation of L-menthol (i.e. (-)-menthol, chemical name is (1R, 2S, 5R)-2-isopropyl-5-methyl Cyclohexanol) method.

Background technique

Natural menthol is mainly L-menthol. L-menthol has a characteristic mint aroma and a strong cooling effect, and the smell is fresher and lighter; L-menthol has stronger physiological activity. , sedation, antiseptic and sterilization, pain treatment and other effects, so it has greater medical value. These properties of L-menthol make it have higher industrial value than other configurations of menthol.

Due to people's increasing concern for quality of life, the demand for L-menthol is also increasing. However, the naturally extracted L-menthol is overly dependent on artificially cultivated mint plants, and is more and more difficult to meet the needs of production and life due to the influence of regions and climates. People prepare L-menthol by synthetic means to meet demand, because the product prepared by chemical method is made up of 8 kinds of isomers of menthol, which are respectively L-menthol ((1R, 2S, 5R)-2- Isopropyl-5-methyl-cyclohexanol), L-isomenthol ((1R,2S,5S)-2-isopropyl-5-methyl-cyclohexanol), L-neoisomenthol ((1S, 2S, 5S)-2-isopropyl-5-methyl-cyclohexanol), L-neomenthol ((1R, 2R, 5S)-2-isopropyl-5-methyl- cyclohexanol), d-menthol ((1S,2R,5S)-2-isopropyl-5-methyl-cyclohexanol), d-isomenthol ((1S,2R,5R)-2- isopropyl-5-methyl-cyclohexanol), d-neo-menthol ((1R,2R,5R)-2-isopropyl-5-methyl-cyclohexanol), d-neo-menthol ((1S,2S,5R)-2-Isopropyl-5-methyl-cyclohexanol). Among them, the content of L-menthol is low, which seriously affects the use effect. The L-menthol product separated from the mixture of 8 kinds of menthol isomers is cheaper than natural sources, and the quality is equivalent, so it is industrialized. A major part of the process is the isolation of L-menthol.

Chemical preparation technology, biological preparation technology and natural raw material extraction technology are the three main aspects of the current research on the preparation of L-menthol. The chemical preparation technology is currently applied in industry, mainly by Japan Takasago Corporation using the invented chiral catalyst (S)-BINAP-Rh to catalyze the asymmetric isomerization of allylamine to synthesize L-menthol. The current annual output of this technology is 1000 tons L-menthol. The extraction process from natural plant sources is to obtain mint crude oil through steam distillation of mint grass. Obtain L-menthol through repeated crystallization, the process is complicated, the energy consumption is high, and the productivity is low. In the research on the preparation of L-menthol by biocatalysis, two kinds of resolution reactions are mainly involved, one is the esterification or transesterification reaction catalyzed by microorganisms or enzymes, and the other is the hydrolysis reaction catalyzed by microorganisms or enzymes . Yang Lirong etc. have developed a kind of method that utilizes Bacillus thuringiensis stereoselective hydrolysis menthol isomer esterification mixture, to prepare L-menthol, the highest conversion rate reaches 74%, selectivity d.e.=90.1% (Yang Lirong, Meng Yan. A preparation method of Bacillus thuringiensis and L-menthol: CN101671639A[P]2009.8.13). In the method for preparing L-menthol by asymmetric hydrolysis, because the polarity difference of the mixture of L-menthol and menthol isomer esterification is not large, the separation process needs to be loaded down with trivial details and expensive processes such as silica gel column chromatography (Xu Jianhe, Zheng Gaowei. Bacillus subtilis esterase and its application in the production of L-menthol: CN 101338287A[P]2008.7.25)

Although some progress has been made in the research on the separation of L-menthol by the above-mentioned biological method, there are generally disadvantages such as low optical purity of the product, low conversion rate, few types of biocatalysts, and high cost of biocatalysts, and the separation and purification process is not easy to achieve. industrial application. If a highly selective microbial catalyst is utilized, the cost of raw materials can be greatly reduced, and the product purity can be improved. At present, there is no report about utilizing Pseudomonas alcaligenes to separate the mixture of menthol isomer esters. method.

Contents of the invention

The object of the present invention is to provide a kind of Pseudomonas alcaligenes and the method and application thereof for preparing L-menthol.

