CN1978659A - Method for preparing L-menthol from stereo-selective hydrolysis of DL fatty acid menthyl ester by whole-cell biological process - Google Patents
Method for preparing L-menthol from stereo-selective hydrolysis of DL fatty acid menthyl ester by whole-cell biological process Download PDFInfo
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Abstract
The invention relates to the method used to hydrolyze DL-fat acid mint ester and produce L-menthol by full cell biological method stereo-selectivity. It supplies the high stereo-selectivity strain, produces full cell, uses the DL-fat acid mint ester as substrate, hydrolyzes and produces L-menthol by full cell biological method stereo-selectivity. The by-product D-fat acid mint ester can be hydrolyzed to high purity D-menthol of which optical purity is 80%e.e-100%e.e; yield is 30%-60% while producing L-menthol of which optical purity is 90%e.e-100%e.e; conversion rate is 40%-50%. Compared with the international L-menthol preparation method, the adopted full cell catalysis can omit fussy enzyme purification, reduce enzyme activity damage, increase enzyme action substrate range and operation stability, and reduce L-menthol production cost.
Description
Technical field
A kind of whole-cell biological method stereo selective hydrolysis DL-fatty acid menthyl ester prepares the method for L-menthol, belongs to biological process resolution of racemic compound technical.
Background technology
Menthol is at present one of chipal compounds of industrial value to be arranged most, in field widespread uses such as medicine, food, makeup and chiral reagent preparations.Natural menthol is influenced by weather, region etc., and output and quality fluctuation are bigger, and synthetic menthol then can overcome the above problems.People have carried out a large amount of research to the route of synthesis of menthol, many be applied to suitability for industrialized production (Fan Cunliang, Yang Zhongbao, " fragrance flavor and cosmetic ", 2002, the 4th phase, p28).Usually the racemic mixture that to adopt these method synthetic are L-menthols, the existence of D-menthol has influenced the aroma and flavor of menthol greatly, and physiologically active, and it is very important therefore menthol to be carried out chiral separation.In recent years, because to the raising of green, environmental protection and the security requirement of food and drug additive, the biology method for splitting has become the main flow trend of chiral separation, the chiral separation of development of new had become the key of menthol chiral separation with enzyme.
At present, at home and abroad supply falls short of demand for the peppermint series product, and the annual requirement of China's menthol also continues to rise.The synthetic method of menthol mainly is external exploitation, domestic independent development is also industrialized also seldom, because natural menthol can't satisfy the demand of modern industry rapid growth, producer is serious day by day to the dependence of synthetic menthol, exploitation and application and development along with enzyme, chemical method synthesizes the menthol racemic modification, splits in conjunction with biocatalysis and carries out the production of L-menthol, and vast potential for future development will be arranged.
Split three kinds of technology of the normal use of DL-menthol enantiomorph in the world: utilize optical activity reagent to split, utilize non-optical active agent to split or the biocatalysis chiral separation.
The DL-menthol generates two kinds of diastereomer compounds that physical properties is different with the optical activity reagent react, and these two kinds of compounds can separate by the method for fractional crystallization, are hydrolyzed respectively then and just obtain purified L-menthol and D-menthol.Optical activity reagent has many kinds, for example: alkaloids, optically active amine such as 1-(Alpha-Naphthyl) ethylamine salt, phthalic acid borneol acetate, optically active acids such as peppermint ethoxyacetic acid, camphor-10-sulfonic acid or the like.
The principle of utilizing non-optical active agent to split is a kind of saturated solution of DL-mixture, when inoculating (introducing crystal seed) with a certain enantiomorph, will induce the crystallization of this kind enantiomorph to separate out, and another kind of enantiomorph is still stayed in the solution.Concrete operations are the solution that makes the DL-mixture earlier, then solution are divided into two equal portions, a D-type crystal seed that adds, and another part adds L-type crystal seed, thereby can isolate D-and L-isomer crystal respectively from two parts of solution.After last two parts of mother liquors merge mixing, be divided into two parts again, repeat top-operation, until most D-and L-isomer are separated.
More than the used optical activity or the non-optical active reagent of method for splitting of two kinds of DL-menthols cost an arm and a leg, be difficult to reclaim, and complex process, need be through repeatedly fractional crystallization separation, easily bring toxicant in process of production into, this all makes the application of synthetic menthol be very restricted.Biocatalysis method for splitting reaction conditions gentleness, environmental protection, efficient have caused people's attention.
