CN101220336A - Saccharomycete with stereoselectivity lipase liveness and application in producing S- type betaxolol hydrochloride with biological split method thereof - Google Patents
Saccharomycete with stereoselectivity lipase liveness and application in producing S- type betaxolol hydrochloride with biological split method thereof Download PDFInfo
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Abstract
The invention discloses a yeast which has the stereoselective lipase activity and the application of the yeast in the preparation of an S-type betaxolol hydrochloride by using a biological separation method. The method selects one yeast with the stereoselective lipase activity by screening from the soil, utilizes an immobilized cell which is obtained by immobilizing the wet bacteria or sodium alginate-activated carbon-polyethylenimine as an enzyme preparation, carries out an enantiomer separation to a substrate which contains acyl group and prepares the S-type betaxolol hydrochloride. The conversion method is simple, the cost is low and the stereoselectivity is better. The usage of the bacterial strain can carry out the chiral separation of Beta-receptor blocker of betaxolol etc., ephedrine hydrochloride, epinephrine, levodropropizine, salbutamol sulfate, captopril, zofenopril and other compounds which contain hydroxy group or acyl group at the chiral center, thereby having important application value for promoting the development process of the chiral drugs of China.
Description
Technical field
The present invention relates to a saccharomycete and application thereof, relate in particular to a strain and have the yeast of stereoselectivity lipase activity and prepare application in the S-betaxolol hydrochloride at biological Split Method.
Background technology
Glaucoma be a kind ofly fall ill rapidly, hazardness causes the common difficult illness in eye of losing one's sight greatly, at any time.Sickness rate rises to some extent in recent years, add up according to the World Health Organization, present global glaucoma patient is about 6,700 ten thousand, and about 4,500,000 people lose eyesight because of glaucoma, in China, age is greater than among 40 years old the crowd, the sickness rate 1%～2% of primary glaucoma, in the whole nation 1,300,000,000 populations, primary glaucoma patient surpasses 7,000,000, glaucoma has become the second largest ophthalmology common disease of China, accounts for 14.36% of ophthalmic diseases.Statistic data shows that glaucoma has become second largest blinding factor.Present glaucomatous clinical treatment is based on operation and pharmacological agent.The just temporary transient intraocular pressure that reduces of operation can only relief of symptoms for the glaucoma of some type, finally still comes to an end with blind.The treatment glaucoma medicine comprises that mainly parasympathomimetic agent, adrenomimetic drug, adrenoceptor retarding agent, carbonic anhydrase inhibitor, height ooze dewatering agent etc.Wherein choice drug adrenoceptor retarding agent such as timolol, betaxolol etc., but these medicines cause bronchospasm, bradyrhythmia, increase cardiac block easily, side effect such as bring high blood pressure down, at present abroad no longer with it as choice drug.
Left-handed betaxolol is the levo form of betaxolol, and it is to β
1The affinity of acceptor is higher than its raceme far away, and this highly selective makes it safer to the patient with heart and lung diseases, and its side effect is also very little, and does not have film stable (toponarcosis) effect and the effect of endogenous sympathomimetic amine.Left-handed betaxolol is to the β of eye ciliated epithelium
2Acceptor also has higher avidity, can reduce the generation of aqueous humor and reduces intraocular pressure, also can avoid multiple injury by retardance L-voltage-dependent calcium channel protection optic nerve.Experimentation on animals shows that the reduction intraocular pressure effect of left-handed betaxolol generation is than betaxolol 1-2mmHg eager to excel in whatever one does, also have and report that its dextrorotatory form of energy force rate of left-handed betaxolol reduction intraocular pressure is strong by about 25.9%, its validity period is also much longer than racemic modification and dextrorotatory form.Eye is that pungency is arranged with the modal side effect of betaxolol, by result of use is identical but the collyrium that concentration reduces can make this side effect reduce to minimum.The eye drops listing of external existing l-betaxolol hydrochloride, commodity are called " Betoptic S ", are mainly used in chronic open angle glaucoma of treatment or high intraocular pressure patient and reduce intraocular pressure.
Ramesh A.Joshi study group reported the technology for preparing the S-betaxolol with chemical synthesis in 2005, and had applied for several pieces of relevant patents of technology therewith, and these technologies or the expensive reagent of needs perhaps need expensive catalysts.
After microbial transformation was applied to produce, generally combining with microbial transformation with chemical method prepared chiral drug.Biocatalysis resolution reaction stereoselectivity and regioselectivity are strong, and the reaction conditions gentleness can be avoided or reduce and use strong acid, highly basic and some poisonous raw materials, improves operational condition, reduces environmental pollution; Can avoid using the chiral reagent or the chiral catalyst of necessary costliness in the asymmetric synthesis.The biological catalyst low production cost can be mass-produced, and is with short production cycle, is not subjected to seasonal effect, and catalyzer can reuse, and further reduces cost.
