CN102154166B - Pseudomonas alcaligenes, method for preparing 1-menthol by pseudomonas alcaligenes and application of pseudomonas alcaligenes - Google Patents
Pseudomonas alcaligenes, method for preparing 1-menthol by pseudomonas alcaligenes and application of pseudomonas alcaligenes Download PDFInfo
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- menthol
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- NOOLISFMXDJSKH-KXUCPTDWSA-N (-)-Menthol Chemical compound CC(C)[C@@H]1CC[C@@H](C)C[C@H]1O NOOLISFMXDJSKH-KXUCPTDWSA-N 0.000 title claims abstract description 78
- 241000168225 Pseudomonas alcaligenes Species 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 34
- 239000000203 mixture Substances 0.000 claims abstract description 42
- 238000006243 chemical reaction Methods 0.000 claims abstract description 40
- 238000000855 fermentation Methods 0.000 claims abstract description 20
- 230000004151 fermentation Effects 0.000 claims abstract description 20
- 150000002148 esters Chemical class 0.000 claims abstract description 12
- NOOLISFMXDJSKH-AEJSXWLSSA-N (+)-menthol Chemical compound CC(C)[C@H]1CC[C@H](C)C[C@@H]1O NOOLISFMXDJSKH-AEJSXWLSSA-N 0.000 claims abstract description 10
- NOOLISFMXDJSKH-UTLUCORTSA-N (+)-Neomenthol Chemical class CC(C)[C@@H]1CC[C@@H](C)C[C@@H]1O NOOLISFMXDJSKH-UTLUCORTSA-N 0.000 claims abstract description 7
- NOOLISFMXDJSKH-UHFFFAOYSA-N DL-menthol Natural products CC(C)C1CCC(C)CC1O NOOLISFMXDJSKH-UHFFFAOYSA-N 0.000 claims description 59
- 229940041616 menthol Drugs 0.000 claims description 57
- -1 menthol isomer carboxylate Chemical class 0.000 claims description 26
- 239000007788 liquid Substances 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 16
- 239000000872 buffer Substances 0.000 claims description 12
- 108090000790 Enzymes Proteins 0.000 claims description 11
- 102000004190 Enzymes Human genes 0.000 claims description 11
- 229940041514 candida albicans extract Drugs 0.000 claims description 11
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 11
- 239000012138 yeast extract Substances 0.000 claims description 11
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 10
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 10
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 10
- 239000002054 inoculum Substances 0.000 claims description 9
- 239000005018 casein Substances 0.000 claims description 8
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 8
- 235000021240 caseins Nutrition 0.000 claims description 8
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 8
- 229920000053 polysorbate 80 Polymers 0.000 claims description 8
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 7
- 239000001110 calcium chloride Substances 0.000 claims description 7
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 238000006555 catalytic reaction Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 239000007979 citrate buffer Substances 0.000 claims description 3
- 230000009467 reduction Effects 0.000 claims description 3
- 238000009834 vaporization Methods 0.000 claims description 3
- 230000008016 vaporization Effects 0.000 claims description 3
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 2
- 150000007933 aliphatic carboxylic acids Chemical class 0.000 claims description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical group OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 claims description 2
- 238000000638 solvent extraction Methods 0.000 claims description 2
- 239000000758 substrate Substances 0.000 abstract description 21
- 239000001963 growth medium Substances 0.000 abstract description 2
- 230000008020 evaporation Effects 0.000 abstract 1
- 238000001704 evaporation Methods 0.000 abstract 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 27
- FKRCODPIKNYEAC-UHFFFAOYSA-N ethyl propionate Chemical compound CCOC(=O)CC FKRCODPIKNYEAC-UHFFFAOYSA-N 0.000 description 22
- 238000002360 preparation method Methods 0.000 description 17
- 239000000047 product Substances 0.000 description 16
- 239000007789 gas Substances 0.000 description 10
- 230000009466 transformation Effects 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 9
- 238000000605 extraction Methods 0.000 description 9
- 230000010355 oscillation Effects 0.000 description 8
- 244000246386 Mentha pulegium Species 0.000 description 7
- 235000016257 Mentha pulegium Nutrition 0.000 description 7
- 235000004357 Mentha x piperita Nutrition 0.000 description 7
- 238000004587 chromatography analysis Methods 0.000 description 7
- MTZQAGJQAFMTAQ-UHFFFAOYSA-N ethyl benzoate Chemical compound CCOC(=O)C1=CC=CC=C1 MTZQAGJQAFMTAQ-UHFFFAOYSA-N 0.000 description 7
- OBNCKNCVKJNDBV-UHFFFAOYSA-N ethyl butyrate Chemical compound CCCC(=O)OCC OBNCKNCVKJNDBV-UHFFFAOYSA-N 0.000 description 7