The principle of biocatalysis chiral separation is some isomer in the racemize enantiomorph to be had the stereoselectivity of height with some special microorganism or enzyme, and inoperative or effect is very little, thereby utilize the speed of speed of reaction to realize the technology of Chiral Separation to another isomer.Rising the eighties in the world the biocatalysis chiral separation of DL-menthol has been carried out a large amount of research, mainly is to utilize enzymatic asymmetric esterification of commercialization or transesterification, or utilizes the enzymatic asymmetric hydrolysis reaction of commercialization.Because of chiral separation not only relates to the activity of enzyme, stereoselectivity also is a very important investigation index, and this has improved the requirement to enzyme.
About enzyme catalysis asymmetric hydrolysis method the following report is arranged:
Japanology scholar (Jp 56015690,1981 for Yamada H, Tani Y et al.) has reported the earliest and has utilized Gloeophyllum sepiarium IFO 6267 esterases hydrolysis DL-menthyl acetates, but product yield is low, only about 20%.The same year, (Omata T such as Omata, Iwamoto N et al., European Journalof Applied Microbiology and Biotechnology, 1981,11 (4), p199) utilize again immobilization Rhodotorula-Minuta Var Texensis cell in water saturated normal heptane successful stereo selective hydrolysis DL-succsinic acid menthyl ester, and prepared the L-menthol.Nineteen ninety, Murase etc. (Jp 02299596,1990 for Murase H, Yoshikawa K et al.) Ochrobactrum anthropi esterases selective hydrolysis DL-menthyl acetate, but enzymic activity is very low, only reaches 21% transformation efficiency behind the reaction 65h.
The method of utilizing at present biological method for splitting to produce the L-menthol in the world mainly contains:
South Africa CSIR organizes (Chaplin J A, Gardiner N S et al., WO 0236795,2002) utilize Pseudomonas fluorescens lipase selectivity transesterification fractionation DL-menthol to prepare the L-menthol, molecular balance transformation efficiency low (about 20%), the reaction product complexity, comprise L-menthyl acetate, organic solvent, unconverted steric isomer, excessive esterifying agent and byproduct of reaction, bring difficulty for the separation of L-menthyl acetate, separable programming is loaded down with trivial details and product purity is not high; And utilize alkali lye hydrolysis L-menthyl acetate, transformation efficiency is low, less than 50%.
Germany Haarmann﹠amp; (the Gatfield I L of Reimer company, Hilmer J M et al., US 6706500,2002) utilize recombinant C andida Rugosa lipase selective hydrolysis DL-phenylformic acid menthyl ester to prepare the L-menthol, this enzyme stability in the aqueous solution is not high, is difficult to recycle, though can improve the stability of enzyme after the employing immobilization, but the enzyme deactivation that immobilization causes is serious, high reactivity yield only 43%; And the concentration of substrate of enzymatic hydrolysis reaction is not high, the highest by about 0.8%.
Summary of the invention
(1) technical problem that will solve
The object of the present invention is to provide a kind of whole-cell biological method stereo selective hydrolysis DL-fatty acid menthyl ester to prepare the method for L-menthol, when obtaining highly purified L-menthol, by product D-fatty acid menthyl ester can be by the D-menthol of hydrolysis for producing high-purity.
(2) technical scheme
The present invention is at first from microorganism, screened can high-efficient solid selective hydrolysis DL-fatty acid menthyl ester bacterial classification.
The approach that designs this method for transformation is as follows:
Main raw and reagent
Bacterial strain uses therefor and source
Zhizopchin (Rhizopus chinensis) CCTCC M201021, Chinese patent ZL03113274.X is open the sixth of the twelve Earthly Branches, Chinese typical culture collection center,
The Chinese common micro-organisms of Rhizopus oryzae (Rhizopus oryzae) AS 3.41 DSMZ,
Rhizopus oryzae (Rhizopus oryzae) AS 3.253 state common micro-organisms DSMZs,
Candida parapsilosis (Candida parapsilosis) CICC 1676 Chinese industrial microbial strains preservation centers,
Candida parapsilosis (Candida parapsilosis) CICC 1627 Chinese industrial microbial strains preservation centers,
Fold candida (Candida rugosa) CICC 31281 Chinese industrial microbial strains preservation centers,
The Chinese common micro-organisms of Pseudomonas fluorescens (Pseudomonas fluorescens) AS 1.823 DSMZ,
The Chinese common micro-organisms of Pseudomonas fluorescens (Pseudomonas fluorescens) AS 1.55 DSMZ,
Or onion cloth gram Salmonella (Burkholderia cepacia) ATCC 25416 U.S. typical case DSMZ.
