CN100334198C - Serration and its use in preparation of chiral precurser for dielzepin - Google Patents
Serration and its use in preparation of chiral precurser for dielzepin Download PDFInfo
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- CN100334198C CN100334198C CNB2004100670461A CN200410067046A CN100334198C CN 100334198 C CN100334198 C CN 100334198C CN B2004100670461 A CNB2004100670461 A CN B2004100670461A CN 200410067046 A CN200410067046 A CN 200410067046A CN 100334198 C CN100334198 C CN 100334198C
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- acid methyl
- glycidic acid
- methyl esters
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Abstract
The present invention relates to a Serratia sp. ECU1010 capable of generating stereoselectivity ester hydrolase and the application of the Serratia sp. ECU1010 to the preparation of diltiazem chiral precursors. Lyoli pase produced after the Serratia sp. ECU1010 of the present invention is fermented and cultured can be used for disassembling racemate (+/-)-methoxyphenyl glycidic acid methyl ester, so chiral precursor-(-)-methoxyphenyl glycidic acid methyl ester which is the key for the production of diltiazem, namely a cardiovascular medicine, is obtained. Experiments indicate that the serratia lipase has the advantages of high catalysis efficiency, strong selectivity, good product optical purity, easy recovery, etc.
Description
Technical field
The present invention relates to a strain Serratia and an application thereof, specifically, relate to Serratia and the application method in the preparation diltiazem chiral precursor thereof that the stereoselectivity ester hydrolase is produced in a strain.
Technical background
Odizem (diltiazem) is the flat class calcium ion channel blocker of the synthetic benzo thiophene seventies, because this medication is imitated definitely, toxic side effect is little, for a long time, is widely used in stenocardia and hypertensive long-term treatment.Adopt the synthetic diltiazem of chemo-enzymatic process can simplify synthetic route, improve yield, reduce cost.In the chemo-enzymatic process production technique, general lipase-catalyzed fractionation (±)-p-methoxyphenyl glycidic acid methyl esters that adopts, wherein unwanted (+)-isomer is by the hydrolysis of enzyme selectivity ground, and useful (-)-p-methoxyphenyl glycidic acid methyl esters is intactly remained, and is used for synthetic single enantiomer (+)-Odizem as starting raw material.More existing documents and patent have related to the technology of this respect, comparatively typically have:
(1) European patent EP 362 556 (its All factors being equal, preference will be give to patent is JP 221016/88) discloses the employing several microorganisms in nineteen ninety, as Achromobacter cycloclasters IAM 1013, Alcaligenes faecalis OUT 8030, and Serratia marcescens ATCC 27117 etc., the method of catalysis (±)-p-methoxyphenyl glycidic acid methyl esters stereo selective hydrolysis, but the concentration of substrate of reaction is very low, and aftertreatment is comparatively complicated, and yield is not high yet.
(2) U.S. Pat P 5,274,300 (1989-2-10 application, 1993-12-28 authorize) disclose and have a kind ofly utilized microbe-derived commercial enzyme preparation (for example Lipase OF and Lipase MAP etc.) catalysis to split the method for (±)-p-methoxyphenyl glycidic acid methyl esters and analog thereof in heterogeneous enzyme mebrane reactor system.Be primarily characterized in that water that will contain enzyme and the organic solvent that contains substrate place the both sides of hollow-fibre membrane to react mutually respectively, and utilize sodium bisulfite (NaHSO
3) with the addition reaction of aldehyde, eliminate the disadvantageous effect that the by product (p-methoxy phenylacetaldehyde) after hydrolysate (p-methoxyphenyl glycidic acid) decarboxylation may cause enzyme.The main drawback of this method is that the cost of used commercial enzyme is higher, and optical purity of products is good inadequately, and needs complicated membrane reactor equipment.
(3) document J Ferment Bioeng 1994, and 78:59-63 has reported a kind of more feasible microorganism enzymatic method for splitting.The extracellular lipase that this method is produced microorganism Serratia marcescens Sr41 8000 fermentations is fixed on the hollow-fibre membrane, and the hydrolysis that is used for catalysis (±)-p-methoxyphenyl glycidic acid methyl esters splits.The weak point of this method is to upload on the membrane reactor after still needing earlier enzyme to be extracted, its technology and equipment more complicated, and enzyme is very unstable in reactor, and the transformation period under 22 ℃ has only 127 hours, and its production efficiency progressively reduces with the reactor operation.In addition, this arts demand uses special ability to be subjected to the hollow-fibre membrane of organic solvent, and since in the reaction process substrate and product to stride the membrane mass transfer resistance bigger, therefore limited the performance of enzyme catalysis efficient to a certain extent.