- 235000001050 hortel pimenta Nutrition 0.000 description 7
- 229940070765 laurate Drugs 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 230000003534 oscillatory effect Effects 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 229940070710 valerate Drugs 0.000 description 7
- 239000000306 component Substances 0.000 description 6
- POULHZVOKOAJMA-UHFFFAOYSA-M dodecanoate Chemical compound CCCCCCCCCCCC([O-])=O POULHZVOKOAJMA-UHFFFAOYSA-M 0.000 description 6
- 230000032050 esterification Effects 0.000 description 6
- 238000005886 esterification reaction Methods 0.000 description 6
- 244000005700 microbiome Species 0.000 description 6
- 238000004321 preservation Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 230000035479 physiological effects, processes and functions Effects 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 239000003054 catalyst Substances 0.000 description 4
- 238000004817 gas chromatography Methods 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 235000006679 Mentha X verticillata Nutrition 0.000 description 3
- 235000002899 Mentha suaveolens Nutrition 0.000 description 3
- 235000001636 Mentha x rotundifolia Nutrition 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000005265 energy consumption Methods 0.000 description 3
- 239000012533 medium component Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- VVJKKWFAADXIJK-UHFFFAOYSA-N Allylamine Chemical compound NCC=C VVJKKWFAADXIJK-UHFFFAOYSA-N 0.000 description 2
- 241000193388 Bacillus thuringiensis Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 2
- 229940097012 bacillus thuringiensis Drugs 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005809 transesterification reaction Methods 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 108091007473 BsubE Proteins 0.000 description 1
- 241000985905 Candidatus Phytoplasma solani Species 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 230000003260 anti-sepsis Effects 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 150000007942 carboxylates Chemical class 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 229960004873 levomenthol Drugs 0.000 description 1
- 239000001525 mentha piperita l. herb oil Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 235000019477 peppermint oil Nutrition 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 239000003507 refrigerant Substances 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 238000001256 steam distillation Methods 0.000 description 1
- 230000000707 stereoselective effect Effects 0.000 description 1
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Images
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
Technical field
The present invention relates to the method for a kind of Pseudomonas alcaligenes (Pseudomonas alcaligenes) and preparation MENTHOL (i.e. (-)-menthol, chemistry (1R, 2S, 5R)-2-sec.-propyl by name-5-methyl-cyclohexanol).
Background technology
Natural menthol is mainly MENTHOL, the characteristic peppermint fragrance of MENTHOL tool and strong refrigerant effect, and smell is more fresh brisk; MENTHOL has stronger physiologically active, and it has the effects such as sterilizing and itch-relieving, excited analgesia, calmness, antisepsis and sterilization, treatment pain, therefore has larger medical value.These character of MENTHOL make it have higher industrial value than the menthol of other configurations.
Because people are also growing to the demand of MENTHOL for the growing interest of quality of life.But the MENTHOL of natural extract is depended on tame mint plants unduly, is subjected to region, climatic influences, more and more is difficult to satisfy produce and needs of life.People prepare MENTHOL by synthetic means and satisfy the demands, because the product of chemical method preparation is comprised of 8 kinds of isomer of menthol, be respectively MENTHOL ((1R, 2S, 5R)-2-sec.-propyl-5-methyl-cyclohexyl alcohol), L-isomenthol ((1R, 2S, 5S)-2-sec.-propyl-5-methyl-cyclohexyl alcohol), L-neoisomenthol ((1S, 2S, 5S)-2-sec.-propyl-5-methyl-cyclohexyl alcohol), L-neomenthol ((1R, 2R, 5S)-2-sec.-propyl-5-methyl-cyclohexyl alcohol), d-menthol ((1S, 2R, 5S)-2-sec.-propyl-5-methyl-cyclohexyl alcohol), d-isomenthol ((1S, 2R, 5R)-2-sec.-propyl-5-methyl-cyclohexyl alcohol), d-neoisomenthol ((1R, 2R, 5R)-2-sec.-propyl-5-methyl-cyclohexyl alcohol), d-neomenthol ((1S, 2S, 5R)-2-sec.-propyl-5-methyl-cyclohexyl alcohol).Wherein the content of MENTHOL is lower, have a strong impact on result of use, isolated MENTHOL product from the mixture of 8 kinds of menthol isomer is cheap lower than the natural origin, every quality is suitable, so a key link of course of industrialization is separated MENTHOL exactly.