In order to achieve the above purpose, the inventor isolated and purified a new strain capable of selectively transesterifying L-menthol esters for the first time. After 16S rDNA and physiological and biochemical identification, the strain was Pseudomonas alcaligenes, named Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99). The bacterial strain was preserved in the China General Microorganism Culture Collection Management Center, the preservation date was December 3, 2010, and the preservation number was CGMCC No.4405.

Pseudomonas alcaligenes CGMCC No.4405 can be used as a catalyst for the preparation of L-menthol by separating the esterified mixture of menthol isomers.

The method utilizing Pseudomonas alcaligenes CGMCC No.4405 to prepare L-menthol comprises the steps:

(1) adding Pseudomonas alcaligenes with a preservation number of CGMCC No.4405 to the culture medium for fermentation;

(2) Catalyze the mixture of menthol isomer esters according to the following first scheme, second scheme or third scheme to obtain reaction solution, the mixture of said menthol isomer esters contains L-menthol ester Compounds, and containing L-isomenthol esters, L-neomenthol esters, L-neomenthol esters, d-menthol esters, d-isomenthol esters, d-neoisomenthol Any one or more of esterified compounds and d-neomenthol esterified compounds:

The first scheme: the mixture of the menthol isomer esterification is added to the fermented liquid obtained in step (1), and the reaction liquid is obtained after the reaction;

The second scheme: centrifuging the fermentation broth obtained in step (1) to obtain a supernatant, adding the mixture of menthol isomer esters to the supernatant, and reacting to obtain a reaction solution;

Scheme B The third scheme: dehydrating the fermented broth obtained in step (1) to obtain a solid preparation of the fermented broth, and then adding the solid preparation of the fermented broth and the mixture of menthol isomer esters to a pH of 5-5 React in the buffer solution of 10, obtain reaction solution;

(3) The reaction solution is separated to obtain L-menthol.

Further, in step (2) of the present invention, the mixture of the menthol isomer esterification refers to each menthol isomer and aliphatic carboxylic acid or aromatic carboxylic acid in the menthol isomer mixture. A mixture of esters formed.

Further, in the third scheme of step (2) of the present invention, the buffer solution is phosphate buffer, citric acid buffer or tris-hydrochloric acid buffer; the solid preparation of the fermentation broth is relatively The mass concentration of the buffer solution is 1g/L-100g/L.

Further, in the step (2) of the present invention, the components of the medium for the fermentation culture are: casein 1-10g/L, Tween-801-15g/L, yeast extract 1-8g/L, ammonium sulfate 0～10g/L, dipotassium hydrogen phosphate 1～6g/L, calcium chloride 0～0.5g/L and anhydrous magnesium sulfate 0～1g/L; the pH of the culture medium is 5～10; The inoculum amount of the single cell bacteria relative to the culture medium is 1%-10% (volume); the fermentation time is 12-48 hours; the fermentation temperature is 15-50°C.

Further, in step (3) of the present invention, the separation means that the reaction solution is evaporated under reduced pressure and then extracted with an organic solvent to obtain L-menthol.

Compared with chemical preparation methods, biological preparation methods and traditional natural raw material extraction methods, the inventive method has the following advantages:

(1) Adopting the traditional method of extracting L-menthol from natural peppermint plants, not only the production process efficiency is relatively low, energy consumption is large, but also easily affected by the planting yield of peppermint plants; and the chemical preparation method is easy to inactivate because the catalyst itself is expensive , and need to use a large amount of organic solvents and additives, the production cost and environmental cost are all higher; and in contrast, the present invention uses Pseudomonas alcaligenes LM99 (Pseudomonasalcaligenes LM99) to selectively hydrolyze menthol iso The L-menthol ester in the mixture of conformation esters not only has the advantages of higher selectivity and conversion rate, but also can greatly reduce the use of organic solvents in the process, and is environmentally friendly.

(2) In the industrialized resolution and preparation of L-menthol methods, the mixture of menthol isomers is used as the initial raw material, and most of the methods now adopt the pair of d-menthol and L-menthol The enantiomers were first isolated from the mixture of menthol isomers and then resolved. However, the present invention directly adopts the mixture of menthol isomers synthesized by chemical method as the substrate, undergoes simple esterification reaction, no pre-separation process is needed, the method is simple, and has obvious advantages in terms of equipment investment and operating cost.