People such as Giuseppe DB have reported that in nineteen ninety-five chemo-enzymatic process prepares the method for S-betaxolol, and what they were used for the reaction of catalysis chiral separation is some microbial lipases.That wherein effect is best is pseudomonas lipase A K, and this is reflected in the tertiary butyl methyl ether medium and reacts, and the reaction system flash-point is low, and is inflammable, has potential safety hazard; Enzyme and substrate weight ratio 1: 7, the catalyzer cost is very high; Their optical purity only can reach 80% (resolution yield 50%), by just making the ee value reach 90% to its hydrochloride recrystallization.The reaction stereoselectivity of other micro bioenzyme catalysis is very different, and enzyme-catalyst weight ratio is 1: 1, and the catalyzer cost is higher.In addition, they do not report the stability of enzyme, do not have the report of cells involved or enzyme immobilization yet, and are still far away apart from industrialization demands.The screening stereoselectivity is strong, catalytic efficiency is high and the good stability microbial enzyme, and to explore the novel process of the low production S-betaxolol hydrochloride of catalysis cost on this basis and have independent intellectual property right be the active demand of China present pharmaceutical industry institute.
Summary of the invention
The object of the present invention is to provide that a kind of stereoselectivity is strong, catalytic efficiency is high and the yeast of good stability; Another purpose is that with this yeast be catalyzer, provides it to prepare application in the S-type betaxolol hydrochloride at biological Split Method.
Yeast among the present invention can produce has stereoselective lipase, it is characterized in that can contain acylated compound to chiral centre carries out the enantiomorph fractionation.This yeast is cement rhodotorula bacterium (Rhodotorulamucilaginosa) DQ832198, China Committee for Culture Collection of Microorganisms common micro-organisms center (Datun Road, Chaoyang District, Beijing City), deposit number CGMCC.No.2257 have been preserved in on November 20th, 2007.
This saccharomycetic screening may further comprise the steps:
(1) is that sole carbon source carries out enrichment culture and plate isolation with the substrate, obtains to act on the bacterial classification of substrate.
(2), contain new compound and optical value in the screening converted product and be negative bacterial classification by converted product is carried out TLC and polarimetry.
(3) structure that is tested and appraised product determines conversion of substrate to be the bacterial classification of expection product at last.
(4) bacterial classification is identified.
The present invention screens the saccharomycetic concrete screening method with stereoselectivity lipase activity from soil as follows: at first from the local soil sampling of difference, then soil sample is used enrichment medium enrichment culture secondary; Enrichment culture liquid suitably dilutes dull and stereotyped cultivation of back coating selectivity, will separate good and have the bacterium colony of transparent circle to choose to the inclined-plane; Slant strains cultivated add substrate behind the certain hour and transform, extract product then and measure, and the structure of product is identified.
Converted product can be analyzed with polarimetry, tlc (TLC) and high performance liquid chromatography (HPLC).Optical value is measured in 589nm; It is stationary phase that TLC measures with the silica GF254 thin layer plate, and (10: 1v/v) be developping agent, iodine vapor is a developer to chloroform-methanol, adopts the dual-wavelength lamellar scanning method to carry out; The HPLC condition determination is: with chirality Chiralcel OJ-H post is stationary phase, and normal hexane-Virahol (85: 15) is a moving phase, flow velocity 1mL/min, and the detection wavelength is 273nm.
The HPLC condition determination of product betaxolol is: stationary phase: the OD-H post, moving phase: hexane/Virahol/diethylamine 6/4/0.5, flow velocity 0.5mL/min detects wavelength 254nm, sample size 20 μ L.
The ee value of converted product is calculated by the peak area of two isomer on the sample HPLC collection of illustrative plates, and formula is: ee
s(%)=(peak area
S-peak area
R)/(peak area
S+ peak area
R) * 100.
For having stereoselective enzyme, stereoselectivity is an important techniques index, this index E value representation, and the E value is big more, and the stereoselectivity of bacterial classification is good more.E=ln[(1-c) (1-ee
s)]/ln[(1-c) (1+ee
s)]=ln[1-c (1+ee
p)]/ln[1-c (1-ee
p)], ee wherein
p=([P]-[Q])/([P]+[Q]), ee
S=([B]-[A])/([B]+[A]), c=ee
S/ (ee
S+ ee
p).A, B are respectively two kinds of enantiomorphs of substrate; P, Q are respectively two kinds of enantiomorphs of product.
Product structure is identified with MS, nuclear-magnetism method.
The conversion capability of determining bacterial classification according to the optical value and the thin layer result of converted product.The converted product of excellent species should have the bigger specific rotation light value of negative absolute value, the thin layer result should confirm that it has produced the novel substance that is different from substrate; MS result should prove that the novel substance of its generation is that the substrate deacetylate produces; its ee value is also higher, and the E value of entire reaction is also higher.Converted product with above-mentioned character is separated, obtain the pure acetyl product that takes off, and determine its structure by NMR.