Chemical preparation technology, biological technology of preparing and natural matter extractive technique are 3 main aspects that prepare at present MENTHOL research.The chemical preparation technology is at present in industrial application, mainly is the Japanese STOL company chiral catalyst (S) that the utilizes invention-asymmetric isomerization of BINAP-Rh catalysis allylamine synthetic MENTHOL that obtains, and this technology is produced 1000 tons of MENTHOLs at present per year.That Herba Menthae Haplocalycis is made crude peppermint oil bu through steam distillation from natural phant source extraction process.Obtain MENTHOL through periodic crystallisation, process is complicated, and energy consumption is high, and productivity is low.Biological catalysis prepares in the research of MENTHOL, is mainly concerned with two kinds of resolution reactions, and a kind of is to utilize microorganism or enzymatic esterification or transesterification, and another kind is to utilize microorganism or enzymatic hydrolysis reaction.Yang Lirong etc. have developed a kind of method of utilizing the mixture of bacillus thuringiensis stereo selective hydrolysis menthol isomer esterification, with the preparation MENTHOL, peak rate of conversion reaches 74%, selectivity d.e.=90.1% (Yang Lirong, Meng Yan. the preparation method of a kind of bacillus thuringiensis and MENTHOL: CN101671639A[P] 2009.8.13).Utilize asymmetric hydrolysis to prepare in the method for MENTHOL, because the mixture polarity difference of MENTHOL and the esterification of menthol isomer is little, sepn process need to be by process (Xu Jianhe, the Zheng Gaowei of the loaded down with trivial details costlinesses such as silica gel column chromatography.Bacillus subtilis esterase and for the production of the application of MENTHOL: CN 101338287A[P] 2008.7.25)
Although separating the research of MENTHOL, above-mentioned biological method has made some progress, but the optical purity of ubiquity product is not high, low conversion rate, the biological catalyst kind is few and biological catalyst high in cost of production shortcoming, and the separation and purification process is difficult for realizing, has restricted industrialized application.If utilize the microbial catalyst of highly selective, then can greatly reduce raw materials cost, improve product purity, have not yet to see the method that splits relevant for the mixture that utilizes Pseudomonas alcaligenes (Pseudomonas alcaligenes) to menthol isomer carboxylate.
Summary of the invention
The methods and applications that the purpose of this invention is to provide a kind of Pseudomonas alcaligenes and preparation MENTHOL thereof.
For achieving the above object, first separation and purification of contriver can be produced the new bacterial strain of selectivity transesterification MENTHOL carboxylate to a strain.Identify through 16S rDNA and Physiology and biochemistry, this bacterial strain is that Pseudomonas alcaligenes belongs to (Pseudomonas alcaligenes), called after Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99).This bacterial strain is preserved in Chinese common micro-organisms culture presevation administrative center, and preservation date is on December 3rd, 2010, and preserving number is CGMCC No.4405.
Pseudomonas alcaligenes CGMCC No.4405 can be used for as the catalyzer for preparing MENTHOL by the mixture that separates the esterification of menthol isomer.
The method of utilizing Pseudomonas alcaligenes CGMCC No.4405 to prepare MENTHOL comprises the steps:
(1) be that the Pseudomonas alcaligenes of CGMCC No.4405 joins and carries out fermentation culture in the substratum with preserving number;
(2) by following the first scheme, alternative plan or third party's case the mixture of menthol isomer carboxylate is carried out catalysis, obtain reaction solution, the mixture of described menthol isomer carboxylate contains the MENTHOL carboxylate, and contain in L-isomenthol carboxylate, L-neoisomenthol carboxylate, L-neomenthol carboxylate, d-menthol ester compound, d-isomenthol carboxylate, d-neoisomenthol carboxylate, the d-neomenthol carboxylate any or appoint several:
The first scheme: the mixture of described menthol isomer carboxylate is joined in the fermented liquid that step (1) obtains, obtain reaction solution after the reaction;
Alternative plan: the centrifugal supernatant liquor that obtains of fermented liquid that step (1) is obtained, the mixture of described menthol isomer carboxylate is added in the described supernatant liquor, obtain reaction solution after the reaction;
Option b third party's case: the fermented liquid that step (1) is obtained carries out the solid preparation that processed obtains fermented liquid, after the solid preparation of this fermented liquid joined pH with the mixture of menthol isomer carboxylate react in 5～10 the buffered soln, obtain reaction solution;
(3) described reaction solution is separated obtain MENTHOL.