(3) The invention utilizes screened microorganisms to prepare L-menthol, which can be produced by self-fermentation, has stable quality, stable storage, convenient transportation, can greatly reduce raw material costs, and has great application prospects.

(4) the present invention adopts the method for pre-decompression evaporation to separate the esterification product of menthol from L-menthol, which greatly simplifies the separation process, can directly prepare high-purity L-menthol, reduces the energy consumption of separation, and has industrialization prospects.

(5) adopt Pseudomonas alcaligenes LM99 of the present invention to carry out asymmetric catalytic reaction, generate L-menthol purity more than 95%, conversion rate up to 90%; And unreacted substrate can be directly reclaimed , re-racemization into a mixture of the esterification of the substrate menthol isomers, which improves the utilization of the substrate.

Preservation information for biological material samples:

Preserved samples of biological material: Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99);

Preservation unit: General Microbiology Center of China Committee for Culture Collection of Microorganisms (abbreviation: CGMCC);

Address of depository unit: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing (postal code: 100101);

Deposit date: December 3, 2010;

Deposit registration number: CGMCC No.4405.

Description of drawings

Fig. 1 is the gas chromatogram of the reaction solution of Pseudomonas alcaligenes LM99 catalyzed reaction, there are 8 peak signals in the figure, and from left to right are: L-menthol, L-isomenthyl propionate, d- Isomentha propionate, DL-neomentha propionate, L-mentha propionate, d-mentha propionate, L-neeomentha propionate, d-neomentha propionate; 8 signal peaks The peak times of the peaks are 16.440 minutes, 22.323 minutes, 22.665 minutes, 24.323 minutes, 24.757 minutes, 25.323 minutes, 27.540 minutes, and 27.790 minutes.

Fig. 2 is the change curve of conversion rate and product diastereomeric excess value under different reaction times.

Detailed ways

In the embodiment, in the process of catalyzing the mixture of menthol isomer esterification products, the method for measuring the substrate conversion rate and product diastereomeric excess value (d.e.p.) of the reaction solution is specifically as follows (wherein, the The substrate is a mixture of menthol isomer esters):

During the reaction, 5 mL of the reaction liquid and 5 mL of ethyl acetate were shaken and extracted, and then centrifuged (10000×g, 5 min) to obtain the ethyl acetate phase, which was analyzed by chiral gas chromatography. The chiral gas chromatography analysis system is as follows:

Detection instrument: GC-950 gas chromatograph;

Chiral gas phase column: CP-CYCLODEXTRINβ-2,3,6-M-19

(50m×0.25mm×0.25μm);

Detection conditions: column temperature 130°C, detector and injector temperature 260°C and 250°C respectively, carrier gas (N ₂ ) 25mL/min, air 3mL/min, hydrogen 7mL/min.

Calculation formula:

Substrate conversion rate (%)=(initial substrate concentration-residual substrate concentration)/initial substrate concentration×100%;

de _s = c×de _p /(1-c)×100%

de _p (%)=[(A _R -A _S )/(A _S +A _R )]×100%

Among them, c is the conversion rate of the substrate, de _p is the excess value of the product diastereomer, and wherein _AS and _AR are the peak areas of (L)-menthol and non-(L)-menthol in the sample measured by gas chromatography Sum.

Embodiment 1: Screening and identification of strains

In this example, soil was collected in the Hangzhou area to screen for Pseudomonas alcaligenes LM99 (Pseudomonasalcaligenes LM99). Methods as below:

Enrichment culture: medium composition (g/L): peptone 5.0, yeast extract 3.0, L-menthol propionate 10.0, ammonium sulfate 4.0, dipotassium hydrogen phosphate 10, magnesium sulfate 0.2, pH 7.0. 121°C Sterilize for 20 minutes. Weigh 1 g of fresh soil sample and add it to a 250 mL shaker flask filled with 50 ml enrichment medium, and place it in a shaker at 30°C and 200 r/min for cultivation. According to the inoculum volume of 1%, transfer was performed every other day, and repeated three times.

