CN101906408B - Enzyme preparation for synthesizing D-aryl glycine through biocatalysis and preparation method and application thereof - Google Patents
Enzyme preparation for synthesizing D-aryl glycine through biocatalysis and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an enzyme preparation for synthesizing D-aryl glycine through biocatalysis, which consists of activated cells of arthrobacter aurescens producing hydantoinase and activated cells of agrobacterium radiobacter producing carbamoylase. The invention also relates to a preparation method and application of the enzyme preparation for synthesizing the D-aryl glycine through the biocatalysis. The enzyme preparation can be used for synthesizing the D-aryl glycine or derivatives thereof through the catalysis of heterogeneous hydrolysis of hydantoin. Through the enzyme preparation, the production cost is reduced greatly and the yield of products is improved.
Description
Technical field
The invention belongs to the microbial fermentation field, particularly zymin of a kind of synthesizing D-aryl glycine through biocatalysis and its preparation method and application.
Background technology
The D-aryl amino acid (as, the D-aryl glycine) is the very important alpha-non-natural amino acid of a class, since its configuration and natural amino acid just in time phase can be used on the contrary medicine (as, antagonist), the synthetic and preparation of peptide and sweeting agent etc., along with the further exploitation to its using value, the demand in market is at rapid growth in recent years.
The preparation method of traditional D-aryl amino acid (such as, D-aryl glycine) comprised chemosynthesis and Split Method, but it need to waste the enantiomer of half, causes the shortcomings such as production efficiency is low, production capacity is low, energy consumption is high.
In biocatalysis D-aryl glycolylurea hydrolysis method, traditional technology is " bacterium two enzymes " method, such as Chinese patent or patent application 01105347,200710191968 etc. described.But employed a kind of microorganism itself will be taken into account two kinds of enzymes in this technology, is difficult to the substrate of enduring high-concentration and keeps continuous enzyme catalysis in industrial production, therefore is difficult to add the substrate of saturation concentration and realizes heterogeneous enzyme catalysis.Even therefore these patent applications are authorized, also abandoned patent right because being not suitable for actual industrial production.
Chinese patent application 200610048209.0 and 200710062118.7 discloses the method for the synthetic D-aryl glycine or derivatives thereof of a kind of heterogeneous enzyme catalysis hydrolysis 5-aryl glycolylurea, replace traditional " bacterium two enzymes " method with " two bacterium, two enzymes " method, wherein use gold Arthrobacter (Arthrobacter aurescens) LZ98 or its mutant bacteria UV-LZ98 to produce hydantoin enzyme (Hydantoinase) and use agrobacterium radiobacter (Agrobacterium radiobacter) LZ99 or its mutant bacteria UV-LZ99 to produce carboxamide amine lytic enzyme, the zymin that produces thus certain enzymic activity is come so that corresponding synthetic method is achieved.Yet, two strain bacterial strain purchase costs of the preparation zymin of using in the method are higher, especially LZ99 and mutant bacteria UV-LZ99 are not only expensive, and the carboxamide amine lytic enzyme that produces stability when plant-scale lengthy fermentation is inadequate, the fermentation weather resistance is not enough, and the heterogeneous enzyme catalysis that has greatly limited this patent is hydrolyzed the industrial applications that 5-aryl glycolylurea synthesizes the D-aryl glycine.
Summary of the invention
The present invention is for overcoming problems of the prior art, zymin of a kind of synthesizing D-aryl glycine through biocatalysis and its preparation method and application is provided, it can replace the existing zymin for preparing the method for D-aryl glycine for heterogeneous enzyme catalysis hydrolysis 5-aryl glycolylurea, production cost is reduced greatly, and method simple, be easy to industrial applications.
The present invention is achieved by the following technical solutions:
First aspect of the present invention, the zymin of synthesizing D-aryl glycine through biocatalysis, it is comprised of the active cells of the gold Arthrobacter that produces hydantoin enzyme and the active cells of the agrobacterium radiobacter that produces carbamyl hydrolysis enzyme.
The zymin of described synthesizing D-aryl glycine through biocatalysis is characterized in that, the initial enzymic activity of the hydantoin enzyme that described gold Arthrobacter produces is greater than 0.32U, and the initial activity of the carbamyl hydrolysis enzyme that described agrobacterium radiobacter produces is greater than 10U.
The zymin of described synthesizing D-aryl glycine through biocatalysis, the initial hydantoin enzyme activity of described gold Arthrobacter is 0.32U～0.45U.
The zymin of described synthesizing D-aryl glycine through biocatalysis, described gold Arthrobacter are gold Arthrobacter 3256 active cellss, and described agrobacterium radiobacter is agrobacterium radiobacter 3255 active cellss.