By to colonial morphology, somatic cells shape and size, have or not spore and mycelia to form and the observation of modes of reproduction is carried out preliminary evaluation to bacterial classification.Through analysis, carry out determining of bacterial classification to this bacterial strain 16S DNA.Determine that it is cement rhodotorula (Rhodotorula mucilaginosa) DQ832198, deposit number CGMCC.No.2257.
Can carry out immobilization to the bacterial strain that screens with sodium alginate-activated carbon-polyvinyl alcohol, carry out bio-transformation with this immobilized cell as catalyzer, immobilized cell can reuse.
The present invention prepares the bioconversion method of S-betaxolol hydrochloride intermediate, may further comprise the steps:
(1) cultivation of bacterium producing multi enzyme preparation.
(2) in conjunction with the chemical process synthetic compound (1) of these research and development.
(3) be conversion of substrate with compound (1).
(4) substrate is added transform in the yeast culture liquid and obtain intermediate product.
(5) synthesize the S-betaxolol hydrochloride by the intermediate product that obtains in conjunction with the technology of this research department's exploitation.
In step (4), can transform with resting cell, also can transform with immobilized cell.
It below is detailed description of the present invention.
1, the preparation of substrate
The preparation of compound (1)
a：K
2CO
3，CH
3CN，C
3H
5OCl，b：CH
3OH，C
3H
7NH
2 c：Et
3N，DMAP，(CH
3CO)
2O EtOAc
Get a certain amount of p-hydroxyphenylethanol, add an amount of acetonitrile it is dissolved fully, add epoxy chloropropane and Anhydrous potassium carbonate, 75 ℃ of oil baths, back flow reaction 6h, cooling, suction filtration is removed K
2CO
3And with washing with acetone precipitation several times, merging filtrate, underpressure distillation is to substantially dry, and with obtaining etherificate product 1 behind the sherwood oil recrystallization, 4-(2,3 glycidoxy) phenylethyl alcohol.
A certain amount of etherificate product is dissolved in the methyl alcohol, slowly adds Isopropylamine while stirring, under room temperature, react 7h, complete to the thin layer detection reaction.Underpressure distillation removes desolvates and remaining Isopropylamine; With the chloroform dissolving, use saturated NaHCO again
3With the saturated aqueous common salt extraction,, promptly get aminate 4-[2-hydroxyl-3-[(methylethyl with the organic phase evaporate to dryness) amino] the propoxy-phenylethyl alcohol.
Get a certain amount of aminate, use an amount of acetic acid ethyl dissolution, add aceticanhydride (pressing three times of equivalents of raw material adds); with DMAP is catalyzer, in anhydrous pyridine and acetic anhydride product is carried out acetylize, under the room temperature condition; 0.5h react completely, add suitable quantity of water with stopped reaction, use saturated NaHCO
3Extracted organic phase three times, saturated NaCl solution extraction three times is used anhydrous Na then
2SO
4Dry.Promptly obtain the substrate (compound 1) of microbial transformation.
2, the preparation of immobilized cell
Had bibliographical information now and prepared immobilized cell with sodium alginate, aluminium alginate, carrageenin, carrageenin-gelatin, gelatin-glutaraldehyde.The present invention prepares immobilized cell with sodium alginate (CA)-gac (C) plural gel, and method is as follows:
With a certain amount of sodium alginate (1～5%), distilled water and gac (0～5%) heated and boiled together, cool to room temperature; The bacterial strain that a certain amount of the present invention is filtered out joins in the above-mentioned PVA mixture that configures and stirs; With syringe it is at the uniform velocity splashed into (mixing also fully stirs into emulsus) in 1～10% borax and the calcium chloride mixed solution, filter, clean, promptly get even immobilized cell globule.
Polymine (PEI) enhanced processing method is as follows:
The polyethyleneimine: amine aqueous solution of preparation 0.1%～5% adds 10～50mmol/L calcium chloride, and adjust pH to 5～10 add equivalent CA-C or PVA-CA-C embedded particles, concussion 10～48h.Taking-up is washed with sterilized water, handles 10～60min with glutaraldehyde again, cleans with sterilized water.
3, bioconversion reaction
Thalline of the present invention is inoculated in substratum (corn steep liquor 0.5～5%, peptone 0.1～5%, glycerine 0.5～5%, initial pH 4～10) in, in 20 ℃～50 ℃ cultivation 12～48h, add substrate (1) 0.5～12g/L then, transform 5～45h in 20 ℃～50 ℃ again.Converted product is measured transformation efficiency and product ee value with the HPLC method.
Except that the adding substrate directly transforms in bacterial culture fluid, after also microorganism cells can being filtered out, add the buffered soln of pH4～10, utilize resting cell to transform.Can in transformation system, can add chloroform, hexane or toluene when utilizing conversion of resting cells to improve conversion rate.
Transform in packed bed reactor with immobilized cell: with pack into the reaction column of φ 10～50 * 100～500 of immobilized cell, by 20～50 ℃ of insulations of thermostat container.When carrying out continuous conversion reaction, fill immobilized cell gel 50～500g in packed bed reactor, concentration of substrate is 1～10g/L, and substrate solution is flowed into by reaction column top, and reacted conversion fluid is collected from reactor lower part.The product that transforms is measured transformation efficiency and product ee value with the HPLC method with this understanding.