Further, the present invention is in step (2), and the mixture of described menthol isomer carboxylate refers to the mixture of the ester that each menthol isomer in the menthol isomer mixture and aliphatic carboxylic acid or aromatic carboxylic acid form.
Further, the present invention is in third party's case of step (2), and described buffered soln is phosphoric acid buffer, citrate buffer solution or tris-HCI buffer; The solid preparation of described fermented liquid is 1g/L～100g/L with respect to the mass concentration of buffered soln.
Further, the present invention is in step (2), and the composition of the substratum of described fermentation culture is: casein 1～10g/L, tween-80 1～15g/L, yeast extract paste 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 substratum is 5～10; Described Pseudomonas alcaligenes is 1%～10% (volume) with respect to the inoculum size of substratum; Fermentation time is 12～48 hours; Leavening temperature is 15～50 ℃.
Further, the present invention is in step (3), and described separation refers to described reaction solution is carried out first reduction vaporization, obtains MENTHOL with organic solvent extraction afterwards.
The inventive method has the following advantages with respect to chemical preparation process, biological preparation method and traditional natural matter extracting method:
(1) adopt tradition to extract the method for MENTHOL from natural mint plants, not only the production process efficiency ratio is lower, energy consumption is large, and easily is subject to the impact of mint plants plantation output; And chemical preparation process is because the expensive easily inactivation of catalyzer itself, and needs use a large amount of organic solvent and auxiliary agent, and is all higher on production cost and Environmental costs; And by contrast, the present invention adopts Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99), MENTHOL carboxylate in the mixture of selective hydrolysis menthol isomer carboxylate, not only have advantages of than highly selective and transformation efficiency, and can a large amount of uses that reduce organic solvents on the technique, environmentally friendly.
(2) industrialized fractionation prepares in the MENTHOL method, all be to adopt the mixture of menthol isomer as initial feed, and most of method all adopt d-menthol and this a pair of enantiomer of MENTHOL at first separated from the mixture of menthol isomer again and split now.And the present invention directly adopts the synthetic menthol isomer mixture that obtains of chemical method as substrate, through simple esterification, no longer needs in advance sepn process, and method is simple, and is with the obvious advantage aspect facility investment and process cost.
(3) the present invention utilizes the microorganism of screening to prepare MENTHOL, the preparation of can fermenting voluntarily, and steady quality, storage-stable, convenient transportation can reduce raw materials cost greatly, has vast application prospect.
(4) the present invention's method of adopting reduction vaporization is in advance separated the carboxylate of menthol with MENTHOL, has greatly simplified sepn process, and MENTHOL that can the direct preparation of high-purity degree has reduced the energy consumption of separating, and possesses industrial prospect.
(5) adopt Pseudomonas alcaligenes Pseudomonas alcaligenes LM99 of the present invention to carry out asymmetric catalysis, generate MENTHOL purity more than 95%, transformation efficiency Gao Keda 90%; And unreacted substrate can directly reclaim, and racemization becomes the mixture of substrate menthol isomer esterification again, has improved substrate utilization ratio.
The preservation information of biological material specimens:
The biological material specimens of preservation: Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99);
Depositary institution: (be called for short: CGMCC) at China Committee for Culture Collection of Microorganisms common micro-organisms center;
Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica (postcode: 100101);
Preservation date: on December 3rd, 2010;
Preservation registration number: CGMCC No.4405.
Description of drawings
Fig. 1 is the chromatography of gases figure of the reaction solution of Pseudomonas alcaligenes LM99 catalyzed reaction, have 8 peak-to-peak signals among the figure, be followed successively by from left to right: MENTHOL, the different peppermint propionic ester of L-, the different peppermint propionic ester of d-, the new peppermint propionic ester of DL-, L-peppermint propionic ester, d-peppermint propionic ester, the strange peppermint propionic ester of L-, the strange peppermint propionic ester of d-; The appearance time of 8 fignal centers is followed successively by 16.440 minutes, 22.323 minutes, 22.665 minutes, 24.323 minutes, 24.757 minutes, 25.323 minutes, 27.540 minutes, 27.790 minutes.
Fig. 2 is the change curve of transformation efficiency and product diastereomeric excess value under the differential responses time.