Second aspect of the present invention, the preparation method of the zymin of described synthesizing D-aryl glycine through biocatalysis, it may further comprise the steps:
The gold Arthrobacter that a. will produce hydantoin enzyme is inoculated in the liquid nutrient medium that contains corn steep liquor, molasses, aqueous sodium hydroxide solution, sodium-chlor, 2-thiouracil, sal epsom, manganous sulfate and Secondary ammonium phosphate, cultivates in 30～34 ℃ of stir-activatings.Wherein, stirring velocity is 60rpm, and soak time is 12～18h, preferred 14～16h; Air pressure is 0.18～0.23Mpa, is preferably 0.2Mpa; Air flow quantity is 20～26L/h; Reactor pressure is 0.04～0.06Mpa, is preferably 0.05Mpa; Then this nutrient solution is flocculated with chitosan aqueous solution, filter results solid zymin 1;
It is aqueous sodium hydroxide solution 15～25 weight parts, sodium-chlor 40～60 weight parts, 2-thiouracil 27～37 weight part sal epsom 8～10 weight parts, manganous sulfate 1.2～1.8 weight parts and Secondary ammonium phosphate 3～4 weight parts of 25～35% (w/w) that described liquid nutrient medium contains corn steep liquor 360～460 weight parts, molasses 380～480 weight parts, concentration, and adds water and be settled to 8000 weight parts;
The agrobacterium radiobacter that b. will produce carbamyl hydrolysis enzyme is inoculated in the liquid nutrient medium that contains corn steep liquor, glucose, peptone, sodium-chlor, manganous sulfate, Secondary ammonium phosphate and pectinose, cultivate in 32～38 ℃ of stir-activatings, wherein stirring velocity is 60rpm; Soak time is 15～22h, and preferred 16～18h most preferably is 17h; Air pressure is 0.18～0.23Mpa, is preferably 0.2Mpa; Air flow quantity is 18～22L/h; Reactor pressure is 0.04～0.06Mpa, is preferably 0.05Mpa; Adding sodium hydroxide adjusting pH in described substratum is 6.5～7.8, is preferably 6.8～7.3, most preferably is 7.0, then this nutrient solution is flocculated with chitosan aqueous solution, filters results solid zymin 2;
Described liquid nutrient medium contains corn steep liquor 200～400 weight parts, glucose 180～260 weight parts, peptone 80～100 weight parts, 25g sodium-chlor 20～30 weight parts, manganous sulfate 1.6～2.0 weight parts, Secondary ammonium phosphate 6.8～7.6 weight parts and pectinose 12～16 weight parts; And described liquid nutrient medium is added water be settled to 8000 weight parts;
C. the solid zymin 2 that the solid zymin 1 that step a is made and step b make is mixed, and makes the zymin of synthesizing D-aryl glycine through biocatalysis.The weight proportion of wherein said solid zymin 1 and described solid zymin 2 is different and different with substrate, and usually controlling the two ratio is 1～5: 1～50, and preferred ratio is 1～5: 1～20, and preferred ratio is 1～2: 1～5.
The 3rd aspect of the present invention, the zymin of described synthesizing D-aryl glycine through biocatalysis, it is used for the method for the synthetic D-aryl glycine of the heterogeneous hydrolysis glycolylurea of enzyme catalysis.
The method of the synthetic D-aryl glycine of the heterogeneous hydrolysis glycolylurea of described enzyme catalysis may further comprise the steps:
1. in the enzymic catalytic reaction tank, add entry and 5-aryl glycolylurea, and to adjust the pH value be 6.5～7.8, the add-on of aryl glycolylurea accounts for 5～21% (w/w) of water and 5-aryl glycolylurea gross weight, gets mixing solutions;
Step 1. in, the material of proportioning will form suspension liquid thus, ie in solution is hypersaturated state, has the solid material of 5-aryl glycolylurea to exist.
2. under the condition that stirs, add the zymin of the arbitrary described synthesizing D-aryl glycine through biocatalysis of claim 1～5 in the enzymic catalytic reaction tank, at 25～40 ℃ of lower stirring reactions, until can't detect 5-aryl glycolylurea, get the D-aryl glycine;
The zymin of wherein said synthesizing D-aryl glycine through biocatalysis account for the mixing solutions of step in 1. gross weight 1～10%, be preferably and be 2-8%; 8～66 hours reaction times;
3. isolate D-aryl glycine solid by 200 mesh sieves, through recrystallization, obtain D-aryl glycine crystal;
Step 3. in, owing to having thalline and tiny solid phase prod crystallization, therefore preferred by the separation of 200 mesh sieves, the solid that is retained down is the larger D-aryl glycine crystal of granularity (thick product), and thalline and broken short grained solid product enter into film separating system by sieve aperture.
4. extract the D-aryl glycine that dissolves in the enzymic catalytic reaction solution by holding back 5000～50,000 molecular weight ultrafiltration membrance filters and evaporation concentration, pass through again recrystallization, get D-aryl glycine crystal.
The solution of gained is saturated D-aryl glycine behind the D-aryl glycine crystal owing to isolating, and in order further to improve productive rate, has therefore adopted step 4..Be about to the liquid that 3. step can flow through sieve and remove thalline by membrane filtration, to the concentrated D-aryl glycine crystal that obtains of filtrate, through recrystallization, obtain D-aryl glycine crystal.