4, S-type betaxolol hydrochloride is synthetic
In converted product, add amount of methanol and excessive sodium hydroxide, reacted 5～50 minutes.Remove methyl alcohol then, obtain deacetylated product with the ethyl acetate extraction water.
Add toluene, phenyl aldehyde and tosic acid in deacetylated product, charge into argon gas, oil bath refluxes down.Wash with water after the cooling,, under reduced pressure boil off toluene and get amino alcohol derivative the organic layer dried over sodium sulfate.
Amino alcohol derivative is dissolved in the dry DMF, adds NaH, normal temperature reacted 1～30 minute down.In 0 ℃～-50 ℃ reaction 1～10h, react 1～5h then at normal temperatures behind the adding brooethyl cyclopropane.Reaction solution is poured in an amount of saturated solution of sodium bicarbonate, removed NaOH and use chloroform extraction again.Underpressure distillation is removed chloroform and is obtained the S-betaxolol.
The S-betaxolol is dissolved with an amount of Virahol, drip the normal concentrated hydrochloric acid of twice, Virahol is removed in underpressure distillation, use the saturated sodium bicarbonate solution washed product, wash water layer (3*20ml) with chloroform, chloroform is removed in decompression distillation down, obtains weak yellow liquid, carry out recrystallization with sherwood oil-acetone mixed system again, promptly get S-type betaxolol hydrochloride.
The invention has the advantages that: 1, the invention provides a kind of yeast with stereoselectivity lipase activity, utilize this bacterial classification to carry out beta-blocker, ephedrine hydrochloride, suprarenin, levodropropizine, salbutamol sulfate, Robert Caputo profit, zofenopril etc. such as betaxolol and contain the chiral separation of acylated compound, have important use to be worth the process that promotes China's chiral drug exploitation at chiral centre; 2, with regard to synthesis technique the relative chemical method, with the yeast with stereoselectivity lipase activity is Catalyst Production S-type betaxolol hydrochloride, biological catalyst is cheap, production conversion reaction conditions gentleness, environmental friendliness and bioconversion reaction speed is fast, stereoselectivity good has application promise in clinical practice.
Description of drawings
Fig. 1 process route chart of the present invention;
The HPLC collection of illustrative plates of Figure 22 0# bacterial strain converted product;
The HPLC collection of illustrative plates of Figure 32 7# bacterial strain converted product;
The HPLC collection of illustrative plates of Fig. 4 racemize betaxolol;
The HPLC collection of illustrative plates of Fig. 5 S-betaxolol product.
Embodiment
For the present invention is illustrated better, as follows for embodiment.
Collect 15 by the soil sample of grease contamination near restaurant, urban district, Zhengzhou, the oily factory, take by weighing various soil sample kind 10g, be suspended in respectively in the 0.9%NaCl solution, remove bigger impurity particle, inoculation enrichment medium ((NH with 8 layers of filtered through gauze
4)
2SO
40.2%; K
2HPO
40.2%; NaCl 0.05%; MgSO
47H
2O 0.05%; Substrate 3%; PH nature), cultivate and get the 10mL nutrient solution behind the 71h and use again with quadrat method enrichment secondary again.Coating selectivity flat board (it contains agar 2% to composition with enrichment culture).Obtain 52 energy through primary dcreening operation and on the flat board that with the substrate is sole carbon source, grow, and produce the bacterial strain of transparent circle, it is chosen switching LB substratum (yeast extract paste 0.5%, peptone 1%, NaCl 1%, agar 2%, pH 7.5) slant preservation made, and be used for conversion test.
Respectively with 52 inoculation of above-mentioned primary dcreening operation gained to transforming substratum (yeast extract 0.2%, peptone 0.5%, glucose 1%, the pH nature) in, add substrate (concentration 0.3%～0.7%) after cultivating 24h, transform again and cultivate 48h, extract product and carry out TLC and polarimetry.Choosing the converted product optical value in conjunction with document is that negative value and absolute value are bigger, and TLC result shows that 13 bacterial strains that have new product to generate carry out multiple sieve, sieve result such as table 1 again.
Table 1 bacterial classification sieves TLC and optically-active result again
| Numbering | Optical value | TLC result | Numbering | Optical | TLC result | |
| 3 6 13 14 19 20 21 | -1.9 -1.0 -2.5 -3.8 -4.4 -12.5 -2.3 | + + + + + + + | 24 27 28 42 45 52 | -0.4 -7.5 -0.7 -1.9 -1.0 -2.5 | + + + + + + |
TLC result have product with+note, do not have product in-.
Asymmetric hydrolysis takes place in substrate 1 in this test under the catalysis of microbial enzyme, and the product that has the bacterial classification hydrolysis substrate of stereoselectivity enzymic activity to be produced has opticity, and the product optical value is that negative value and the bigger bacterial classification of absolute value are required for the present invention wanting.Bacterial strain 20# and 27# meet requirement of the present invention as shown in Table 1.