Summary of the invention
The objective of the invention is to, provide a strain can produce the microorganism strains Serratia sp.ECU1010 of (+)-p-methoxyphenyl glycidic acid methyl esters stereospecificity lytic enzyme and produce enzyme substratum and culture condition accordingly, and disclose the method that a kind of enzymatic Split Method prepares the synthetic precursor (-) of chiral drug Odizem-p-methoxyphenyl glycidic acid methyl esters.
The present invention is achieved by the following scheme:
(1) screen the microorganism that can efficiently split (±)-p-methoxyphenyl glycidic acid methyl esters from the bacterial strain of soil and the preservation of this laboratory institute, the result obtains the microorganism strains Serratia sp.ECU1010 that (+)-p-methoxyphenyl glycidic acid methyl esters lytic enzyme is produced in a strain.
(2) bacterial strain Serratia sp.ECU1010 is carried out fermentating enzyme-producing condition optimization, carry out enzyme after the immobilization as biological catalyst with its resolvase or with carrier as " ViewSonic lucky C " (Eupergit C, a kind of commercialization Resins, epoxy of German Degussa company).
(3) biological catalyst is placed water-organic solvent (as isopropyl ether or toluene) two-phase reaction system, the resolution reaction of catalysis (±)-p-methoxyphenyl glycidic acid methyl esters, through after the conventional processing, reclaim and obtain diltiazem chiral precursor (-)-p-methoxyphenyl glycidic acid methyl esters.
(1) bacterial strain
From the bacterial strain of soil and the preservation of this laboratory institute, through primary dcreening operation, multiple sieve and separation and purification, obtain a strain and produce the bacterial strain Serratia sp.ECU1010 of (+)-p-methoxyphenyl glycidic acid methyl esters lytic enzyme, hydrolysis (±)-p-methoxyphenyl glycidic acid methyl esters has obtained (-)-p-methoxyphenyl glycidic acid methyl esters.Therefore, the present invention selects Serratia Serratia sp.ECU1010 as the enzymatic production bacterial strain, this bacterial strain now is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC, No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City), and preserving number is: CGMCCNO.1219; The preserving number date: on September 14th, 2004; Classification name: Serratia (Serratia sp).
(2) strain characteristic (Serratia sp.ECU1010)
1, size and form: be tyrothricin, about 0.9～2.0 μ m of thalline length, straight warp: 0.5～0.8 μ m has all flagellums and can help its motion;
2, be fit to growing environment: can be 10～40 ℃ of temperature, survive in pH5～9, NaCl concentration 0～7% (w/v) environment;
3, cultural characters: some Serratias can produce red pigments, yet it is also very common not produce the bacterial strain of pigment, and nearly about 20% can produce red pigments, usually in room temperature or only just can form pigment in room temperature.Serratiasp.ECU1010 can produce red pigments, but product pigment ability and culture condition are closely related, the pigment instability of producing, and bacterial strain is after 4 ℃ of preservation for some time, and its redness can be taken off.
(3) cultivation of bacterial strain
1, slant culture: peptone 0.5wt%, yeast extract paste 0.5wt%, K
2HPO
40.2wt%, NaCl 0.05wt%, MgSO
47H
2O 0.05wt%, FeSO
47H
2O 0.001wt%, agar 2wt%, pH are 6.5, with the glycerine suspension inoculation of freezing preservation to slant medium, 30 ℃ of constant temperature culture 2 days;
2, shake-flask culture: tween-80 0.1～1.5wt%, dextrin 0～3.0wt%, peptone 0～4.5wt%, corn steep liquor 0～3wt%, ammonium sulfate 0～1.0wt%, K
2HPO
40.2wt%, NaCl 0.05wt%, MgSO
47H
2O 0.05wt%, FeSO
47H
2O 0～0.01wt%, CaCl
20～0.1wt%, pH is 5～8, shakes bottled liquid measure 10～30v/v%, inoculum size 2～10v/v%, 20～40 ℃ of culture temperature, shaking speed 100～200rpm, shake-flask culture 1～3 day; Produce enzyme and can reach 4000～10000 U/L.
3, fermentation in the jar: substratum is identical with shake-flask culture.At the 5L fermentor tank substratum 3L that packs into, after 121 ℃ of sterilizations, insert the 200ml seed culture fluid, in 25～35 ℃, air flow is that (be equivalent to 0.5～2.0vvm), mixing speed is 200～400 rev/mins to 1.5～6.0L/min then, jar, centrifugal collection fermented liquid supernatant hour are put in fermentation to 12～36.