Preferably, 4. the method steps of the synthetic D-aryl glycine of described heterogeneous enzyme catalysis hydrolysis glycolylurea can also adopt following steps:
Separate the part that sees through sieve aperture by the ultrafiltration membrance filter screening first, namely get the solution that is dissolved with the D-aryl glycine in the enzymic catalytic reaction tank, its film dialyzate is evaporated concentrated the thick product of another part D-aryl glycine, obtains D-aryl glycine crystal through recrystallization again.
D-aryl glycine in the method for the synthetic D-aryl glycine of described heterogeneous enzyme catalysis hydrolysis glycolylurea is D-PG, D-pHPG, the D-fluorophenyl glycine, the D-chlorobenzene glycine, D-bromobenzene glycine, D-iodobenzene glycine, D-methoxy phenylglycine, D-pyridine series glycine, D-naphthalene series glycine, D-quinoline series glycine D-pyrimidine glycine, D-pyrazine glycine, D-pyridazine glycine, D-indoles glycine, D-thiophene glycine, D-furans glycine, D-pyrroles's glycine, D-pyrazoles glycine, D-imidazoles glycine, D-thiazole glycine, a kind of in D-oxazole glycine and the D-isoxazole glycine.
Further, the D-aryl glycine in the described method for preparing the synthetic D-aryl glycine of heterogeneous enzyme catalysis hydrolysis glycolylurea is D-PG; The preparation method of described D-PG is:
1. in the enzymic catalytic reaction tank, add entry 30kg and 5-phenyl hydantoin because of 2.8kg, and adjustment pH value is 6.8;
2. the zymin 2.8kg that under the condition that stirs, adds arbitrary described synthesizing D-aryl glycine through biocatalysis of claim 1-5 to the enzymic catalytic reaction tank, at 38 ℃ of lower stirring reactions, until can't detect the 5-phenyl hydantoin because of, and intermediate product N-carboxamide phenylglycine content is less than or equal to 0.25% (w/w), show to react and substantially finish, make D-PG;
3. separate and separate out the D-PG solid, through recrystallization, obtain the D-PG crystal product;
4. extract the D-PG that dissolves in the enzymic catalytic reaction solution by ultrafiltration membrance filter and evaporation concentration, get another part, be i.e. the D-PG product.
In the zymin of described synthesizing D-aryl glycine through biocatalysis, described gold Arthrobacter and agrobacterium radiobacter are viable bacteria, are preferably the bacterium of activation culture.Therefore, the zymin of the synthesizing D-aryl glycine through biocatalysis that provides of preferred first aspect present invention is comprised of the active cells of the gold Arthrobacter that produces hydantoin enzyme and the active cells that produces the agrobacterium radiobacter of carbamyl hydrolysis enzyme.In the present invention, " active cells " expression somatic cells is through activation culture, so that cell has the enzymic activity of the enzyme of corresponding purified form, namely hydantoin enzyme activity and carbamyl hydrolysis enzyme are active.Therefore, the zymin of described synthesizing D-aryl glycine through biocatalysis can keep long enzymic activity in commercial process.Although the zymin of described synthesizing D-aryl glycine through biocatalysis is insoluble (comprising thalline), but according to inventor's long-term observation, the coarse crystal particle that is used in conjunction with the standby D-aryl glycine of heterogeneous legal system of the present invention is more much bigger than thalline and fragment thereof, therefore can easily extract the coarse crystal particle by the method for sieving separating, and can not introduce too much thalline impurity to affect purity, can not separate forfeiture yet and fall the coarse crystal particle and cause productive rate to descend.
In a first aspect of the present invention, the initial hydantoin enzyme activity of gold Arthrobacter is greater than 0.30U, the initial carbamyl hydrolysis enzyme of agrobacterium radiobacter is greater than 9U, preferred initial hydantoin enzyme activity is greater than 0.32U, also preferred initial carbamyl hydrolysis enzyme is greater than 10U, it is 9.5U～15U that the best is selected initial carbamyl hydrolysis enzyme, and it is 10U～12U that the best is selected initial carbamyl hydrolysis enzyme.Because in commercial process, the activity of enzyme can reduce gradually.Therefore, itself need to have certain catalytic capability the thalline in a first aspect of the present invention, and this catalytic capability need to be quantitatively controlled in suitability for industrialized production.Like this, the present invention feeds intake the front initial activity of measuring for thalline as the catalytic capability index after using activation, and its concrete measuring method can be carried out according to mode listed in the specific embodiment of the invention.The inventor finds, because used the agrobacterium radiobacter that produces carbamyl hydrolysis enzyme instead, catalytic efficiency and stability are improved, the intermediate product accumulation is few, therefore the gold Arthrobacter that produces hydantoin enzyme need not very good, and its initial hydantoin enzyme activity can be preferably 0.32～0.45U.Like this, can use commercially available more cheap gold Arthrobacter, such as gold Arthrobacter 3256.In addition, be easier to owing to producing the agrobacterium radiobacter screening of carbamyl hydrolysis enzyme, therefore can not use expensive LZ series bacterial classification, and use more cheap commercially available agrobacterium radiobacter, such as agrobacterium radiobacter 3255.In the specific embodiment of the present invention, preferred zymin is comprised of agrobacterium radiobacter 3255 and gold Arthrobacter 3256, and preferred zymin is comprised of agrobacterium radiobacter 3255 active cellss and gold Arthrobacter 3256 active cellss.
