CN103981228B - A kind of lipase-catalyzed benzene glycolylurea is prepared the method for N-carbamyl phenylglycine - Google Patents
A kind of lipase-catalyzed benzene glycolylurea is prepared the method for N-carbamyl phenylglycine Download PDFInfo
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Abstract
The invention discloses a kind of lipase-catalyzed benzene glycolylurea and prepare the method for N-carbamyl phenylglycine, described method is: taking meso benzene glycolylurea as raw material, taking isopropyl alcohol as solvent, taking lipase as catalyst, in the buffer solution of chitosan-containing, form pH value and be 6.4～6.8 reaction system, under 40～50 DEG C of conditions, carry out stirring reaction, after reacting completely, by reactant liquor separation and purification, obtain N-carbamyl phenylglycine; The present invention is using lipase as hydrolase, and shitosan and lipase coexist, and makes lipase have good hydrolysing activity in PH values 6.4～6.8 scopes, and the productive rate of N-carbamyl phenylglycine reaches 41.0～49.5% of material quantity; The inventive method not only environmental pollution is little, and product purity is high, be produced into low, and have industrial applications be worth.
Description
(1) technical field
The present invention relates to a kind of preparation method of N-carbamyl phenylglycine, particularly a kind of fatFat enzymatic hydrolysis benzene glycolylurea is prepared the method for N-carbamyl phenylglycine.
(2) background technology
Benzene glycolylurea, as raw material, is hydrolyzed its amido link with hydantoin enzyme and prepares N-carbamyl phenylglycine,Technology is comparative maturity, not only has both at home and abroad a large amount of research reports, and has that industrialization is actual answersWith, be the critical process step of producing phenylglycine, N-carbamyl phenylglycine is also in keyMesosome. This technical matters process has fewer environmental impacts, and product purity is high, promising. But,The major defect that this technology exists is that hydantoin enzyme production cost is high, thereby poor in economic benefit,Actual industrial production difficulty. For this reason, reducing its production cost is the problem that urgently needs solution.
(3) summary of the invention
The present invention seeks to reduce benzene using hydantoinase hydrolysis amido link and prepare N-carbamyl phenylglycineFinancial cost, substitute hydantoin enzyme with lipase, the amido link of hydrolysis benzene glycolylurea obtains N-carbamylBase phenylglycine, the production cost of lipase is significantly less than hydantoin enzyme, and wide material sources can obviously reduceFinancial cost.
The technical solution used in the present invention is:
The invention provides the side that a kind of lipase-catalyzed benzene glycolylurea is prepared N-carbamyl phenylglycineMethod, described method is: taking meso benzene glycolylurea as raw material, taking isopropyl alcohol as solvent, taking lipase asCatalyst forms pH value and is 6.4～6.8 reaction system, 40～50 in the buffer solution of chitosan-containingUnder DEG C condition, carry out stirring reaction (preferably stirring under 200～300rpm), after having reacted,By reactant liquor separation and purification, obtain N-carbamyl phenylglycine; The buffer solution of described chitosan-containingThat the acetic acid aqueous solution of shitosan and volumetric concentration 1% is mixed into chitosan mass concentrationAfter the chitosan solution of 2.5～3.6mg/ml, regulate pH value to 5.6～6.2, and then add final concentrationThe disodium-hydrogen and the sodium dihydrogen phosphate that are 0.025mol/L, stir, and makes chitosan-containingBuffer solution; The consumption of described lipase is 1200～2150U/L reaction system, described meso benzeneThe initial concentration of glycolylurea is 7.0～8.8g/L reaction system, and the volume final concentration of described isopropyl alcohol is49～62%, the consumption of the buffer solution of described chitosan-containing is counted 0.8～2.0g/L with the quality of shitosanReaction system.
Further, preferred described lipase is porcine pancreatic lipase (active is 2500U/g) or false silk fermentFemale lipase (activity is 5000U/g).