Main agents
D-menthol, L-menthol, DL-menthol are all purchased the Sigma-Aldrich company in the U.S..
It is typical thing that the DL-fatty acid menthyl ester is selected the DL-menthyl acetate.
Determining of DL-menthol and DL-menthyl acetate analytical procedure
L-menthol, D-menthol, L-menthyl acetate, D-menthyl acetate standard specimen are analyzed by the chirality gas-chromatography (Varian3900) that has fid detector on cp-chirasil Dex CB chromatographic column (purchasing the Varian company in the U.S.).The retention time of L-menthol is 7.3min, and the retention time of D-menthol is 8.3min, and the retention time of L-menthyl acetate is 8.8min, and the retention time of D-menthyl acetate is 8.5min.
Determine concrete chromatographic condition: chiral column cp-chirasil Dex CB (25m * 0.25mm * 0.25 μ m); Splitting ratio 1: 50; 270 ℃ of injector temperatures; 270 ℃ of detector temperatures; Carrier gas H
2The post heating schedule keeps 8min for 100 ℃, and 4 ℃/min is warming up to 140 ℃, keeps 2min.L-menthol, D-menthol, L-menthyl acetate, D-menthyl acetate all can reach baseline separation under this condition.
The optical purity of product is estimated by mapping excessive value (%e.e.).
Reaction after product e.e.
pValue %e.e.
p=[(S
L-S
D)/(S
L+ S
D)] * 100%
Reaction back substrate e.e.
sValue %e.e.
s=[(S
D'-S
L')/(S
L'+S
D')] * 100%
Reaction conversion ratio transformation efficiency (%)=[(e.e.
s+ e.e.
0)/(e.e.
s+ e.e.
p)] * 100%
S
L: the peak area of reaction back L-menthol; S
D: the peak area of reaction back D-menthol;
S
L': the peak area of reaction back L-menthyl acetate; S
D': the peak area of reaction back D-menthyl acetate;
E.e.
0: substrate e.e. value when reacting initial.
The cultivation of bacterial strain and the preparation of full cell
(1) substratum of bacterial strain is formed:
The mould medium composition is counted with g/100ml: maltose 0.5～3, peptone 0.5～4, MgSO
47H
2O0.01～0.1, K
2HPO
40.2～1.0, sweet oil 1.0～2.0, soybean cake powder 1.0～5.0, pH4～6.
The microzyme culture medium composition is counted with g/100ml: glucose 3.5～5.0, yeast extract paste 0.15～0.5, KH
2PO
40.1～1.5, MgSO
47H
2O 0.01～0.25, and NaCl 0.001～0.025, FeSO
47H
2O0.001～0.025, pH6.0～7.5.
The bacteria culture medium composition is counted with g/100ml: glucose 0.5～2.0, extractum carnis 0.1～1.0, Tryptones 0.1～1.0, Fe
2SO
47H
2O 0.001～0.05, K
2HPO
40.1～0.5, NaCl 0.1～1.0, pH7.0～8.0.
(2) preparation of full cell:
Is that 5%～30% 250ml shakes in the bottle in 20～35 ℃, 100～300 rev/mins shaking culture 24～72 hours with inoculation at liquid amount; It is centrifugal cultivate to finish the back, collects thalline physiological saline washed twice, and lyophilize obtains stem cell and is used for stereo selective hydrolysis and reacts.
The reaction of microorganism cells catalysis stereo selective hydrolysis
Whole-cell catalytic stereo selective hydrolysis reaction conditions is: be reflected at cell concn and count 0.5～20 with g/100ml, concentration of substrate counts 0.1～15 with g/100ml, control reaction buffer pH value 6.0～8.5,20～40 ℃ of temperature of reaction, in 12～48 hours reaction times, reaction buffer can be selected Tris-hydrochloric acid, phosphate buffered or Citrate trianion buffering for use.