In order further to improve defective of the prior art, we are through arduous experiment, find when the strain cell that uses suitable activation culture is directly prepared the zymin of synthesizing D-aryl glycine through biocatalysis of the present invention (namely adopting " two bacterium " of the present invention method), even wherein adopted the carboxamide amine lytic enzyme in more conventional existing " two bacterium, two enzymes " method of carbamyl hydrolysis enzyme (Carbamylase) replacement, also can satisfy the demand of the synthetic D-aryl glycine of heterogeneous enzyme catalysis hydrolysis 5-aryl glycolylurea, therefore can not use the bacterial strain such as costlinesses such as mutant bacteria UV-LZ99 to extract corresponding carboxamide amine lytic enzyme, thereby finally be mixed with the zymin of synthesizing D-aryl glycine through biocatalysis.More unexpectedly be that when adopting method activation strain cell of the present invention and prepare the zymin of synthesizing D-aryl glycine through biocatalysis, even the producing bacterial strain of hydantoin enzyme also can use comparatively routine and the more cheap bacterial strain of price.The use of above bacterial strain is when reducing cost, do not affect stability and the weather resistance of the synthetic D-aryl glycine of the heterogeneous hydrolysis of plant-scale enzyme catalysis 5-aryl glycolylurea, also can directly use equipment and the fermentation flow process of original heterogeneous enzyme catalysis, need not new equipment cost and drop into, also need not to retrain technician on the production line to grasp other fermentation flow processs.
The beneficial effect that the present invention compared with prior art has is:
1. use in the synthetic D-aryl glycine of heterogeneous enzyme catalysis hydrolysis 5-aryl glycolylurea with original " two bacterium, two enzymes " method and compare, the present invention has used enzyme instead and has used activation instead and compound method, and speed of response of the present invention is fast, energy-conservation, productive rate is high, constant product quality etc.
2. " two bacterium " method need not to extract the step of enzyme, and the reactivation process of enzyme (bacterium) and the cultivation amplification process of bacterium cell unite two into one, and have simplified the step of actual production, have reduced production cost.
3. the bacterial classification cost is low, the bacterial classification that need not multiple costliness matches (even the activity of hydantoin enzyme is lower, do not affect final fermentation efficiency) yet, and new activation and compound method are so that the zymin that obtains after the preparation is lasting, stable aborning, need not as " two enzymes, two bacterium " method, need in long reaction process, the monitoring enzymic activity also add in good time, can be amplified to easily in the production of technical scale level, be particularly suitable for suitability for industrialized production.
4. substrate strong adaptability, need not be as existing " two bacterium, two enzymes " method, the enzyme that D-pHPG can use agrobacterium radiobacter LZ99 fermentation to produce then needs the enzyme of the agrobacterium radiobacter UV-LZ99 that filters out in addition to other D-aryl glycines.
5. directly use cell and do not used lyoenzyme, can avoid the step of mixed fermentation and enzyme extraction, only need activated spawn to get final product, albumen impurity problem for long-term puzzlement crystal purity, we find that after deliberation cell paste and the crude product that crystallizes out with heterogeneous method vary in size, just can separate simply very convenient suitability for industrialized production with 200 commercially available mesh sieves.
Embodiment
The present invention is described in detail below in conjunction with specific embodiment.
Embodiment one
1, the preparation of hydantoin enzyme preparation
In the 10L fermentor tank, add respectively 416g corn steep liquor, 428g molasses, 20mL 30% (w/w) aqueous sodium hydroxide solution, 50g sodium-chlor, 32g 2-thiouracil, 10g sal epsom, 15g manganous sulfate, 3.6g Secondary ammonium phosphate and water to volume and reach 8L, sterilization 0.5h under 0.1～0.12Mpa pressure, 120～125 ℃ of conditions, when being cooled to 30.5 ℃, will be in advance in the 250mL shaking flask cultured gold Arthrobacter 3256 (in available from Shijiazhuang day biological technology limited liability company) be inoculated in the fermentor tank.Be that 0.2Mpa, air flow quantity are under the prerequisite of 20～26L/h in air pressure subsequently, control reactor pressure is that 0.05Mpa, temperature are 33 ℃, stirs fermentation 15h.Then, fermented liquid with 1% (w/w) chitosan 400g flocculation, is settled out and contains the hydantoin enzyme cell, the hydantoin enzyme preparation that filtration under diminished pressure must wet is gold Arthrobacter 3256 cells of activation.