Further, the deacetylation of preferred described shitosan is greater than 90%(preferably 91.5%～92.1%).
Further, the preferred described conversion reaction time is 52～60 hours, after conversion reaction finishes,With liquid chromatography (chromatographic condition: C18 post, pH5.2 acetic acid-sodium acetate of volume ratio 99:1 is slowRushing liquid and methyl alcohol is mobile phase, flow velocity 0.5 ml/min, and 30 DEG C of column temperatures, sample size 5 microlitres,UV-detector, wavelength 254nm) detection reaction liquid, benzene glycolylurea is that standard items are quantitatively measuredProduct inversion quantity.
Further, the consumption of preferred described lipase is 1500-1700U/L reaction system, described inThe initial concentration of racemization benzene glycolylurea is 7.7-7.9g/L reaction system, the volume final concentration of described isopropyl alcoholFor 55-57%, the consumption of the buffer solution of described chitosan-containing is counted 1.0-1.8g/L with the quality of shitosanReaction system.
Further, the method for preferred described reactant liquor separation and purification is: after reaction finishes, by reactant liquorUnder 0.1 atmospheric pressure, 50 DEG C of vacuum reclaim isopropyl alcohol, obtain concentrate, in concentrate, add oilEther extracts, and after stratification, gets petroleum ether layer and enters with the ethanol water of volumetric concentration 30%Row extraction, gets ethanol water phase, and under 0.1 atmospheric pressure, 50 DEG C of vacuum reclaim ethanol dry,To N-carbamyl phenylglycine.
Further, lipase-catalyzed benzene glycolylurea of the present invention is prepared N-carbamyl phenylglycineMethod recommend carry out as follows: (1) is dissolved in meso benzene glycolylurea in isopropyl alcohol, in makingRacemization benzene glycolylurea solution; (2) acetic acid aqueous solution of shitosan and volumetric concentration 1% being mixed into shell gathersAfter the chitosan solution of the mass concentration 2.5～3.6mg/ml of sugar, with the NaOH water of 1.0M concentrationSolution regulates pH value to 5.6～6.2, and then adds final concentration to be the phosphoric acid one of 0.025mol/LHydrogen sodium and sodium dihydrogen phosphate, stir, and makes the buffer solution of chitosan-containing, then by lipase with containThe buffer solution of shitosan is mixed and made into lipase mixed liquor; (3) in then being prepared by step (1)After the lipase mixed liquor that racemization benzene glycolylurea solution is prepared with step (2) mixes, use 1.0MThe sodium hydrate aqueous solution of concentration regulates pH value to 6.4～6.8, forms reaction system, 40～50DEG C, carry out stirring reaction under 200～300rpm condition, after reacting completely, by reactant liquor separation and purification,Obtain N-carbamyl phenylglycine; The deacetylation of described shitosan is greater than 90%; Described fatThe consumption of enzyme is 1500～1700U/L reaction system (preferably 1600U/L), described meso benzene seaThe initial concentration of cause is 7.7～7.9g/L reaction system (preferably 7.8g/L reaction system), described isopropylThe volume final concentration of alcohol is 55～57%(preferably 56%), the consumption of the buffer solution of described chitosan-containingCount 1.0～1.8g/L reaction system (preferably 1.3g/L) with the quality of shitosan; Described lipase isPorcine pancreatic lipase or lipase from candida sp.
Prepare N-carbamyl phenylglycine taking benzene glycolylurea as material, enzyme method, there is environmental pollution little,The advantage that product purity is high.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: the present invention does with lipaseFor hydrolase, with alkaline polysaccharide--shitosan and lipase coexist to regulate the suitable hydrolysis of lipasePH value, makes lipase have good hydrolysing activity in PH values 6.4～6.8 scopes, pig pancreas fatFat enzyme reaches 49.0～49.5% of material quantity to the productive rate of N-carbamyl phenylglycine, Candida fatFat enzyme reaches 41.0～41.5% of material quantity to the productive rate of N-carbamyl phenylglycine; The inventive methodNot only keep environmental pollution little, the advantage that product purity is high, and also lipase source is wide, and price is low,Industrial production maturation, obviously reduces the production cost of this technique, has industrial applications and is worth. WithTime also widened the range of application of lipase.