Transform and finish back extraction product, then each component of product is determined its structure by NMR and MS by the silicagel column separation and purification.The result shows that the relevant data of two kinds of products (2) that bacterial strain 20# and 27# conversion of substrate generate and (3) is as follows.
The hydrogen spectrum data of product (2) are as follows:
7.14，6.8(each2H，d，J＝8.5Hz，Ar-H)；3.82(2H.t，J＝6.6Hz，H-1)；2.82(2H，t，J＝6.6Hz，H-2)；3.60(1H，dd，J＝1.44Hz，J＝14.0Hz，H-3
1)；3.33(1H，dd，J＝7.5Hz，J＝14.0Hz，H-3
2)；4.46(1H，Ws，H-4)；4.10(1H，dd，H-5
1)；4.03(1H，dd，H-5
2)；3.70(1H，m，H-6)；1.26，1.22(each3H，d，J＝6.76Hz，J＝6.56Hz，CH
3*2)；2.24(6H，s，-COCH
3)
High resolution positive ion ESI-MS m/z:338.1958[M+H]
+360.1774[M+Na]
+
Its specific rotation light value is negative value, by [α] of the specific rotation light value of the separating obtained compound of 20# bacterial strain converted product (2)
D 20Be-2700 (C=1, CH
3OH), by [α] of the specific rotation light value of the separating obtained compound of 27# bacterial strain converted product (2)
D 20Be-711,, learn the S-type product that this compound needs for the present invention thus because the optical value of structurally similar compounds all is a negative value.
The hydrogen spectrum data of product (3) are as follows:
7.14，6.80(each 2H，d，J＝8.5Hz，Ar-H)；3.81(2H.t，J＝6.6Hz，H-1)；2.81(2H，t，J＝6.6Hz，H-2)；3.60(1H，dd，J＝1.44Hz，J＝14.0Hz，H-31)；3.43(1H，dd，J＝7.5Hz，J＝14.0Hz，H-3
2)；4.46(1H，Ws，H-4)；4.10(1H，dd，H-5
1)；4.03(1H，dd，H-5
2)3.60(1H，m，H-6)；1.26，1.21(each3H，d，J＝6.76Hz，J＝6.56Hz，CH
3*2)；2.20(3H，s，-COCH
3)
High resolution positive ion ESI-MS m/z:296.1876[M+H]
+318.1689[M+Na]
+
[α] by the specific rotation light value of the separating obtained compound of 20# bacterial strain converted product (3)
D 20Be+116, by [α] of the specific rotation light value of the separating obtained compound of 27# bacterial strain converted product (3)
D 20Be+285.
By NMR and MS result as can be known: the structure of compound (2) and compound (3) is as follows:
The structural formula of microbial transformation product
The HPLC of two bacterial strain converted products the results are shown in Figure 2 and Fig. 3.
Fig. 2 and Fig. 3 have confirmed that further compound (2) is the S-type product of wanting required for the present invention.
Measure ee value, the transformation efficiency of product and calculate the E value thus with the HPLC method.The E of 20# bacterial strain is 10.6, and the E of 27# bacterial strain is 5.3.Hence one can see that: the 20# bacterial strain is better than the 27# bacterial strain.Select the 20# bacterial strain to be preserved in slant preservation substratum (glucose 1%, peptone 0.5%, yeast extract paste 0.2%, agar 2%) at last.
By to colonial morphology, thalline shape and size, have or not spore and mycelia to generate and the observation of modes of reproduction is carried out preliminary evaluation to bacterial classification.The result is as follows:
Cell has circle (5 μ m) and oval (5 * 6 μ m), no spore, and no pseudohypha is mainly fragmentation, and minority is budding.
The form difference of this bacterial strain on different substratum, concrete form is as follows:
YEPD (one) substratum: lawn is less, pinkiness, and smooth surface, glossy, more moistening, the edge is more transparent, and is more neat, corrugationless, easily picking is thick, and is tasteless, and cell has circle (5 μ m) and oval (5 * 6 μ m).
Malt extract medium: lawn is less, is bright pink look, surface folding, and tarnish, relatively drier, the edge is because the influence of fold is not too neat, no spore, no pseudohypha, easy picking is thick, and is tasteless, cell circle (3.5～4 μ m) on the high side.
The PDA substratum: lawn is bigger, pinkiness, and smooth surface, glossy, more moistening, edge color is more shallow, and is transparent slightly, relatively more neat, no spore, no pseudohypha, easily picking is thick, and is tasteless, cell ellipse (4.5 * 6.5 μ m) on the high side.
Bean sprouts medium: lawn is bigger, pinkiness, and smooth surface, glossy, more moistening, color is dark and consistent, and the edge is more neat, no spore, no pseudohypha, easily picking is thick, and is tasteless, cell ellipse (2 * 3～3 * 4 μ m) on the high side.