Simultaneously, get in addition the 1mL fermented liquid and measure the enzymic activity of hydantoin enzyme: with the 1mL fermented liquid with the centrifugal 30min of 4800r/min, abandon supernatant liquor and the gained cell is resuspended in the physiological saline, make cumulative volume reach 1mL, do not extract enzyme and directly in this cell suspension, add the phosphate buffer solution (pH8.0) that 9mL contains 2.2% (w/w) 4-Hydroxyphenyl hydantoin, in 38 ℃ of lower slowly concussion reaction 30min, then add 0.1mL 6N hydrochloric acid mixing termination reaction, then centrifugal 30min.Get supernatant liquor, measure the wherein concentration of D-N-carboxamide D-pHPG with HPLC.The enzyme of 1 hydantoin enzyme unit alive (U) is defined as the amount of the D-N-carboxamide D-pHPG that the contained cell of every 1mL fermented liquid can produce in every 1min.According to three fermentation results, the hydantoin enzyme activity is respectively 0.37U, 0.42U and 0.39U, show with the method directly stable activation go out the cell of enzymatic activity high.
2, the preparation of carbamyl hydrolysis enzyme preparation
In the 10L fermentor tank, add respectively 360g corn steep liquor, 220g glucose, 90g peptone, 25g sodium-chlor, 1.8g manganous sulfate, 7.2g Secondary ammonium phosphate, 14g pectinose and water to volume 8L, and to regulate pH with sodium hydroxide be 6.8.Sterilization 0.5h under 0.1～0.12Mpa pressure, 12.～125 ℃ of conditions, be cooled to 30.5 ℃, will be in advance in the 250mL shaking flask cultured agrobacterium radiobacter 3255 (in available from Shijiazhuang day biological technology limited liability company) be inoculated in the fermentor tank.Be that 0.2Mpa, air flow quantity are under the prerequisite of 18～22L/h at pressure subsequently, control reactor pressure 0.05Mpa, temperature are 35 ℃, stir fermentation 17h.Then, with fermented liquid with 1% (w/w) chitosan 400g flocculation, thereby be settled out cell, the carbamyl hydrolysis enzyme preparation that filtration under diminished pressure must wet is agrobacterium radiobacter 3255 cells of activation
Simultaneously, get in addition the 1mL fermented liquid and measure the enzymic activity of carbamyl hydrolysis enzyme: with the 1mL fermented liquid with the centrifugal 30min of 4800r/min, abandon supernatant liquor and the gained cell is resuspended in the physiological saline, make cumulative volume reach 1mL, do not extract enzyme and directly in this cell suspension, add the phosphate buffer solution (pH8.0) that 9mL contains 2.2% (w/w) D-N-carboxamide D-pHPG, in 38 ℃ of lower slowly concussion reaction 30min, then add 0.1mL 6N hydrochloric acid mixing termination reaction, then centrifugal 30min.Get supernatant liquor, measure the wherein concentration of D-pHPG with HPLC.The enzyme of 1 carbamyl hydrolysis enzyme unit alive (U) is defined as the amount of the D-pHPG that the contained cell of every 1mL fermented liquid can produce in every 1min.According to three fermentation results, the enzymic activity of carbamyl hydrolysis enzyme is respectively 10.1U, 11.7U and 10.6U, show with the method directly stable activation go out the cell of enzymatic activity high.
3, the zymin of synthesizing D-aryl glycine through biocatalysis
Wet hydantoin enzyme preparation and the wet carbamyl hydrolysis enzyme preparation of getting above preparation mix, and namely get two zymin--zymins of synthesizing D-aryl glycine through biocatalysis.
Embodiment two,
The application of the zymin of synthesizing D-aryl glycine through biocatalysis--catalyzed reaction example
1, D-is to the preparation of phenylglycine
In the enzymic catalytic reaction tank of 50L, add first 30kg water, then drop into the 5-phenyl hydantoin because of 2.8kg, adding hydrochloric acid or sulfuric acid adjustment pH is 6.8, the wet carbamyl hydrolysis enzyme zymin 1.5kg of preparation in the wet hydantoin enzyme preparation 1.5kg of preparation and embodiment one step 2 under the condition that stirs, adding again by embodiment one step 1, mix, the control temperature is 38 ℃, stirring reaction.
When reaction proceeds to the 4h left and right sides, then there is the D-PG crystal to separate out, to sample therebetween and use the high performance liquid chromatography detection reaction, the concentration of D-PG is substantially constant at about 1.6% in the reaction solution.Treat liquid chromatography almost can't check substrate 5-phenyl hydantoin because of, and intermediate N carboxamide phenylglycine content is when being lower than 0.25%, stopped reaction.The transformation efficiency of benzene glycolylurea is more than 99.6%.
Material in this 50L enzymic catalytic reaction tank is crossed 200 mesh sieves, to realize separating between solid product and small somatic cells and the solution, obtain the coarse crystal of D-PG.Take water as solvent, just can obtain D-PG 1.48kg by the acid-base chemical conversion recrystallization, this is first part's product; Screening separates the liquid portion obtain and further extracts: at first liquid is filtered by holding back 5000 molecular weight ultra-filtration membranes, filtrate is concentrated through reduction vaporization, during the D-PG crystallization is arranged.Filter and collect the D-PG crystallization, adopt again the identical recrystallization method of first part's product, just can obtain second section product 0.51kg.Merge two portions D-PG, altogether purity be more than 99.8%, quality is the product of 1.99kg, total recovery 83%.