Embodiment
In an embodiment, carry out in the catalytic process at the mixture to menthol isomer carboxylate, reaction solution is carried out the measuring method (wherein, described substrate is the mixture of menthol isomer carboxylate) specific as follows of substrate conversion efficiency, product diastereomeric excess value (d.e.p):
In reaction process, 5mL reaction solution and 5ml ethyl acetate oscillation extraction, then centrifugal (10000 * g, 5min) obtain the ethyl acetate phase, carry out the Chiral gas chromatography analysis.The system that this Chiral gas chromatography is analyzed is as follows:
Detecting instrument: GC-950 gas chromatograph;
Chirality gas phase post: CP-CYCLODEXTRIN β-2,3,6-M-19
(50m×0.25mm×0.25μm)；
Testing conditions: 130 ℃ of column temperatures, detector and sampler temperature are respectively 260 ℃ and 250 ℃, carrier gas (N
2) 25mL/min, air 3mL/min, hydrogen 7mL/min.
Calculation formula:
Substrate conversion efficiency (%)=(initial substrate concentration-residue concentration of substrate)/initial substrate concentration * 100%;
d.e.
s＝c×d.e.
p/(1-c)×100％
d.e.
p(％)＝[(A
R-A
S)/(A
S+A
R)]×100％
Wherein, c is substrate conversion efficiency, d.e.
pBe product diastereomeric excess value, wherein A
S, A
RBe respectively the peak area sum of (L)-menthol and non-(L)-menthol in the gas-chromatography institute test sample product.
Embodiment 1: the screening of bacterial classification and evaluation
The present embodiment gathers soil at In Hangzhou Region of Zhe Jiang Province, carries out the screening of Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99).Method is as follows:
Enrichment culture: medium component is (g/L): peptone 5.0, and yeast extract paste 3.0, MENTHOL propionic ester 10.0, ammonium sulfate 4.0, dipotassium hydrogen phosphate 10, sal epsom 0.2, pH are 7.0.121 ℃ of sterilization 20min.Take by weighing fresh soil sample 1g and join in the 250mL shaking flask that the 50ml enrichment medium is housed, place 30 ℃, 200r/min shaking table to cultivate.Inoculum size by 1% is every other day once transferred triplicate.
Primary dcreening operation is dull and stereotyped: medium component is (g/L): yeast extract paste 1.0, and ammonium sulfate 2.0, dipotassium hydrogen phosphate 2.0, sodium-chlor 2.0, sal epsom 0.5, MENTHOL propionic ester 20.0, agar 20.0, pH are 7.0.120 ℃ of sterilization 20min.Adopt method of scoring, with the evenly line on plate culture medium of enrichment bacterium liquid, cultivated 1～2 day for 30 ℃.The thalline mark of hydrolysis transparent circle will be arranged, place 4 ℃ of Refrigerator stores.
Sieve again shaking flask: medium component is (g/L): peptone 5.0, and glucose 5.0, yeast extract paste 5.0, sweet oil 50.0, ammonium sulfate 5.0, dipotassium hydrogen phosphate 2.0, sodium-chlor 1.0, sal epsom 0.2, pH are 7.0.120 sterilization 20min.Single bacterium colony access 20mL that primary dcreening operation is obtained sieves in the substratum again, 30 ℃, growth is after 24 hours under the 200rpm, get fresh fermented liquid 5ml, add the mixture of menthol isomer carboxylate, the mixture concentration of menthol isomer carboxylate is 20g/L, 30 ℃ of oscillatory reactions 24 hours, add ethyl acetate oscillation extraction hydrolysate, be used for Chiral gas chromatography and analyze, with substrate selective higher all higher bacterial strain preservation is for subsequent use with product diastereomeric excess value.
Strain identification: 16S rDNA identifies and Physiology and biochemistry is identified.
Through screening, be numbered the bacterial strain of LM99, its diastereomer is the highest to value, and has relatively preferably transformation efficiency, and the gas phase spectrogram is seen Fig. 1.As can be seen from Figure 1, the product MENTHOL goes out the peak position at 16.440min.Bacterial strain LM99 is done 16S rDNA PCR sequencing analysis and Physiology and biochemistry experiment (seeing Table 1), sequencing result is shown in SEQ No.1, result by sequencing analysis and Physiology and biochemistry experiment can determine that the bacterial strain that filters out is that Pseudomonas alcaligenes belongs to this bacterial strain called after Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99).