Analytical results to this bacterial strain 16S DNA shows that its 16S DNA has 320 base pairs, (Rhodotorula mucilaginosa) is in full accord for the gene of this bacterial strain and cement rhodotorula, retrieving this bacterial strain through gene pool is cement rhodotorula (Rhodotorula mucilaginosa) DQ832198, deposit number CGMCC.No.2257.
The bacterial classification CGMCC.No.2257 of preservation is inserted in the substratum (corn steep liquor 2%, peptone 0.5%, glycerine 2%, initial pH 6), and the bottled 110mL of 250mL triangle (promptly 45%) adds 1g substrate (1), 28 ℃ of conversion 12h in 32 ℃ after cultivating 16h.Reaction solution equal volume of ethyl acetate fermented liquid three times are used saturated NaHCO mutually with ethyl acetate
3Give a baby a bath on the third day after its birth time, saturated NaCl solution gives a baby a bath on the third day after its birth time, more once with the distillation washing, saturated NaHCO
3Use the ethyl acetate back extraction mutually once with saturated NaCl mutually, merge organic phase, and use anhydrous Na
2SO
4Drying gets product (2) 0.42g, productive rate 47.5%, and product ee value is 95%.Product (3) 0.58g, productive rate 68%.Wherein product (2) is our purpose product, prepares the S-betaxolol hydrochloride by product (2) by method noted earlier, and its ee value is 95%, and specific rotatory power reaches [α]
D 20Be-19.7 (C=1, CH
3OH).
The HPLC collection of illustrative plates of racemize betaxolol and S-betaxolol such as Fig. 3 and Fig. 4.The HPLC collection of illustrative plates can further prove the laevo-oonfiguration of product.
In embodiment 3, obtain thalline after filtration behind the preservation spawn culture of the present invention, the phosphoric acid buffer of thalline with PH=6 washed three times, in identical damping fluid, transform then by condition among the embodiment 3, the result is similar to example 3, advantage is that easier extraction of converted product and cell can reuse, with with a collection of cell transformation 9 times, the transformation ability is not fallen as follows, shows that the enzyme stability in this thalline is good.
With the phosphoric acid buffer packing of pH 6 sterilization, the cooling back adds the 3g/L substrate, adds the toluene of different amounts then, and centrifugal cell average mark is to each bottle, in 28 ℃, 210r/min transforms.
The speed of cell transformation is very not slow when not adding organic solvent in the reaction system, and top speed is 0.3min
-1, required product is 45% at the peak rate of conversion of reaction 6h.After adding toluene in the reaction system, the permeability of cell obtains changing, and enzyme-to-substrate can fully contact, and the transformation efficiency of substrate is improved.Add the product yield of toluene when reaction 1h and reach maximum value 66%, top speed is 0.65min
-1
Result in the reaction system behind the different amount of the adding toluene conversion 0.5h shows that conversion rate is the highest when adding 15% toluene in the reaction system.The stereoselectivity index E value and the aqueous phase of conversion reaction are basic identical.
Replace toluene at embodiment 5 usefulness chloroforms, other condition is identical.
After adding chloroform in the reaction system, also increased the permeability of cell, but effect does not have toluene obvious, top speed is 0.12min
-1, reaction 3h product yield reaches maximum value 55%, changes not obvious later in time.The stereoselectivity index E value of conversion reaction is with basic identical at aqueous phase.
Replace toluene at embodiment 5 usefulness normal hexanes, other condition is identical.
After adding normal hexane in the reaction system, also increased the permeability of cell, but effect does not have toluene obvious, almost reach the highest during reaction 3h, top speed is 0.08min
-1, product yield reaches 61%, changes not obvious later in time.The stereoselectivity index E value of conversion reaction is with basic identical at aqueous phase.
By immobilized cell physical strength such as table 2 and the table 3 of method noted earlier with the differing materials preparation.
The withstand voltage properties of table 2. different fixing cell
| Process for fixation | CA | Aluminium alginate | Carrageenin | Gelatin | Carrageenin-gelatin | PVA-CA | PVA-CA-C |
| Physical strength (g/) | 16.6 | 15.2 | 65.7 | 54.2 | 60 | 120 | 35.1 |
Annotate: data are measured by the counterweight pressurization in the table
The anti-vibration performance of table 3. different fixing cell
| Embedding method | Carrageenin | Gelatin | OK a karaoke club-glue name glue | CA | Aluminium alginate | PVA- CA | PVA-CA- C |
| Particle remains intact the time (h) | 0.5 | 0.4 | 0.6 | 1.0 | 0.2 | 1.2 | 1.0 |
All better by CA, PVA-CA and PVA-CA-gac gained particle as seen from the above table than the physical strength and the resistance to pressure of carrageenin, gelatin and carrageenin-gelatin.
There is certain influence in the consumption of activated carbon to the permeability of embedded particles, the results are shown in Table 4.