2, the preparation of D-PG derivative
Adopt preparation method and the parameter identical to the preparation of phenylglycine with above-mentioned 1D-, wherein replace 5-benzene glycolylurea 2.8kg with 5-4-Hydroxyphenyl hydantoin 4.5kg, finally obtain purity and be the D-pHPG crystal 3 .1kg more than 99.7%.
Adopt preparation method and the parameter identical to the preparation of phenylglycine with above-mentioned 1D-, wherein with 5-anisole glycolylurea 3.2kg is replaced 5-benzene glycolylurea 2.8kg, finally obtain purity and be D-more than 99.8% to anisole glycine crystal 2 .3kg.
Adopt preparation method and the parameter identical to the preparation of phenylglycine with above-mentioned 1D-, wherein with 5-fluorobenzene glycolylurea 3.8kg is replaced 5-benzene glycolylurea 2.8kg, finally obtain purity and be D-more than 99.8% to fluorophenyl glycine crystal 2 .6kg.
Adopt preparation method and the parameter identical to the preparation of phenylglycine with above-mentioned 1D-, wherein with 5-chlorobenzene glycolylurea 3.5kg is replaced 5-benzene glycolylurea 2.8kg, finally obtain purity and be the D-p-chlorophenylglycine crystal 2 .4kg more than 99.8%.
Adopt preparation method and the parameter identical to the preparation of phenylglycine with above-mentioned 1D-, wherein with 5-bromobenzene glycolylurea 4.1kg is replaced 5-benzene glycolylurea 2.8kg, finally obtain purity and be crystal D-more than 99.8% to bromobenzene glycine crystal 2 .9kg.
Adopt with example two in 1 identical preparation method and parameter, wherein with 5-to iodobenzene glycolylurea 2.8kg replacement 5-benzene glycolylurea 2.8kg, finally obtain purity and be D-more than 99.7% to iodobenzene glycine crystal 2 .1kg.
3, the preparation of D-3-pyridine glycine
In the enzymic catalytic reaction tank of 50L, add first 32kg water, under the condition that stirs, drop into 5-(3-pyridyl) glycolylurea 3.8kg, adding hydrochloric acid or sulfuric acid adjustment pH is 6.3, add again the wet carbamyl hydrolysis enzyme zymin 3.0kg by 2 preparations among the 1 wet hydantoin enzyme preparation 1.8kg for preparing and the embodiment one among the embodiment one, mix, in 33 ℃ of lower stirring reactions.
When reaction proceeds to the 6h left and right sides, then there is D-3-pyridine glycine crystal to begin to separate out, then regularly sampling and use the high performance liquid chromatography detection reaction, treat that liquid chromatography almost can't check substrate 5-(3-pyridyl) glycolylurea, and the intermediate N carboxamide-when 3-pyridine glycine content is lower than 0.20% (w/w), stopped reaction.The transformation efficiency of 5-(3-pyridyl) glycolylurea is more than 99.6%.
Reaction mixture to realize separating between solid product and thalline and the solution, obtains the coarse crystal of D-3-pyridine glycine through 200 mesh sieves, take water as solvent, just can obtain D-3-pyridine glycine 1.87kg by the acid-base chemical conversion recrystallization, and this is first part's product; To further extract through the liquid that obtains of sieve: at first liquid is filtered by the ultra-filtration membrane of holding back 10,000 molecular weight, filtrate is concentrated through reduction vaporization, during D-3-pyridine glycine crystallization is arranged.Filter and collect the crystallization of D-3-pyridine glycine, adopt again the identical recrystallization method of first part's product, just can obtain second section product 0.81kg.Merge two portions D-3-pyridine glycine, altogether purity be more than 99.2%, quality is the product of 2.68kg, total recovery 82%.
4, the preparation of D-2-naphthalene glycine
In the enzymic catalytic reaction tank of 50L, add first 37kg water, under the condition that stirs, drop into 5-(2-naphthyl) glycolylurea 3.8kg, adding hydrochloric acid or sulfuric acid adjustment pH is 7.1, add again the wet carbamyl hydrolysis enzyme zymin 2.3kg by 2 preparations among the wet hydantoin enzyme preparation 1.5kg of embodiment one/1 preparation and the embodiment one, mix, in 38 ℃ of lower stirring reactions.
When reaction proceeds to the 7h left and right sides, then there is D-2-naphthalene glycine crystal to begin to separate out, then regularly sampling and use the high performance liquid chromatography detection reaction, when treating that liquid chromatography almost can't check substrate 5-(2-naphthyl) glycolylurea and corresponding intermediate, stopped reaction.The transformation efficiency of 5-(2-naphthyl) glycolylurea is about 99.0%.