The Physiology and biochemistry experimental result of table 1 Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99)
Embodiment 2:
Select the fermentation culture based component to be: casein 10g/L, tween-80 9g/L, yeast extract paste 3g/L, ammonium sulfate 8g/L, dipotassium hydrogen phosphate 6g/L regulates pH to 7.0.The seed liquor of Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99) is inoculated in the above-mentioned fermention medium of 50mL with 1% (v/v) inoculum size, place 50 ℃, fermentation is after 20 hours in the 200r/min constant-temperature table, take out the 5mL fermented liquid and make thick enzyme, thick enzyme is with the concentration of 1g/L, (content of each isomer is the mixture of menthol isomer propionic ester: d-isomenthol propionic ester 12.3%, L-isomenthol propionic ester 12.3%, d-menthol propionic ester 24.4%, MENTHOL propionic ester 24.4%, d-neoisomenthol propionic ester 13.3%, L-neoisomenthol 13.3%) concentration with 1g/L joins in the 5ml phosphate buffered saline buffer (pH of buffer=7.0), place 35 ℃, oscillatory reaction 10h in the 200r/min constant-temperature table, after finishing, reaction adds 5ml ethyl acetate oscillation extraction, carry out again gas chromatographic analysis, the transformation efficiency that obtains final substrate is 51.1%, product diastereomeric excess value 95.3%.
Embodiment 3:
Select the fermentation culture based component to be: casein 8g/L, tween-80 3g/L, yeast extract paste 1g/L, ammonium sulfate 4g/L, dipotassium hydrogen phosphate 4g/L, calcium chloride 0.5g/L, sal epsom 0.5g/L regulates pH to 8.0.The seed liquor of Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99) is inoculated in the above-mentioned fermention medium of 50mL with 12% (v/v) inoculum size, place 18 ℃, fermentation is after 40 hours in the 200r/min constant-temperature table, (content of each isomer is the mixture of menthol isomer butyric ester: d-isomenthol butyric ester 10.3%, L-isomenthol butyric ester 10.3%, d-menthol butyric ester 26.4%, MENTHOL butyric ester 26.4%, d-neoisomenthol butyric ester 13.3%, L-neoisomenthol butyric ester 13.3%) joins in the 5ml phosphoric acid buffer in (pH=10.0) with the concentration of 60g/L, place 45 ℃, oscillatory reaction 60h in the 200r/min constant-temperature table, after finishing, reaction adds 5ml ethyl acetate oscillation extraction, carry out again gas chromatographic analysis, as shown in Figure 2: the transformation efficiency that obtains final substrate is 79.4%, product diastereomeric excess value 99%.
Embodiment 4:
Select the fermentation culture based component to be: casein 5g/L, tween-80 15g/L, yeast extract paste 10g/L, ammonium sulfate 12g/L, dipotassium hydrogen phosphate 7g/L, calcium chloride 1g/L, sal epsom 1g/L regulates pH to 6.0.The seed liquor of Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99) is inoculated in the above-mentioned fermention medium of 50mL with 10% (v/v) inoculum size, place 30 ℃, fermentation is after 48 hours in the 200r/min constant-temperature table, take out the 5mL fermented liquid and make thick enzyme, thick enzyme is with the concentration of 30g/L, (content of each isomer is the mixture of menthol isomer valerate: d-isomenthol valerate 15.3%, L-isomenthol valerate 15.3%, d-menthol valerate 24.4%, MENTHOL valerate 24.4%, d-neoisomenthol valerate 10.3%, L-neoisomenthol valerate 10.3%) concentration with 40g/L joins in the 5ml citrate buffer (pH of buffer=5.0), place 20 ℃, oscillatory reaction 50h in the 200r/min constant-temperature table, after finishing, reaction adds 5ml ethyl acetate oscillation extraction, carry out again gas chromatographic analysis, the transformation efficiency that obtains final substrate is 30%, product diastereomeric excess value 93.8%.
Embodiment 5:
Select the fermentation culture based component to be: casein 3g/L, tween-80 20g/L, yeast extract paste 8g/L, ammonium sulfate 2g/L, dipotassium hydrogen phosphate 1g/L, calcium chloride 0.2g/L, sal epsom 1.5g/L regulates pH to 10.0.The seed liquor of Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99) is inoculated in the above-mentioned fermention medium of 50mL with 5% (v/v) inoculum size, place 15 ℃, fermentation is after 12 hours in the 200r/min constant-temperature table, take out the 5mL fermented liquid and make thick enzyme, thick enzyme is with the concentration of 100g/L, (content of each isomer is the mixture of menthol isomer benzoic ether: d-isomenthol benzoic ether 12.3%, L-isomenthol benzoic ether 12.3%, d-menthol benzoic ether 20.4%, MENTHOL benzoic ether 20.4%, d-neoisomenthol benzoic ether 17.3%, L-neoisomenthol benzoic ether 17.3%) concentration with 50g/L joins in the 5ml tris-HCI buffer (pH of buffer=9.0), place 60 ℃, oscillatory reaction 1h in the 200r/min constant-temperature table, after finishing, reaction adds 5ml ethyl acetate oscillation extraction, carry out again gas chromatographic analysis, the result is as shown in table 2, the transformation efficiency of final substrate is 90%, product diastereomeric excess value 90%.