Table 4. gac is to the influence of embedded particles permeability and physical strength
| Gac (%) | 0 | 0.5 | 1 | 2 |
| Transformation time | 12 | 11.5 | 11 | 10 |
Annotate: contain PVA 10% in the embedded material, CA 2%, and transformation time is that transformation efficiency reaches 50% required time.
As seen add gac speed of reaction is improved, but,, can select 0.5% so the consumption of gac can not be too many along with the increase particulate physical strength of gac progressively reduces.But after phosphoric acid buffer soaked, the particle physical strength obviously descended through the immobilized cell of aforesaid method, added substrate conversion particle off-bottom after a quarter, and not tolerate P-levels acid buffered soln of these particles is described.
After polymine is handled, through all raisings greatly of performance of CA-C and the immobilized cell tolerate P-levels acid of CA-PVA-C buffered soln.Little through the immobilized cell machinery of CA-PVA-C Strength Changes, and because the PVA permeability is poor especially, its conversion rate is slow especially.Be significantly improved through the immobilized cell physical strength of CA-C, the immobilization particle changing effect of the reinforcement of usefulness 0.6%PEI is best, suitable with the free cell basically, and it is excellent to transform the back particle.
When concentration of substrate is 3g/L, the residence time, the transformation efficiency of substrate was 80% when being 15h, and purpose product ee value is＞95%.When the residence time one timing, substrate conversion efficiency can reduce along with the increase of substrate (1) concentration, and when increasing concentration of substrate, the residence time is wanted corresponding increase.
For volume productivity, volume productivity reduces along with the prolongation of the residence time.In the residence time one timing, increase concentration of substrate and can improve volume productivity, but if concentration of substrate is too high, the transformation efficiency of substrate is very low, substrate can't make full use of.
At concentration of substrate is 3g/L, and the residence time is under the condition of 15h, and this reactor still keeps stable at continuous operation after 40 days.
Claims (10)
1. the microbial strains from soil, screened of a strain, it is characterized in that, this bacterial strain is cement rhodotorula (Rhodotorulamucilaginosa) DQ832198, can produce stereoselectivity lipase, through China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number: CGMCC.No.2257.
2. the application of cement rhodotorula CGMCC.No.2257 in preparation S-type betaxolol hydrochloride according to claim 1 is characterized in that, as follows:
(1) cement rhodotorula CGMCC.No.2257 is cultivated under the following conditions, ferments, obtain cell culture fluid or wet thallus or immobilized cell, with it as zymin;
(a) slant medium is: glucose 0.5～5%, peptone 0.1～5%, yeast extract paste 0.1～5%, agar 2%;
(b) seed and fermention medium are: glucose 0～5%, peptone 0～5%, corn steep liquor 1～5%, peptone 0.1～5%, glycerine 1～5%, initial pH 5～10;
(c) culture condition is: 20～50 ℃ of temperature, time 5～48h, liquid amount coefficient 10～60%;
(2) above-mentioned zymin is inoculated in consists of: corn steep liquor 0.5～5%, peptone 0.1～5%, glycerine 0.5～5%, in initial pH 4～10 substratum, cultivate 12～48h in 20 ℃～50 ℃, (2-acetoxy-3-(N-(sec.-propyl)-N-acetylamino) propoxy-phenylethyl alcohol acetic ester 0.5～12g/L is as substrate to add 4-then, transform 5～50h in 20 ℃～50 ℃, converted product in the middle of generating, middle converted product is measured transformation efficiency and product ee value with the HPLC method;
(3) synthetic S-type betaxolol hydrochloride:
In middle converted product, add amount of methanol and excessive sodium hydroxide, reacted 5～50 minutes, remove methyl alcohol then, obtain deacetylated product with the ethyl acetate extraction water;
Add toluene, phenyl aldehyde and tosic acid in deacetylated product, charge into argon gas, oil bath refluxes down, washes with water after the cooling, with the organic layer dried over sodium sulfate, under reduced pressure boils off toluene and gets amino alcohol derivative;
Amino alcohol derivative is dissolved in the dry DMF, add NaH, normal temperature reacted 1～30 minute down, react 1～10h in 0 ℃～-50 ℃ after adding the brooethyl cyclopropane, react 1～5h then at normal temperatures, reaction solution is poured in an amount of saturated solution of sodium bicarbonate, removed NaOH and use chloroform extraction again, underpressure distillation is removed chloroform and is obtained the S-betaxolol;
The S-betaxolol is dissolved with an amount of Virahol, drip the normal concentrated hydrochloric acid of twice, Virahol is removed in underpressure distillation, use the saturated sodium bicarbonate solution washed product, wash water layer with chloroform, chloroform is removed in decompression distillation down, obtains weak yellow liquid, carry out recrystallization with sherwood oil-acetone mixed system again, promptly get S-type betaxolol hydrochloride.