Adopting the optical purity of the L-menthol that full cell stereo selective hydrolysis DL-fatty acid menthyl ester obtains is 90%e.e.～100%e.e., transformation efficiency 40%～50%.
The good stability of described stereo selective hydrolysis reaction, reaction half-time reaches 240 hours.
When preparation L-menthol, the by product D-fatty acid menthyl ester that obtains is by the D-menthol of hydrolysis for producing high-purity, and optical purity is 80%e.e.～100%e.e., and productive rate is 30%～60%.
The used full cell of stereo selective hydrolysis can be reused 4 batches, and each centrifugal gained thalline in back that uses is recycled and reused for hydrolysis reaction with fresh phosphoric potassium damping fluid washed twice, can keep excellent catalytic performance.
(3) beneficial effect
The present invention obtains a series of highly-solid selectively bacterial strains by screening.Carry out asymmetric hydrolysis DL-fatty acid menthyl ester with such bacterial strain and prepare the L-menthol, optical purity can reach more than the 90%e.e..Compare with the preparation method of relevant L-menthol in the world, adopt whole-cell catalytic to save loaded down with trivial details enzyme purification, immobilization process, reduced the enzyme loss of living, improved the substrate scope and the operational stability of enzyme effect, and reduced the production cost of L-menthol.
The present invention and methods involving comparison in the world, when preparation L-menthol, the by product D-fatty acid menthyl ester that obtains can prepare highly purified D-menthol by hydrolysis, and its optical purity can reach more than the 80%e.e..
The present invention adopts different full cell microorganism stereo selective hydrolysis DL-fatty acid menthyl esters to prepare optically pure L-menthol; The substrate operating restraint is wider, can be 0.1～15 (in g/100ml); The reaction stability height, the transformation period can reach 240 hours.
The present invention is applied to microbe whole-cell in the chiral separation research of menthol first, for the chiral separation of menthol and analog and realize that finally large-scale industrial production provides thinking.
The present invention splits for novel chiral has the meaning of outbalance with the exploitation in enzyme source, has embodied advantage and the vital role of biological process in chipal compounds is produced.
Embodiment
The preparation of embodiment 1DL-menthyl acetate
In being housed, the 100ml there-necked flask of condensation reflux device and thermometer adds 0.02mol menthol and 0.02mol diacetyl oxide, and adding 0.4mmol tosic acid is made catalyzer, it is 10ml that the adding normal heptane is made reaction solvent to reaction volume, stirs and heating reflux reaction 4h.Be cooled to below 40 ℃ after reaction finishes, add 5%NaHCO
3The excessive acid that neutralizes, to no longer producing till the bubble, standing demix, the saturated common salt water washing of the phase of anhydrating, organic phase, dividing anhydrates then uses anhydrous MgSO mutually
4Dried overnight.Filter, the mixed solution rotary evaporation promptly gets DL-menthyl acetate purely except that desolvating.The purity of gas chromatographic detection DL-menthyl acetate is 99.9%, and productive rate (in menthol) is 98.5%.
Embodiment 2 different strains are to the hydrolysis situation of DL-menthyl acetate
In the buffer solution of potassium phosphate of 1ml, 50mmol/L (pH7.0), add 13mg dry mycelium and 10mg DL-menthyl acetate, the difference oscillatory reaction is 24 hours on 30 ℃ constant temperature shaking table, after the reaction, mixture bactofugation body, supernatant liquor ethyl acetate extraction product and substrate, chirality gas chromatographic analysis product e.e. value and transformation efficiency.The conversion situation such as the table 1 of each bacterial strain.
Table 1
| Microorganism strains | e.e. p(％) | Transformation efficiency (%) |
| Rhizopus chinensis CCTCC M201021 Rhizopus oryzae AS3.41 Rhizopus oryzae AS3.253 Candida parapsilosis CICC 1676 Candida parapsilosis CICC 1627 Candida rugosa CICC 31281 Pseudomonas fluorescens AS 1.823 Pseudomonas fluorescens AS 1.55 Burkholderia cepacia ATCC 25416 | 98.6 97.1 97.6 96.2 95.9 97.1 98.4 95.2 95.1 | 43.6 42.1 44.8 41.6 46.3 49.9 44.1 46.6 41.4 |
The full cell asymmetric hydrolysis of embodiment 3 usefulness CCTCC M201021 DL-menthyl acetate
In the buffer solution of potassium phosphate of 1ml, 50mmol/L (pH7.0), add 13mg dry mycelium and 10mg DL-menthyl acetate, the difference oscillatory reaction is 12,16,20,24,36,48 hours on 30 ℃ constant temperature shaking table, after the reaction, mixture bactofugation body, supernatant liquor ethyl acetate extraction product and substrate, chirality gas chromatographic analysis product e.e. value and transformation efficiency.Result such as table 2.