(4) enzyme catalysis resolution reaction
1, fermented liquid after the cultivation through the centrifuging and taking supernatant liquor as the enzyme source, with (±)-p-methoxyphenyl glycidic acid methyl esters is substrate, its add-on is 10-200g/L (a reactant cumulative volume), the add-on of enzyme is 10～100U/g substrate, at normal heptane, octane-iso, toluene, isopropyl ether, methyl tertiary butyl ether, organic solvent such as hexanaphthene or chloroform, reaction preferably is hydrolyzed in the two-phase system that isopropyl ether and Tris-HCl damping fluid are formed, oil-water biphase volume ratio is 1/9-9/1 (v/v), be preferably in the 1/2-3/1 scope, temperature of reaction is 20～50 ℃, be preferably 25～40 ℃, the control reaction conversion ratio is 50～52%, and the reaction times is 3～30 hours.
2, the fermented liquid centrifuging and taking supernatant liquor after will cultivating without any pre-treatment, directly adds macromolecule resin [as resin Eupergit C (" the lucky C of ViewSonic ") or silica gel] or diatomite etc. and carries out immobilization.Saved the extraction step of enzyme when carrying out immobilization, simplified operation, the save operation time, reduced the enzyme loss of living simultaneously with hollow-fibre membrane.Prepared immobilized enzyme is as biological catalyst, and reaction under these conditions is hydrolyzed.Immobilized enzyme can repeat repeatedly to use the reaction that is hydrolyzed.
The extraction of (five) (-)-p-methoxyphenyl glycidic acid methyl esters and refining
Collect the organic phase of reaction, earlier with 5% NaHSO
3Solution washing is used 5% NaHCO again
3Solution washing, after anhydrous magnesium sulfate drying is handled, underpressure distillation, crystallization in appropriate solvent (as Virahol) can obtain highly purified crystal (-)-p-methoxyphenyl glycidic acid methyl esters (ee＞99%).
Adopt method of the present invention, technological process is simple, the reaction conditions gentleness; The catalytic activity and the stereoselectivity of used enzyme are higher; The immobilized enzyme stability of preparation is high, but successive reaction is more than 5 batches.(-) that is obtained-p-methoxyphenyl glycidic acid methyl esters optical purity can reach more than the 99%ee.This method has advantage simple and practical, with low cost.Be expected to apply industrial.
Embodiment
To be further elaborated technology contents of the present invention by embodiment below, and its objective is to be better understanding content of the present invention.Therefore, the cited case does not limit protection scope of the present invention.
Embodiment 1
Slant culture: peptone 0.5wt%, yeast extract paste 0.5wt%, K
2HPO
40.2wt%, NaCl 0.05wt%, MgSO
47H
2O 0.05wt%, FeSO
47H
2O 0.001wt%, agar 2wt%, pH6.5.With the glycerine suspension inoculation of freezing preservation to inclined-plane (flat board) substratum, about 1.5 days of 30 ℃ of constant temperature culture.
The product enzyme is cultivated: tween-80 1.0wt%, peptone 3.0wt%, K
2HPO
40.2wt%, NaCl 0.05wt%, MgSO
47H
2O 0.05wt%, FeSO
47H
2O 0.001wt%, pH7.0,250ml shake bottled liquid measure 30ml, inoculum size 2.5v/v%, 25 ℃ of culture temperature, shaking speed 200rpm, shake-flask culture is 2 days under these conditions, produces enzyme and can reach 4780U/L.
The enzyme unit (U) that lives is defined as: under 25 ℃, pH7.0 condition, per minute hydrolysis p-NP acetic ester (1.0mmol/L) generates the required enzyme amount of 1 μ mol p-NP.
Embodiment 2
Slant culture is identical with embodiment 1.Shake the 30ml substratum of packing in the bottle at 250ml, it is composed as follows:
Tween-80 1.0wt%, dextrin 2.0wt%, corn steep liquor 3.0wt%, ammonium sulfate 0.5wt%, K
2HPO
40.2wt%, NaCl 0.05wt%, MgSO
47H
2O 0.05wt%, FeSO
47H
2O 0.001wt%, pH6.5.Cultivate 12 hours nutrient solution will be under 30 ℃, 150rpm condition in advance as seed, changing 4 1L that the 150ml same medium is housed over to by the inoculum size of 5v/v% shakes in the bottle, 30 ℃ of culture temperature, to cultivate 24 hours under the condition of shaking speed 150rpm, production of enzyme is 9480U/L.