Reaction mixture is crossed 200 mesh sieves to realize separating between solid product and somatic cells and the solution, obtains the coarse crystal of D-2-naphthalene glycine, take water as solvent, gets D-2-naphthalene glycine 1.90kg by the acid-base chemical conversion recrystallization, and this is first part's product; To further extract through the partially liq that obtains of sieve: at first liquid is filtered by the ultra-filtration membrane of holding back 10,000 molecular weight, filtrate is concentrated through reduction vaporization, during have D-2-naphthalene glycine crystallization to separate out.Filter and collect D-2-naphthalene glycine crystallization, adopt again the identical recrystallization method of first part's product, just can obtain second section product 0.63kg.Merge two portions D-2-naphthalene glycine, altogether purity be more than 98.8%, quality is the product of 2.53kg, total recovery 75%.
5, the preparation of D-2-quinoline glycine
In the enzymic catalytic reaction tank of 50L, add first 36kg water, under the condition that stirs, drop into 5-(2-quinolyl) glycolylurea 4.2kg, adjusting pH is 6.5, then add the wet carbamyl hydrolysis enzyme zymin 3.2kg by 2 preparations among the 1 wet hydantoin enzyme preparation 1.5kg for preparing and the embodiment one among the embodiment one, mix, in 37 ℃ of lower stirring reactions.
When reaction proceeds to the 6h left and right sides, then there is D-2-quinoline glycine crystal to begin to separate out, then regularly sampling and use the high performance liquid chromatography detection reaction, treat that liquid chromatography almost can't check substrate 5-(2-quinolyl) glycolylurea, and its intermediate N carboxamide-when the content of D-quinoline glycine in reaction solution is lower than 0.3% (w/w), stopped reaction.The transformation efficiency of 5-(2-quinolyl) glycolylurea is about 99.5%.
Reaction mixture is crossed 200 mesh sieves to realize separating between solid product and somatic cells and the solution, obtains the coarse crystal of D-2-quinoline glycine.Take water as solvent, get D-2-quinoline glycine 2.55kg by the acid-base chemical conversion recrystallization, this is first part's product; To further extract through the liquid that obtains of sieve: at first with liquid by holding back the ultrafiltration membrance filter of 20,000 molecular weight, filtrate is concentrated through reduction vaporization, during have D-2-quinoline glycine crystallization to separate out, filter, collect D-2-quinoline glycine crystallization.Adopt again the identical recrystallization method of first part's product, just can obtain second section product 0.63kg, qualified after testing after, merge two portions D-2-quinoline glycine, altogether purity be more than 98.5%, quality is the product of 3.18kg, total recovery 85%.
Claims (7)
1. the zymin of synthesizing D-aryl glycine through biocatalysis is characterized in that, it is comprised of the active cells of the gold Arthrobacter that produces hydantoin enzyme and the active cells of the agrobacterium radiobacter that produces carbamyl hydrolysis enzyme;
The preparation method of the zymin of described synthesizing D-aryl glycine through biocatalysis, it may further comprise the steps:
The gold Arthrobacter that a. will produce hydantoin enzyme is inoculated in the liquid nutrient medium that contains corn steep liquor, molasses, aqueous sodium hydroxide solution, sodium-chlor, 2-thiouracil, sal epsom, manganous sulfate and Secondary ammonium phosphate, cultivate in 30～34 ℃ of stir-activatings, wherein, stirring velocity is 60rpm; Soak time is 12～18h; Air pressure is 0.18～0.23Mpa; Air flow quantity is 20～26L/h; Reactor pressure is 0.04～0.06Mpa; Then this nutrient solution is flocculated with water-soluble chitosan, filter results solid zymin 1;
It is 25～35% aqueous sodium hydroxide solution 15～25 weight parts, sodium-chlor 40～60 weight parts, 2-thiouracil 27～37 weight part sal epsom 8～10 weight parts, manganous sulfate 1.2～1.8 weight parts and Secondary ammonium phosphate 3～4 weight parts that described liquid nutrient medium contains corn steep liquor 360～460 weight parts, molasses 380～480 weight parts, mass concentration, and adds water and be settled to 8000 weight parts;
The agrobacterium radiobacter that b. will produce carbamyl hydrolysis enzyme is inoculated in the liquid nutrient medium that contains corn steep liquor, glucose, peptone, sodium-chlor, manganous sulfate, Secondary ammonium phosphate and pectinose, cultivate in 32～38 ℃ of stir-activatings, wherein stirring velocity is 60rpm; Soak time is 15～22h; Air pressure is 0.18～0.23Mpa; Air flow quantity is 18～22L/h; Reactor pressure is 0.04～0.06Mpa; Adding sodium hydroxide adjusting pH in described substratum is 6.5～7.8, then this nutrient solution is flocculated with water-soluble chitosan, filters results solid zymin 2;
Described liquid nutrient medium contains corn steep liquor 200～400 weight parts, glucose 180～260 weight parts, peptone 80～100 weight parts, 25g sodium-chlor 20～30 weight parts, manganous sulfate 1.6～2.0 weight parts, Secondary ammonium phosphate 6.8～7.6 weight parts and pectinose 12～16 weight parts; And described liquid nutrient medium is added water be settled to 8000 weight parts;
C. the solid zymin 2 that the solid zymin 1 that step a is made and step b make is mixed, and makes the zymin of synthesizing D-aryl glycine through biocatalysis, and the weight proportion of wherein said solid zymin 1 and described solid zymin 2 is 1～5: 1～50.