Table 2
| Reaction times (hour) | e.e. p(％) | Transformation efficiency (%) |
| 12 16 20 24 36 48 | 96.8 97.3 96.7 97.0 92.9 90.4 | 31.8 39.3 44.5 49.9 51.8 52.5 |
Table 2 explanation react after 24 hours near theoretical yield 50%, continues prolongation reaction times to 48 hour, and the product optical purity still can reach more than the 90%e.e..
The full cell asymmetric hydrolysis of embodiment 4 usefulness CCTCC M201021 different concns DL-menthyl acetate
In the buffer solution of potassium phosphate of 1ml, 50mmol/L (pH7.0), add 7mg, 13mg, 67mg, 133mg, 200mg dry mycelium and 5mg, 10mg, 50mg, 100mg, 150mg DL-menthyl acetate respectively, oscillatory reaction is 24 hours on 30 ℃ constant temperature shaking table, after the reaction, mixture bactofugation body, supernatant liquor ethyl acetate extraction product and substrate, chirality gas chromatographic analysis product e.e. value and transformation efficiency.Result such as table 3.
Table 3
| Cell concn (g/100ml) | Substrate DL-menthyl acetate concentration (g/100ml) | e.e. p (％) | Transformation efficiency (%) |
| 0.7 1.3 6.7 13.3 20.0 | 0.5 1.0 5.0 10.0 15.0 | 97.5 96.1 96.4 95.1 93.4 | 49.9 48.7 46.5 40.1 37.1 |
The full cell asymmetric hydrolysis of CCTCC M201021 DL-menthyl acetate is adopted in table 3 explanation, still can keep higher transformation efficiency and selectivity when concentration of substrate is higher, and full cell has higher substrate tolerance.
The full cell asymmetric hydrolysis of CCTCC M201021 DL-menthyl acetate under embodiment 5 differing tempss
In the buffer solution of potassium phosphate of 1ml, 50mmol/L (pH7.0), add 13mg dry mycelium and 10mg DL-menthyl acetate, respectively at oscillatory reaction on 20 ℃, 30 ℃, 40 ℃, 50 ℃ the constant temperature shaking table 24 hours, after the reaction, mixture bactofugation body, supernatant liquor ethyl acetate extraction product and substrate, chirality gas chromatographic analysis product e.e. value and transformation efficiency.Result such as table 4.
Table 4
| Temperature of reaction (℃) | e.e. p(％) | Transformation efficiency (%) |
| 20 30 40 50 | 97.2 96.9 95.8 84.8 | 31.2 48.5 49.2 4.7 |
Table 4 explanation temperature is in the time of 20～40 ℃, and full transformation vigor and enantioselectivity are all higher.When temperature surpassed 50 ℃, the enzyme that works in the full cell was because of high temperature deactivation, and conversion capability descends rapidly.
Embodiment 6 different pH values are the full cell asymmetric hydrolysis of CCTCC M201021 DL-menthyl acetate down
In the buffer solution of potassium phosphate of the different pH values of 1ml, 50mmol/L, add 13mg dry mycelium and 10mg DL-menthyl acetate, oscillatory reaction is 24 hours on 30 ℃ constant temperature shaking table, after the reaction, mixture bactofugation body, supernatant liquor ethyl acetate extraction product and substrate, chirality gas chromatographic analysis product e.e. value and transformation efficiency.Result such as table 5.Table 5
| PH value in reaction | e.e. p(％) | Transformation efficiency (%) |
| 6.0 7.0 7.5 8.0 8.5 | 95.0 95.6 96.0 95.1 92.3 | 46.4 49.0 49.6 43.9 41.7 |
The full cell transformation of table 5 explanation is reflected in pH6.0～8.5 scopes, all has higher product e.e. value and transformation efficiency.