Primary screening plate: medium composition (g/L): yeast extract 1.0, ammonium sulfate 2.0, dipotassium hydrogen phosphate 2.0, sodium chloride 2.0, magnesium sulfate 0.5, L-menthol propionate 20.0, agar 20.0, pH 7.0. Sterilize at 120°C for 20min. Streak the enriched bacterial solution evenly on the plate medium by streaking method, and culture at 30°C for 1-2 days. Mark the cells with hydrolysis transparent circles and store them in a refrigerator at 4°C.

Re-screened shake flask: medium composition (g/L): peptone 5.0, glucose 5.0, yeast extract 5.0, olive oil 50.0, ammonium sulfate 5.0, dipotassium hydrogen phosphate 2.0, sodium chloride 1.0, magnesium sulfate 0.2, pH is 7.0. Sterilize at 120 for 20 minutes. Put the single bacterium colony obtained from the primary screening into 20mL re-screening medium, grow for 24 hours at 30°C and 200rpm, take 5ml of fresh fermentation broth, add the mixture of menthol isomer esters, menthol isomer esters The concentration of the mixture was 20g/L, and the reaction was shaken at 30°C for 24 hours, and ethyl acetate was added to shake and extract the hydrolyzate, which was used for chiral gas chromatography for analysis. Higher strains were preserved for future use.

Identification of strains: 16S rDNA identification and physiological and biochemical identification.

After screening, the strain numbered LM99 had the highest diastereomer pair value and a relatively good conversion rate. The gas chromatogram is shown in Figure 1. As can be seen from Figure 1, the peak position of the product L-menthol is at 16.440min. Perform 16S rDNA PCR sequencing analysis and physiological and biochemical experiments on bacterial strain LM99 (see Table 1). The sequencing results are shown in SEQ No.1. The results of sequencing analysis and physiological and biochemical experiments can determine that the strains screened out are pseudoalkali Bacillus, the strain named Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99).

Table 1 Physiological and biochemical experiment results of Pseudomonas alcaligenes LM99

Example 2:

The components of the selected fermentation medium are: casein 10g/L, Tween-80 9g/L, yeast extract 3g/L, ammonium sulfate 8g/L, dipotassium hydrogen phosphate 6g/L, and adjust the pH to 7.0. The seed solution of Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99) was inoculated into 50 mL of the above-mentioned fermentation medium with an inoculum amount of 1% (v/v), and fermented in a constant temperature shaker at 50 ° C and 200 r/min for 20 hours Afterwards, take out 5mL fermented liquid and make crude enzyme, crude enzyme is with the concentration of 1g/L, the mixture of menthol isomer propionate (the content of each isomer is: d-isomenthol propionate 12.3%, L-Isomenthol Propionate 12.3%, d-Menthol Propionate 24.4%, L-Menthol Propionate 24.4%, d-Neo-Isomenthol Propionate 13.3%, L-Neo-Isomenthol 13.3 %) was added to 5ml of phosphate buffer (buffer pH=7.0) at a concentration of 1g/L, placed in a 35°C, 200r/min constant temperature shaker for 10h, and 5ml of ethyl acetate was added to shake and extract after the reaction was completed. , and then analyzed by gas chromatography, the conversion rate of the final substrate was 51.1%, and the diastereomeric excess value of the product was 95.3%.

Example 3:

The selected fermentation medium components are: casein 8g/L, Tween-803g/L, yeast extract 1g/L, ammonium sulfate 4g/L, dipotassium hydrogen phosphate 4g/L, calcium chloride 0.5g/L, magnesium sulfate 0.5g/L, adjust the pH to 8.0. The seed solution of Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99) was inoculated in 50 mL of the above-mentioned fermentation medium with a 12% (v/v) inoculum, and fermented in a constant temperature shaker at 18 ° C and 200 r/min for 40 After 1 hour, the mixture of menthol isomer butyrate (the content of each isomer is: d-isomenthol butyrate 10.3%, L-isomenthol butyrate 10.3%, d-menthol butyrate ester 26.4%, L-menthol butyrate 26.4%, d-neoisomenthol butyrate 13.3%, L-neoisomenthol butyrate 13.3%) were added to 5ml phosphate buffer at a concentration of 60g/L medium (pH=10.0), placed in a 45°C, 200r/min constant temperature shaker for 60 hours of shaking reaction, after the reaction was completed, 5ml of ethyl acetate was added to shake and extract, and then gas chromatography analysis was carried out, as shown in Figure 2: the final bottom was obtained The conversion rate of product was 79.4%, and the diastereomeric excess value of the product was 99%.