(4) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present inventionBe not limited in this:
Shitosan of the present invention is purchased from Zhejiang Province gold shell Biochemie Co., Ltd, and porcine pancreatic lipase is purchased from ShanghaiHundred million glad biological Co., Ltds, isopropyl alcohol is purchased from Hangzhou Shuan Lin chemical reagent factory, meso benzene glycolylurea purchased fromMacheng, hubei Province rapid development bio tech ltd, lipase from candida sp is opened safe new century life purchased from BeijingThing Co., Ltd.
Embodiment 1:
(1) get 99ml distilled water, add the chemical pure acetic acid of 1.0ml, be made into 1.0% volumetric concentrationAqueous acetic acid. In aqueous acetic acid, add the shitosan of 0.3 gram of deacetylation 92.1% (to purchaseFrom Zhejiang Province gold shell Biochemie Co., Ltd), be stirred to shitosan and dissolve completely, allocate 0.36 gram of phosphorus intoAcid one hydrogen sodium and 0.30 gram of sodium dihydrogen phosphate, stirring and dissolving is even, with the hydrogen-oxygen of 1.0mol/L concentrationChanging sodium water solution adjusting pH value is 5.9, makes the buffer solution of chitosan-containing. Enzyme is lived as 2500U/Gram 0.15 gram of porcine pancreatic lipase (purchased from the glad biological Co., Ltd in Shanghai hundred million) add the slow of chitosan-containingRush in liquid, stir, then with 90 revs/min of stirrings 15 minutes, make lipase mixed liquor.(2) in 130ml chemical pure isopropanol (purchased from Hangzhou Shuan Lin chemical reagent factory), add 1.8 gramsMeso benzene glycolylurea (purchased from Macheng, hubei Province rapid development bio tech ltd), is uniformly dissolved, and makesBenzene glycolylurea solution. (3) the lipase mixed liquor of benzene glycolylurea solution being prepared with step (1) mixes allEven, by the sodium hydrate aqueous solution adjusting pH value to 6.6 of 1.0mol/L concentration, form cumulative volume 233mlReaction system, in water-bath, keep 45 DEG C, with 300 revs/min of mechanical agitation, react 56 hoursStop. With liquid chromatography (chromatographic condition: C18 post, the pH5.2 acetic acid of volume ratio 99:1:Sodium-acetate buffer and methyl alcohol are mobile phase, flow velocity 0.5 ml/min, 30 DEG C of column temperatures, sample size5 microlitres, UV-detector, wavelength 254nm) detection reaction liquid, benzene glycolylurea is that standard items are quantitative,There is 0.89 gram of N-carbamyl phenylglycine to generate, reaction yield 49.4%.