The sad menthyl ester of embodiment 7 usefulness Candida rugosa CICC, 31281 full cell asymmetric hydrolysis DL-
In the buffer solution of potassium phosphate of 1ml, 50mmol/L (pH7.0), add 13mg dry mycelium and the sad menthyl ester of 10mg DL-, oscillatory reaction is 24 hours on 30 ℃ constant temperature shaking table, after the reaction, mixture bactofugation body, supernatant liquor ethyl acetate extraction product and substrate, chirality gas chromatographic analysis product e.e. value and transformation efficiency.The optical purity 97.7%e.e. of products therefrom L-menthol, transformation efficiency 43.8%.
The full cells of embodiment 8 use batch
In the buffer solution of potassium phosphate of 1ml, 50mmol/L (pH7.0), add 13mg Pseudomonasfluorescens AS 1.823 dry myceliums and 10mg DL-menthyl acetate, oscillatory reaction is 24 hours on 30 ℃ constant temperature shaking table, after the reaction, mixture bactofugation body, supernatant liquor ethyl acetate extraction product and substrate, chirality gas chromatographic analysis product e.e. value and transformation efficiency.Centrifugal gained thalline repeats above-mentioned resolution reaction with fresh phosphoric potassium buffering washed twice, and reaction conditions is constant, and reaction finishes same product e.e. value and the transformation efficiency measured in back.Result such as table 6.
Table 6
| Reaction batch | e.e. p(％) | Transformation efficiency (%) |
| First second batch the 3rd batch the 4th batch | 94.1 95.5 98.9 97.3 | 46.1 46.6 43.8 40.8 |
Table 6 explanation, Pseudomonas fluorescens AS 1.823 full cell lyophilized powders have good reaction stability, react four times after, transformation efficiency only is declined by less than 12%.
The hydrolysis of embodiment 9D-menthyl acetate prepares the D-menthol
The NaOH solution 100mL of 3.0mol/L is heated to 60 ℃, add conversion reaction then and finish the isolated 0.2mol D-menthyl acetate in back, and high degree of agitation 60 minutes, ethyl acetate extraction product and substrate, the D-menthyl acetate of qualitative GC analysis revealed 49% peak area and the D-menthol of 51% peak area, the e.e. value of D-menthol is 90.1%.
Claims (8)
1, a kind of whole-cell biological method stereo selective hydrolysis DL-fatty acid menthyl ester prepares the method for L-menthol, it is characterized in that through the cultivation of stereoselectivity bacterial strain and the preparation of full cell, with the DL-fatty acid menthyl ester is substrate, cell concn counts 0.5～20 with g/100ml, concentration of substrate counts 0.1～15 with g/100ml, control reaction buffer pH value 6.0～8.5,20～40 ℃ of temperature of reaction, in 12～48 hours reaction times, prepare the L-menthol by whole-cell biological method stereo selective hydrolysis.
2, method according to claim 1 is characterized in that the bacterial strain of the full cell of used preparation is:
Zhizopchin (Rhizopus chinensis) CCTCC M201021 China typical culture collection center,
The Chinese common micro-organisms of Rhizopus oryzae (Rhizopus oryzae) AS 3.41 DSMZ,
The Chinese common micro-organisms of Rhizopus oryzae (Rhizopus oryzae) AS 3.253 DSMZ,
Candida parapsilosis (Candida parapsilosis) CICC 1676 Chinese industrial microbial strains preservation centers,
Candida parapsilosis (Candida parapsilosis) CICC 1627 Chinese industrial microbial strains preservation centers,
Fold candida (Candida rugosa) CICC 31281 Chinese industrial microbial strains preservation centers,
The Chinese common micro-organisms of Pseudomonas fluorescens (Pseudomonas fluorescens) AS 1.823 DSMZ,
The Chinese common micro-organisms of Pseudomonas fluorescens (Pseudomonas fluorescens) AS 1.55 DSMZ,
Go up or onion cloth gram Salmonella (Burkholderia cepacia) ATCC 25416 U.S. typical case DSMZ.