Embodiment 3
Seed culture is identical with embodiment 2.The 3L substratum of packing in the 5L fermentor tank, it is composed as follows: dextrin 2wt%, corn steep liquor 1.5wt%, ammonium sulfate 0.5wt%, K
2HPO
40.2wt%, NaCl 0.05wt%, MgSO
47H
2O0.05wt%, CaCl
20.05wt%, pH6.5.Behind the medium sterilization, insert the pre-seed liquor 200ml that cultivates 12 hours, aerobic fermentation under 30 ℃, 400rpm and 0.5vvm condition, therebetween at 5-11 hour at the uniform velocity stream add tween-80 6g altogether, then drip bubble enemy (the 1wt/v% aqueous solution) and carry out froth breaking if produce foam.Ferment and put jar after 15 hours, recording production of enzyme is 5600U/L.
Embodiment 4
Get the fermented liquid 5ml among the embodiment 1, the centrifuging and taking supernatant adds equal volume toluene, adds 1.40g (±)-p-methoxyphenyl glycidic acid methyl esters again, and insulation reaction on the shaking table of 30 ℃ and 150rpm keeps reaction pH constant in 8.5.When reaction proceeds to 6 hours, transformation efficiency is near 50%, and the analysis yield of gained (-)-p-methoxyphenyl glycidic acid methyl esters is 46.2%, optical purity＞98%ee, and mapping selection rate (E value) surpasses 100.
Embodiment 5
Get the fermented liquid 10ml among the embodiment 1, the centrifuging and taking supernatant adds 2g Resins, epoxy " the lucky C of ViewSonic " (Eupergit C, German Degussa company product), and insulation after 2 hours on the shaking table of 30 ℃ and 150rpm is filtered, the washing immobilized enzyme.It is suspended in the Tris-HCl damping fluid of 5ml pH8.0, add the 5ml isopropyl ether, add 0.104g (±)-p-methoxyphenyl glycidic acid methyl esters again, insulation reaction on the shaking table of 30 ℃ and 150rpm, keep reaction pH constant in 8.0, the reaction times is 10-14 hour.The immobilized enzyme that leaches after will reaction finishing adds new reaction substrate and continues reaction, so reuse 5 times after, vigor does not significantly descend, the optical purity of gained (-)-p-methoxyphenyl glycidic acid methyl esters all＞98%ee, E value is above 100.
Embodiment 6
Get the fermented liquid 100ml among the embodiment 2, the centrifuging and taking supernatant adds 30g Resins, epoxy Eupergit C, and insulation after 2 hours on the shaking table of 30 ℃ and 150rpm is filtered, the washing immobilized enzyme.The gained immobilized enzyme particle is suspended in the Tris-HCl damping fluid of 50ml pH8.0, add 50ml toluene, add 2.08g (±)-p-methoxyphenyl glycidic acid methyl esters again, insulation reaction on the shaking table of 35 ℃ and 150rpm, keep reaction pH constant in 8.5, the reaction times is 10-14 hour.After will reacting end, collect organic phase, the immobilized enzyme that leaches adds new reaction substrate and continues reaction, so reuses 5 times.Collect the organic phase of 5 batch reactions, NaHSO with 5%
3Wash three times, use 5% NaHCO again
3Washing, after anhydrous magnesium sulfate drying was handled, underpressure distillation, crystallization obtained highly purified (-)-p-methoxyphenyl glycidic acid methyl esters crystal (ee＞99%), and yield is 42%.
M.P.：87-88℃；
[α]
D 20:-207.08 (c=1, methyl alcohol).
Embodiment 7
In the three-necked flask that has mechanical stirring and water bath with thermostatic control, add (±)-p-methoxyphenyl glycidic acid methyl esters 1.0mol (208g) and methyl tertbutyl ethereal solution 2.0L, be stirred to whole dissolvings.Get the fermented liquid 1.0L among the embodiment 3 then, bath temperature is transferred to 28 ℃, start mechanical stirring (200 rev/mins), insert pH electrode simultaneously, with the NaOH solution of automatical potentiometric titrimeter dropping 1.0N, pH is in 8.0 ± 0.1 scopes in control, and calculates the transformation efficiency of enzymically hydrolyse according to alkali consumption.As a result, react after 6 hours, transformation efficiency is 51%, stopped reaction is told organic phase with separating funnel, and the muddy part in interface is poured into and is covered with diatomaceous B and carries out suction filtration, merge water and organic phase, water methyl tertiary butyl ether extracting twice (300ml * 2); Organic phase is used a small amount of 5%NaHSO successively
3, 5%NaHCO
3, deionization and saturated common salt water washing, add anhydrous magnesium sulfate drying again after, rotary evaporation reclaims organic solvent, obtains little yellow solid 95.7g, yield is 46%, obtains 82.3g optical purity (＞99.9%ee) clear crystal in methyl alcohol behind the recrystallization.