Table 2
Embodiment 6:
Select the fermentation culture based component to be: casein 1g/L, tween-80 6g/L, yeast extract paste 7g/L, dipotassium hydrogen phosphate 4g/L, calcium chloride 0.7g/L, sal epsom 0.3g/L regulates pH to 7.0.The seed liquor of Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99) is inoculated in the above-mentioned fermention medium of 50mL with 7% (v/v) inoculum size, place 40 ℃, fermentation is after 48 hours in the 200r/min constant-temperature table, take out the 5mL fermented liquid and make thick enzyme, thick enzyme is with the concentration of 80g/L, (content of each isomer is the mixture of menthol isomer propionic ester: d-isomenthol propionic ester 12.3%, L-isomenthol propionic ester 12.3%, d-menthol propionic ester 19.4%, MENTHOL propionic ester 19.4%, d-neoisomenthol propionic ester 18.3%, L-neoisomenthol propionic ester 18.3%) concentration with 35g/L joins in the 5ml phosphate buffered saline buffer (pH of buffer=7.0), place 37 ℃, oscillatory reaction 22h in the 200r/min constant-temperature table, after finishing, reaction adds 5ml ethyl acetate oscillation extraction, carry out again gas chromatographic analysis, the result is as shown in table 3, the transformation efficiency of the final substrate that obtains is 62.4%, product diastereomeric excess value 94.9%.
Table 3
Embodiment 7:
Select the fermentation culture based component to be: casein 12g/L, tween-80 1g/L, yeast extract paste 5g/L, ammonium sulfate 10g/L, dipotassium hydrogen phosphate 2g/L, calcium chloride 0.5g/L, sal epsom 0.8g/L regulates pH to 5.0.The seed liquor of Pseudomonas alcaligenes LM99 (Pseudomonas alcaligenes LM99) is inoculated in the above-mentioned fermention medium of 50mL with 8% (v/v) inoculum size, place 37 ℃, fermentation is after 55 hours in the 200r/min constant-temperature table, centrifugal (10000Xg, 4 ℃) obtain fermented supernatant fluid, (content of each isomer is the mixture of menthol isomer laurate: d-isomenthol laurate 11.3%, L-isomenthol laurate 11.3%, d-menthol laurate 25.4%, MENTHOL laurate 25.4%, d-neoisomenthol laurate 13.3%, the strange peppermint laurate 13.3% of L-) joins in the 5ml fermented supernatant fluid in (pH=5.0 of fermented liquid) with the concentration of 100g/L, place 50 ℃, oscillatory reaction 35h in the 200r/min constant-temperature table, add 5ml ethyl acetate oscillation extraction after should finishing, carry out again gas chromatographic analysis, the transformation efficiency that obtains final substrate is 24.2%, product diastereomeric excess value 97.5%.
＜110〉Zhejiang University
＜120〉methods and applications of a kind of Pseudomonas alcaligenes and preparation l-menthol thereof
<160> 1
<170> PatentIn version 3.1
<210> 1
<211> 1509
<212> DNA
<213> Pseudomonas alcaligenes
<400> 1
tgaacgctag cggcaggcct aacacatgca agtcggggag aaagcagggg accttcgggc 60
cttgcgctat cagatgagcc taggtcggat tagcttctgc ctggtagtgg gggcacaacg 120
tttcgaaagg aacgctaaga ccgcatacgt ctacgggaga aagcagggga cgttcgggcc 180
ttgctatcag atgatggcct aggtcggatt agctagtagg tgaggtaatg gctcacctag 240
gcgacgatcc gtaactggtc tcagaggatg atcagtcaca ctggaactga gacacggtcc 300
agactctacg ggaggcagca gtggggaata ttggtcacac tggaactgag acacggtcca 360
gactcctacg ggaggcagca gtggggaata ttgaaagcac tttaagttgg gaggaagggc 420
agtaagttaa taccttgctg ttttgacgtt accaacagaa taagcaccgg ctaacttcgt 480
gccagcagcc gcggtaatac gaagggtgca agcgttaatc ggaattactg ggcgtaaagc 540
gcgcgtaggt ggttcagcaa gttggaggtg aaatccccgg gctcaacctg ggaactgcct 600
ccaaaactga ggtgcgaaag gacggtagag ggtagtggaa tttcctgtgt agcggtgaaa 660
tgcgtagata taggaaggaa caccagtggc gaaggcgact acctggactg atactgacac 720
tgaggtgcga aaggtgggga gcaaaacagg attagatacc ctggtagtcc acgccgtaaa 780
cgatgtcgac tagccgttgg gatccttgtg atcagatacc ctggtagtcc acgccgtaaa 840