3, method according to claim 1 is characterized in that the preparation of full cell:
(1) substratum of bacterial strain is formed:
The mould medium composition is counted with g/100ml: maltose 0.5～3, peptone 0.5～4, MgSO
47H
2O0.01～0.1, K
2HPO
40.2～1.0, sweet oil 1.0～2.0, soybean cake powder 1.0～5.0, pH 4～6;
The microzyme culture medium composition is counted with g/100ml: glucose 3.5～5.0, yeast extract paste 0.15～0.5, KH
2PO
40.1～1.5, MgSO
47H
2O 0.01～0.25, and NaCl 0.001～0.025, FeSO
47H
2O0.001～0.025, pH6.0～7.5;
The bacteria culture medium composition is counted with g/100ml: glucose 0.5～2.0, extractum carnis 0.1～1.0, Tryptones 0.1～1.0, Fe
2SO
47H
2O 0.001～0.05, K
2HPO
40.1～0.5, NaCl 0.1～1.0, pH7.0～8.0;
(2) preparation of full cell:
Is that 5%～30% 250ml shakes in the bottle in 20～35 ℃, 100～300 rev/mins shaking culture 24～72 hours with inoculation at liquid amount; It is centrifugal cultivate to finish the back, collects thalline physiological saline washed twice, and lyophilize obtains stem cell and is used for stereo selective hydrolysis and reacts.
4, method according to claim 1 is characterized in that the used damping fluid of hydrolysis reaction is Tris-hydrochloric acid, phosphoric acid salt or citrate buffer.
5, method according to claim 1, it is characterized in that adopting the optical purity of the L-menthol that full cell stereo selective hydrolysis DL-fatty acid menthyl ester obtains is 90%e.e.～100%e.e., transformation efficiency 40%～50%.
6, method according to claim 1 is characterized in that the good stability that described stereo selective hydrolysis reacts, and reaction half-time reaches 240 hours.
7, method according to claim 1 is characterized in that the by product D-fatty acid menthyl ester that obtains is by the D-menthol of hydrolysis for producing high-purity when preparation L-menthol, and optical purity is 80%e.e.～100%e.e., and productive rate is 30%～60%.
8, method according to claim 1, it is characterized in that the used full cell of stereo selective hydrolysis can reuse 4 batches, each centrifugal gained thalline in back that uses is recycled and reused for hydrolysis reaction with fresh phosphoric potassium damping fluid washed twice, can keep excellent catalytic performance.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102031237A (en) * | 2010-11-12 | 2011-04-27 | 浙江大学 | Stenotrophomonas and method for preparing 1-menthol and application thereof |
| CN106755142A (en) * | 2017-01-10 | 2017-05-31 | 台州学院 | The method that Rhizopus oryzae thalline whole-cell catalytic prepares L lactic acid |
| CN112159769A (en) * | 2020-09-30 | 2021-01-01 | 江南大学 | Genetically engineered bacterium for producing L-menthol and construction method and application thereof |
| CN112175849A (en) * | 2020-09-30 | 2021-01-05 | 江南大学 | Recombinant yeast with improved L-menthol yield |
| CN112920959A (en) * | 2021-02-05 | 2021-06-08 | 江南大学 | Method for increasing yield of L-menthol in yeast |
-
2006
- 2006-12-08 CN CNA2006100981972A patent/CN1978659A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102031237A (en) * | 2010-11-12 | 2011-04-27 | 浙江大学 | Stenotrophomonas and method for preparing 1-menthol and application thereof |
| CN106755142A (en) * | 2017-01-10 | 2017-05-31 | 台州学院 | The method that Rhizopus oryzae thalline whole-cell catalytic prepares L lactic acid |
| CN106755142B (en) * | 2017-01-10 | 2020-08-28 | 台州学院 | Method for preparing L-lactic acid by rhizopus oryzae thallus whole cell catalysis |
| CN112159769A (en) * | 2020-09-30 | 2021-01-01 | 江南大学 | Genetically engineered bacterium for producing L-menthol and construction method and application thereof |
| CN112175849A (en) * | 2020-09-30 | 2021-01-05 | 江南大学 | Recombinant yeast with improved L-menthol yield |
| CN112159769B (en) * | 2020-09-30 | 2022-10-04 | 江南大学 | Genetically engineered bacterium for producing L-menthol and construction method and application thereof |
| CN112175849B (en) * | 2020-09-30 | 2022-10-18 | 江南大学 | Recombinant yeast with improved L-menthol yield |
| CN112920959A (en) * | 2021-02-05 | 2021-06-08 | 江南大学 | Method for increasing yield of L-menthol in yeast |
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