3. according to the described method of claim 2, it is characterized in that: immobilized cell carries out immobilization with sodium alginate, polyvinyl alcohol and activated carbon described in the step (1), and carry out intensive treatment with polymine, sodium alginate consumption 1～5%, polyvinyl alcohol consumption 0～5%, activated carbon consumption 0～5%, polymine concentration 0.1～5%.
4. according to the described method of claim 2, it is characterized in that: step directly transforms with cell culture fluid in (2), and concentration of substrate is 1～10g/L, and invert point is 20～50 ℃, and transformation time is 5～50h.
5. according to the described method of claim 4, it is characterized in that: add toluene, normal hexane, chloroform organic solvent to improve conversion rate in transformation system, the organic solvent add-on is 1～10%.
6. according to the described method of claim 2, it is characterized in that: transform in the damping fluid of pH 5～10 with resting cell in the step (2), buffer system is Na
2HPO
4-citric acid, Na
2HPO
4-KH
2PO
4, Na
2HPO
4-NaH
2PO
4, K
2HPO
4-KH
2PO
4, concentration of substrate is 1～10g/L, and invert point is 20～50 ℃, and transformation time is 5～50h.
7. according to the described method of claim 2, it is characterized in that: immobilized cell is used in the packed bed reactor and transforms in the step (2), and packed bed reactor is of a size of 10～50 * 100～500mm, by 20 ℃～50 ℃ insulations of thermostat container; Concentration of substrate 1～10g/L, substrate solution is flowed into by reaction column top or bottom, and reacted conversion fluid is collected from the reactor the other end.
8. the application of cement rhodotorula CGMCC.No.2257 in chipal compounds splits according to claim 1 is characterized in that, utilizes this microorganism to carry out the chiral separation that chiral centre contains acyl group, hydroxy kind compound.
9. as the application of cement rhodotorula CGMCC.No.2257 as described in the claim 8 in chipal compounds splits; it is characterized in that described chiral centre contains acyl group, the hydroxy kind compound is beta-blocker, ephedrine hydrochloride, suprarenin, levodropropizine, salbutamol sulfate, Robert Caputo profit, zofenopril.
10. cement rhodotorula CGMCC.No.2257 adds on compound or sloughs application in the acyl group reaction according to claim 1, it is characterized in that, utilizes this microorganism to add on compound or sloughs acyl group.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250785A (en) * | 2011-05-20 | 2011-11-23 | 华南理工大学 | Preparation method of bacterial cell catalyst for catalyzing starasid synthesis reaction |
| CN102952770A (en) * | 2012-11-12 | 2013-03-06 | 华南理工大学 | Preparation method for cell catalyst with high regioselective acylation activity and microbiologic method for synthesizing cytosine arabinoside monoester |
| CN103122319A (en) * | 2012-12-28 | 2013-05-29 | 郑州大学 | Yeast with stereoselectivity and lipase activity and method for preparing S-type rivastigmine by biological splitting of yeast |
| CN104749237A (en) * | 2015-03-27 | 2015-07-01 | 常州大学 | Selective recognition of sodium alginate modified glassy carbon electrodes to tyrosine enantiomers |
| CN105400709A (en) * | 2015-12-21 | 2016-03-16 | 湖南省植物保护研究所 | Rhodotorula mucilaginosa bacterial strain, microbial inoculum, and preparation method and applications thereof |
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2007
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102250785A (en) * | 2011-05-20 | 2011-11-23 | 华南理工大学 | Preparation method of bacterial cell catalyst for catalyzing starasid synthesis reaction |
| CN102250785B (en) * | 2011-05-20 | 2013-10-30 | 华南理工大学 | Preparation method of bacterial cell catalyst for catalyzing starasid synthesis reaction |
| CN102952770A (en) * | 2012-11-12 | 2013-03-06 | 华南理工大学 | Preparation method for cell catalyst with high regioselective acylation activity and microbiologic method for synthesizing cytosine arabinoside monoester |
| CN103122319A (en) * | 2012-12-28 | 2013-05-29 | 郑州大学 | Yeast with stereoselectivity and lipase activity and method for preparing S-type rivastigmine by biological splitting of yeast |
| CN103122319B (en) * | 2012-12-28 | 2015-03-04 | 郑州大学 | Yeast with stereoselectivity and lipase activity and method for preparing S-type rivastigmine by biological splitting of yeast |
| CN104749237A (en) * | 2015-03-27 | 2015-07-01 | 常州大学 | Selective recognition of sodium alginate modified glassy carbon electrodes to tyrosine enantiomers |
| CN104749237B (en) * | 2015-03-27 | 2017-05-10 | 常州大学 | Selective recognition of sodium alginate modified glassy carbon electrodes to tyrosine enantiomers |
| CN105400709A (en) * | 2015-12-21 | 2016-03-16 | 湖南省植物保护研究所 | Rhodotorula mucilaginosa bacterial strain, microbial inoculum, and preparation method and applications thereof |
| CN105400709B (en) * | 2015-12-21 | 2018-12-11 | 湖南省植物保护研究所 | Cement rhodotorula bacterial strain, microbial inoculum and its preparation method and application |
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