cgatgtcgac tagccgttgg aatccttgag attctcaaat gaattgacgg atgcccgcac 900
aagcgtggga gcatgtggtt taattcgaag caacgcgaag aaccttacct ggccttgacg 960
cgctgagaac tttccagaga tggattggtg ccttagggaa ctcagacaca ggtgctgcat 1020
ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc cgccagggct acacaaaccc 1080
ttgtcctgtt accagcacgt tatggtgggc actctaagga gactgccggt gacaaaccgg 1140
aggaaggtgg ggatgacgtc aagtgatcat ggcccttacg gccagggcta cacacgtgct 1200
acaatggtcg gtacaaaggg ttgccaagcc gcgaggtgga gctaatccca taaaaccgat 1260
cgtagtccgg atcgcagtct gcaactcgac tgctcgggaa ctcagacaca ggtgctgcat 1320
ggctgtcgtc agctcgtgtc gtgagatgtt gggcttgtac acaccgcccg tcacaccacg 1380
ggagtgggtt gctccagaag tagctagtct aaccgcaagg gggacggtta ccacggagtg 1440
attcatgact ggggtgaagt cgtaacaagg tagccgtagg tgaactgcgg ctggatcacc 1500
tcctttcta 1509
Claims (4)
1. a Pseudomonas alcaligenes (Pseudomonas alcaligenes), it is characterized in that: its preserving number is CGMCC No.4405.
2. the application of the Pseudomonas alcaligenes of a claim 1 is characterized in that: be used for as the catalyzer for preparing MENTHOL by the mixture that separates menthol isomer carboxylate.
3. a Pseudomonas alcaligenes that utilizes claim 1 prepares the method for MENTHOL, it is characterized in that, comprises the steps:
(1) be that the Pseudomonas alcaligenes of CGMCC No.4405 joins and carries out fermentation culture in the substratum with preserving number; The composition of the substratum of described fermentation culture is: casein 1～10g/L, tween-80 1～15g/L, yeast extract paste 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 substratum is 5～10; Described Pseudomonas alcaligenes with respect to the inoculum size of substratum by volume percentage composition count 1%～10%; Fermentation time is 12～48 hours; Leavening temperature is 15～50 ℃;
(2) by following the first scheme, alternative plan or third party's case the mixture of menthol isomer carboxylate is carried out catalysis, obtain reaction solution; The mixture of described menthol isomer carboxylate refers to the mixture of the ester that menthol isomer mixture and aliphatic carboxylic acid or aromatic carboxylic acid form; Described menthol isomer mixture contains MENTHOL, and contain in L-isomenthol, L-neoisomenthol, L-neomenthol, d-menthol, d-isomenthol, d-neoisomenthol, the d-neomenthol any or appoint several:
The first scheme: the mixture of described menthol isomer carboxylate is joined in the fermented liquid that step (1) obtains, obtain reaction solution after the reaction;
Alternative plan: the centrifugal supernatant liquor that obtains of fermented liquid that step (1) is obtained, the mixture of described menthol isomer carboxylate is added in the described supernatant liquor, obtain reaction solution after the reaction;
Third party's case: the fermented liquid that step (1) is obtained carries out processed and obtains thick enzyme, after will this thick enzyme join pH with the mixture of menthol isomer carboxylate and react in 5～10 the buffered soln, obtain reaction solution; Described buffered soln is phosphoric acid buffer, citrate buffer solution or tris-HCI buffer;
(3) described reaction solution is carried out first reduction vaporization, obtains MENTHOL with organic solvent extraction afterwards.
4. the method for preparing MENTHOL according to claim 3, it is characterized in that: in third party's case of step (2), described thick enzyme is 1g/L～100g/L with respect to the mass concentration of buffered soln.
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| CN101671639A (en) * | 2009-08-13 | 2010-03-17 | 浙江大学 | Method for preparing bacillus thuringiensis and L-menthol thereof |
| CN101818158A (en) * | 2010-03-30 | 2010-09-01 | 中国科学院上海有机化学研究所 | Biosynthetic gene cluster of FR901464 |
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