Example 4:

The selected fermentation medium components are: casein 5g/L, Tween-8015g/L, yeast extract 10g/L, ammonium sulfate 12g/L, dipotassium hydrogen phosphate 7g/L, calcium chloride 1g/L, magnesium sulfate 1g /L, adjust the pH to 6.0. The seed solution of Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99) was inoculated in 50 mL of the above-mentioned fermentation medium with a 10% (v/v) inoculum amount, and fermented in a constant temperature shaker at 30 ° C and 200 r/min for 48 After 1 hour, take out 5mL fermented liquid and make crude enzyme, crude enzyme is with the concentration of 30g/L, the mixture of menthol isomer valerate (the content of each isomer is: d-isomenthol valerate 15.3% , L-Isomenthol Valerate 15.3%, d-Menthol Valerate 24.4%, L-Menthol Valerate 24.4%, d-Neoisomenthol Valerate 10.3%, L-Neoisomenthol Valerate 10.3%) was added to 5ml citrate buffer solution (buffer solution pH=5.0) with the concentration of 40g/L, placed in 20 ℃, 200r/min constant temperature shaker shaker reaction 50h, after the completion of the reaction, add 5ml Vibrating extraction with ethyl acetate, followed by gas chromatography analysis, the conversion rate of the final substrate was 30%, and the diastereomer excess value of the product was 93.8%.

Example 5:

The selected fermentation medium components are: casein 3g/L, Tween-80 20g/L, yeast extract 8g/L, ammonium sulfate 2g/L, dipotassium hydrogen phosphate 1g/L, calcium chloride 0.2g/L, sulfuric acid Magnesium 1.5g/L, adjust the pH to 10.0. The seed solution of Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99) was inoculated in 50 mL of the above-mentioned fermentation medium with a 5% (v/v) inoculum amount, and fermented in a constant temperature shaker at 15 ° C and 200 r/min for 12 After 1 hour, take out 5mL fermented liquid and make crude enzyme, crude enzyme is with the concentration of 100g/L, the mixture of menthol isomer benzoate (the content of each isomer is: d-isomenthol benzoate 12.3%, L-Isomenthol Benzoate 12.3%, d-Menthol Benzoate 20.4%, L-Menthol Benzoate 20.4%, d-Neo-Isomenthol Benzoate 17.3%, L-neoisomenthol benzoate 17.3%) was added to 5ml tris-hydrochloric acid buffer solution (buffer solution pH=9.0) with the concentration of 50g/L, placed in 60 ℃, 200r/min constant temperature Oscillating reaction 1h in shaker, add 5ml ethyl acetate vibration extraction after reaction is finished, carry out gas chromatography analysis again, the result is as shown in table 2, the transformation rate of final substrate is 90%, and product diastereomer excess value is 90% .

Table 2

Embodiment 6:

The selected fermentation medium components are: casein 1g/L, Tween-806g/L, yeast extract 7g/L, dipotassium hydrogen phosphate 4g/L, calcium chloride 0.7g/L, magnesium sulfate 0.3g/L, adjusted pH to 7.0. The seed solution of Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99) was inoculated in 50 mL of the above-mentioned fermentation medium with a 7% (v/v) inoculum, and fermented in a constant temperature shaker at 40 ° C and 200 r/min for 48 After 1 hour, take out 5mL fermented liquid and make crude enzyme, crude enzyme is with the concentration of 80g/L, the mixture of menthol isomer propionate (the content of each isomer is: d-isomenthol propionate 12.3% , L-Isomenthol Propionate 12.3%, d-Menthol Propionate 19.4%, L-Menthol Propionate 19.4%, d-Neo-Isomenthol Propionate 18.3%, L-Neo-Isomenthol Propionate Propionate (18.3%) was added to 5ml of phosphate buffer (buffer pH=7.0) at a concentration of 35g/L, placed in a 37°C, 200r/min constant temperature shaker for 22h, and 5ml of acetic acid was added after the reaction was completed Ethyl ester was shaken and extracted, and then analyzed by gas chromatography. The results are shown in Table 3. The conversion rate of the final substrate obtained was 62.4%, and the diastereomeric excess value of the product was 94.9%.