According to technical scheme disclosed by the invention and embodiment, relevant engineering technical personnel can be easily with the said microbial strains of the present invention and cultivation and conversion process, and the catalytic kinetics that is applied to the chiral substrates (for example 3-arylolycidyl acetoacetic ester and other short chain fatty alcohol ester) of other similar in other reaction system (as microemulsion and ionic liquid) splits with drawing inferences about other cases from one instance.
Claims (6)
1, a strain Serratia bacterial strain Serratia sp.ECU1010, preserving number is: CGMCC No.1219.
As the application of the said bacterial strain of claim 1, it is characterized in that 2, said bacterial strain is used for splitting (±)-p-methoxyphenyl glycidic acid methyl esters.
As the said application of claim 2, it is characterized in that 3, wherein said fractionation (±)-p-methoxyphenyl glycidic acid methyl esters comprises the steps:
(1) the said bacterial strain ECU1010 of claim 1 carries out fermentation culture, and fermented liquid gets the free-fat enzyme after separating;
(2) will react dissolving substrate (±)-p-methoxyphenyl glycidic acid methyl esters with in the immiscible organic solvent of water by the free-fat enzyme of step (1) gained or its fixed compound and racemization substrate (±)-p-methoxyphenyl glycidic acid methyl esters, oil-water biphase volume ratio is 1/9～9/1, temperature of reaction is 20～50 ℃, reacts after 3～30 hours to collect organic phase and promptly get (-)-p-methoxyphenyl glycidic acid methyl esters;
Wherein: substrate (±)-p-methoxyphenyl glycidic acid methyl esters add-on is 10～200g/L reactant cumulative volume, and the add-on of enzyme is 10～100U/g substrate.
As the said application of claim 3, it is characterized in that 4, wherein said organic solvent is a kind of in hexanaphthene, normal heptane, octane-iso, chloroform, toluene, isopropyl ether or the methyl tertiary butyl ether in the step (2).
5, as the said application of claim 3, it is characterized in that, said free-fat enzyme immobilization thing in the step (2) wherein, its carrier is diatomite or macromolecule resin.
As the said application of claim 5, it is characterized in that 6, wherein said carrier is " the lucky C of ViewSonic ".
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101684460B (en) * | 2008-09-22 | 2014-03-12 | 华东理工大学 | Method for preparing high-activity recombination lipoidase |
| CN103305583A (en) * | 2012-03-12 | 2013-09-18 | 江苏华荣生物科技有限公司 | Simple extraction of racemic p-methoxyphenyl glycidic acid methyl ester |
| CN105524896A (en) * | 2015-12-28 | 2016-04-27 | 苏州开元民生科技股份有限公司 | Method used for fermentation enzyme production of Serratia marcescens lipase and improving enzyme activity of Serratia marcescens lipase |
| CN106047837B (en) * | 2016-06-01 | 2019-08-16 | 华东理工大学 | Serratia lipase mutant, recombinant expression transformant, enzyme preparation and application |
| CN108118016A (en) * | 2018-02-08 | 2018-06-05 | 上海化工研究院有限公司 | A kind of Serratia lipase and its application in (±)-MPGM is split |
| CN109468303A (en) * | 2018-11-28 | 2019-03-15 | 华东理工大学 | A kind of carnosine hydrolase, gene, mutant and its application |
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| Title |
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| 不对称催化合成技术及其最新进展 汪秋安，工业催化，第11卷第5期 2003 * |
| 不对称催化合成技术及其最新进展 汪秋安，工业催化，第11卷第5期 2003;地尔硫卓的合成 张孝义，中国医药工业杂志，第27卷第10期 1996 * |
| 地尔硫卓的合成 张孝义，中国医药工业杂志，第27卷第10期 1996 * |
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Assignee: Jiangsu Huarong Biotechology Co., Ltd. Assignor: East China University of Science and Technology Contract record no.: 2011320000463 Denomination of invention: Serration and its use in preparation of chiral precurser for dielzepin Granted publication date: 20070829 License type: Exclusive License Open date: 20050727 Record date: 20110331 |
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