Reactant liquor 232ml 50 DEG C of vacuum under 0.1 atmospheric pressure reclaim isopropyl alcohol, after having reclaimed, addEnter 100ml petroleum ether extraction 15 minutes, after static layering, isolate benzinum, with volumetric concentration30% ethanol water 90ml extraction 15 minutes, isolates ethanol water phase, at 0.1 atmosphereDepress 50 DEG C of vacuum and reclaim ethanol dry, obtain 0.72 gram of N-carbamyl phenylglycine, yield80.9%。
Embodiment 2:
(1) get 99ml distilled water, add the chemical pure acetic acid of 1.0ml, be made into 1.0% volume denseThe aqueous acetic acid of degree. In aqueous acetic acid, add the shitosan of 0.36 gram of deacetylation 91.7%(purchased from Zhejiang Province gold shell Biochemie Co., Ltd), is stirred to shitosan and dissolves completely, allocates 0.36 intoGram disodium-hydrogen and 0.30 gram of sodium dihydrogen phosphate, stirring and dissolving is even, by 1.0mol/L concentrationIt is 5.6 that sodium hydrate aqueous solution regulates pH value, makes the buffer solution of chitosan-containing. Be 2500 by enzyme work0.12 gram of the porcine pancreatic lipase (purchased from the glad biological Co., Ltd in Shanghai hundred million) of units/gram adds containing shell poly-The buffer solution of sugar, stirs, then with 100 revs/min of stirrings 15 minutes, makes lipase and mixLiquid. (2) in 100ml chemical pure isopropanol (purchased from Hangzhou Shuan Lin chemical reagent factory), allocate 1.4 intoGram meso benzene glycolylurea (Macheng, hubei Province rapid development bio tech ltd), is uniformly dissolved, and makes benzeneGlycolylurea solution. (3) the lipase mixed liquor of benzene glycolylurea solution being prepared with step (1) mixes,By the sodium hydrate aqueous solution adjusting pH value to 6.4 of 1.0mol/L concentration, form cumulative volume 203mlReaction system, in water-bath, keep 40 DEG C, with 200 revs/min of mechanical agitation, react 52 hoursStop. With liquid chromatography (chromatographic condition: C18 post, the pH5.2 acetic acid of volume ratio 99:1:Sodium-acetate buffer and methyl alcohol are mobile phase, flow velocity 0.5 ml/min, 30 DEG C of column temperatures, sample size5 microlitres, UV-detector, wavelength 254nm) detection reaction liquid, benzene glycolylurea is that standard items are quantitative,There is 0.69 gram of N-carbamyl phenylglycine to generate, reaction yield 49.3%.
Reactant liquor 202ml 50 DEG C of vacuum under 0.1 atmospheric pressure reclaim isopropyl alcohol, after having reclaimed, addEnter 100ml petroleum ether extraction 15 minutes, after static layering, isolate benzinum, with volumetric concentration30% ethanol water 90ml extraction 15 minutes, isolates ethanol water phase, at 0.1 atmosphereDepress 50 DEG C of vacuum and reclaim ethanol dry, obtain 0.55 gram of N-carbamyl phenylglycine, yield79.7%。
Embodiment 3:
(1) get 99ml distilled water, add the chemical pure acetic acid of 1.0ml, be made into 1.0% volume denseThe aqueous acetic acid of degree. In aqueous acetic acid, add the shitosan of 0.26 gram of deacetylation 91.5%(Zhejiang Province gold shell Biochemie Co., Ltd), is stirred to shitosan and dissolves completely, allocates 0.36 gram of phosphorus intoAcid one hydrogen sodium and 0.30 gram of sodium dihydrogen phosphate, stirring and dissolving is even, with the hydrogen-oxygen of 1.0mol/L concentrationChanging sodium water solution adjusting pH value is 6.2, makes the buffer solution of chitosan-containing. Be 2500 lists by enzyme workPosition/gram 0.13 gram of porcine pancreatic lipase (the glad biological Co., Ltd in Shanghai hundred million) add glycan buffer solution,Stir, then with 90 revs/min of stirrings 20 minutes, make lipase mixed liquor. (2) at 160mlIn chemical pure isopropanol (Hangzhou Shuan Lin chemical reagent factory), allocate 2.0 grams of meso benzene glycolylurea (Hubei intoRapid development bio tech ltd, Macheng), be uniformly dissolved, make benzene glycolylurea solution. (3) by benzene seaThe shitosan mixed liquor of preparing with step (1) because of solution mixes, with the hydrogen of 1.0mol/L concentrationAqueous solution of sodium oxide regulates pH value to 6.8, forms the reaction system of cumulative volume 263ml, in water-bath50 DEG C of middle maintenances, with 250 revs/min of mechanical agitation, react termination in 60 hours. With liquid chromatography(chromatographic condition: C18 post, the pH5.2 acetic acid of volume ratio 99:1: sodium-acetate buffer and methyl alcoholFor mobile phase, flow velocity 0.5 ml/min, 30 DEG C of column temperatures, sample size 5 microlitres, ultraviolet light detectsDevice, wavelength 254nm) detection reaction liquid, benzene glycolylurea is that standard items are quantitative, has 0.98 gram of N-ammoniaFormoxyl phenylglycine generates, productive rate 49.0%.