table 3

Embodiment 7:

The selected fermentation medium components are: casein 12g/L, Tween-80 1g/L, yeast extract 5g/L, ammonium sulfate 10g/L, dipotassium hydrogen phosphate 2g/L, calcium chloride 0.5g/L, sulfuric acid Magnesium 0.8g/L, adjust the pH to 5.0. The seed liquid of Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99) was inoculated in 50 mL of the above-mentioned fermentation medium with an inoculum size of 8% (v/v), and fermented in a constant temperature shaker at 37 ° C and 200 r/min for 55 After hours, centrifugal (10000 * g, 4 ℃) obtains fermentation supernatant, the mixture of menthol isomer laurate (the content of each isomer is: d-isomenthol laurate 11.3%, L-isomenthol laurate Alcohol laurate 11.3%, d-menthol laurate 25.4%, L-menthol laurate 25.4%, d-neoisomenthol laurate 13.3%, L-neoisomenthol laurate 13.3%) Add the concentration of 100g/L to 5ml of fermentation supernatant (pH of fermentation broth = 5.0), place in 50°C, 200r/min constant temperature shaker and shake for 35h. After completion, add 5ml of ethyl acetate to shake and extract , and then analyzed by gas chromatography, the conversion rate of the final substrate was 24.2%, and the diastereomeric excess value of the product was 97.5%.

<110> Zhejiang University

<120> A kind of Pseudomonas alcaligenes and the method and application thereof for preparing l-menthol

<160> 1

<170> PatentIn version 3.1

<210> 1

<211> 1509

<212> DNA

<213> Pseudomonas alcaligenes

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the

Claims (4)

1. A kind of Pseudomonas alcaligenes (Pseudomonas alcaligenes), is characterized in that: its preservation number is CGMCC No.4405. 2. The application of Pseudomonas alcaligenes according to claim 1, characterized in that it is used as a catalyst for preparing L-menthol by separating the mixture of menthol isomer esters. 3. a method utilizing Pseudomonas alcaligenes to prepare L-menthol according to claim 1, is characterized in that, comprises the steps: (1) Add Pseudomonas alcaligenes with the preservation number CGMCC No.4405 to the medium for fermentation; the components of the medium for fermentation are: casein 1-10g/L, Tween-80 1～15g/L, yeast extract 1～8g/L, ammonium sulfate 0～10g/L, dipotassium hydrogen phosphate 1～6g/L, calcium chloride 0～0.5g/L and anhydrous magnesium sulfate 0～1g/L L; the pH of the culture medium is 5～10; the inoculum size of the Pseudomonas alcaligenes relative to the culture medium is 1%～10% by volume percentage; the fermentation time is 12～48 hours; the fermentation temperature is 15～50℃; (2) According to the following first scheme, second scheme or third scheme, the mixture of menthol isomer esterification is catalyzed to obtain a reaction solution; the mixture of menthol isomer esterification means menthol isomerization A mixture of esters formed from a body mixture and an aliphatic carboxylic acid or an aromatic carboxylic acid; the menthol isomer mixture contains L-menthol, and contains L-isomenthol, L-neoisomenthol, L-neoisomenthol Any one or more of menthol, d-menthol, d-isomenthol, d-neoisomenthol, d-neomenthol: The first solution: adding the mixture of menthol isomer esterification products to the fermentation broth obtained in step (1), and reacting to obtain a reaction solution; The second solution: centrifuging the fermented liquid obtained in step (1) to obtain a supernatant, adding the mixture of menthol isomer esters to the supernatant, and obtaining a reaction liquid after reaction; The third plan: dehydrating the fermented liquid obtained in step (1) to obtain the crude enzyme, and then adding the crude enzyme and the mixture of menthol isomer esterification into a buffer solution with a pH of 5-10 for reaction, Obtain a reaction solution; the buffer solution is a phosphate buffer, a citric acid buffer or a tris-hydrochloric acid buffer; (3) Evaporate the reaction solution under reduced pressure, and then extract it with an organic solvent to obtain L-menthol. 4. The method for preparing L-menthol according to claim 3, characterized in that: in the third scheme of step (2), the mass concentration of the crude enzyme relative to the buffer solution is 1g/L～100g/L L.

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