2. the zymin of synthesizing D-aryl glycine through biocatalysis as claimed in claim 1, it is characterized in that, the initial enzymic activity of the hydantoin enzyme that described gold Arthrobacter produces is greater than 0.32U, and the initial activity of the carbamyl hydrolysis enzyme that described agrobacterium radiobacter produces is greater than 10U.
3. the zymin of synthesizing D-aryl glycine through biocatalysis as claimed in claim 1 is characterized in that, the initial hydantoin enzyme activity of described gold Arthrobacter is 0.32U～0.45U.
4. the zymin of synthesizing D-aryl glycine through biocatalysis as claimed in claim 1 is characterized in that, described gold Arthrobacter is gold Arthrobacter 3256 active cellss, and described agrobacterium radiobacter is agrobacterium radiobacter 3255 active cellss.
5. the zymin of synthesizing D-aryl glycine through biocatalysis as claimed in claim 1 is characterized in that, described zymin is used for the synthetic D-aryl glycine of the heterogeneous hydrolysis glycolylurea of enzyme catalysis;
The method of the synthetic D-aryl glycine of the heterogeneous hydrolysis glycolylurea of described enzyme catalysis may further comprise the steps:
1. in the enzymic catalytic reaction tank, add entry and 5-aryl glycolylurea, and to adjust the pH value be that the ratio that the add-on of 6.5～7.8,5-aryl glycolylurea accounts for water and 5-aryl glycolylurea total mass is 5～21%, get mixing solutions;
2. under the condition that stirs, in the enzymic catalytic reaction tank, add the arbitrary described zymin of claim 1～5, at 25～40 ℃ of lower stirring reactions, until can't detect 5-aryl glycolylurea, make the D-aryl glycine; Described zymin account for the mixing solutions of step in 1. gross weight 1～10%, 8～66 hours reaction times;
3. go out D-aryl glycine solid by 200 mesh sieve precipitation separations, through recrystallization, obtain D-aryl glycine crystal.
6. the zymin of synthesizing D-aryl glycine through biocatalysis as claimed in claim 1, it is characterized in that, described D-aryl glycine is D-PG, D-pHPG, the D-fluorophenyl glycine, the D-chlorobenzene glycine, D-bromobenzene glycine, D-iodobenzene glycine, D-methoxy phenylglycine, D-pyridine series glycine, D-naphthalene series glycine, D-quinoline series glycine D-pyrimidine glycine, D-pyrazine glycine, D-pyridazine glycine, D-indoles glycine, D-thiophene glycine, D-furans glycine, D-pyrroles's glycine, D-pyrazoles glycine, D-imidazoles glycine, D-thiazole glycine, a kind of in D-oxazole glycine and the D-isoxazole glycine.
7. the zymin of synthesizing D-aryl glycine through biocatalysis as claimed in claim 6 is characterized in that, described D-aryl glycine is D-PG, and the preparation method of described D-PG is:
1. in the enzymic catalytic reaction tank, add entry 30kg and 5-phenyl hydantoin because of 2.8kg, and adjustment pH value is 6.8;
2. the zymin 2.8kg that under the condition that stirs, adds arbitrary described synthesizing D-aryl glycine through biocatalysis of claim 1-5 to the enzymic catalytic reaction tank, at 38 ℃ of lower stirring reactions, until can't detect the 5-phenyl hydantoin because of, and intermediate product N-carboxamide phenylglycine content makes D-PG in mass ratio less than or equal to 0.25%;
3. precipitation separation goes out the D-PG solid, through recrystallization, obtains the D-PG crystal;
4. extract the partly soluble D-PG of solution in the enzymic catalytic reaction tank by ultrafiltration membrance filter and evaporation concentration, get D-PG.
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| CN1928103A (en) * | 2006-08-30 | 2007-03-14 | 石家庄经济技术开发区中天化工有限责任公司 | Method of producing D-p-hydroxy-phenyl glycine by heterogeneous enzyme catalysis method |
| CN101104862A (en) * | 2007-06-06 | 2008-01-16 | 河北科技大学 | Method for synthesizing D-arylglycine by using heterogeneous enzyme to catalytically hydrolyzing 5-arylhydantoin |
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| CN1928103A (en) * | 2006-08-30 | 2007-03-14 | 石家庄经济技术开发区中天化工有限责任公司 | Method of producing D-p-hydroxy-phenyl glycine by heterogeneous enzyme catalysis method |
| CN101104862A (en) * | 2007-06-06 | 2008-01-16 | 河北科技大学 | Method for synthesizing D-arylglycine by using heterogeneous enzyme to catalytically hydrolyzing 5-arylhydantoin |
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