Reactant liquor 262ml 50 DEG C of vacuum under 0.1 atmospheric pressure reclaim isopropyl alcohol, after having reclaimed, addEnter 100ml petroleum ether extraction 15 minutes, after static layering, isolate benzinum, with volumetric concentration30% ethanol water 90ml extraction 15 minutes, isolates ethanol water phase, at 0.1 atmosphereDepress 50 DEG C of vacuum and reclaim ethanol dry, obtain 0.79 gram of N-carbamyl phenylglycine, yield80.6%。
Embodiment 4:
(1) get 99ml distilled water, add the chemical pure acetic acid of 1.0ml, be made into 1.0% volume denseThe aqueous acetic acid of degree. In aqueous acetic acid, add the shitosan (Zhejiang of 0.3 gram of deacetylation 92.1%Jiang Jin shell biochemistry corporation,Ltd.), be stirred to shitosan and dissolve completely, allocate 0.36 gram of phosphoric acid one intoHydrogen sodium and 0.30 gram of sodium dihydrogen phosphate, stirring and dissolving is even, with the NaOH of 1.0mol/L concentrationIt is 5.9 that the aqueous solution regulates pH value, makes the buffer solution of chitosan-containing. By enzyme work be 5000 units/Gram 0.10 gram of lipase from candida sp (biological Co., Ltd of safe new century is opened in Beijing) allocate shitosan intoBuffer solution, stirs, then with 90 revs/min of stirrings 20 minutes, makes lipase mixed liquor.(2) in 130ml chemical pure isopropanol (Hangzhou Shuan Lin chemical reagent factory), allocate in 2.0 grams and disappearRevolve benzene glycolylurea (Macheng, hubei Province rapid development bio tech ltd), be uniformly dissolved, make benzene glycolylurea moltenLiquid. (3) the lipase mixed liquor of benzene glycolylurea solution being prepared with step (1) mixes, and usesThe sodium hydrate aqueous solution of 1.0mol/L concentration regulates pH value to 6.8, forms cumulative volume 233ml'sReaction system keeps 45 DEG C in water-bath, with 300 revs/min of mechanical agitation, reacts 60 hours eventuallyOnly. With liquid chromatography (chromatographic condition: C18 post, the pH5.2 acetic acid of volume ratio 99:1: vinegarAcid sodium buffer solution and methyl alcohol are mobile phase, flow velocity 0.5 ml/min, 30 DEG C of column temperatures, sample size 5Microlitre, UV-detector, wavelength 254nm) detection reaction liquid, benzene glycolylurea is that standard items are quantitative,There is 0.82 gram of N-carbamyl phenylglycine to generate, productive rate 41.0%.
Reactant liquor 232ml 50 DEG C of vacuum under 0.1 atmospheric pressure reclaim isopropyl alcohol, after having reclaimed, addEnter 100ml petroleum ether extraction 15 minutes, after static layering, isolate benzinum, with volumetric concentration30% ethanol water 90ml extraction 15 minutes, isolates ethanol water phase, at 0.1 atmosphereDepress 50 DEG C of vacuum and reclaim ethanol dry, obtain 0.66 gram of N-carbamyl phenylglycine, yield80.5%。
Comparative example 1
Get 100ml distilled water, allocate 0.36 gram of disodium-hydrogen and 0.30 gram of sodium dihydrogen phosphate into, stirMix and be uniformly dissolved, regulate the pH value of chitosan solution with the sodium hydrate aqueous solution of 1.0mol/L concentrationBe 5.9, make buffer solution. The porcine pancreatic lipase that is 2500U/ gram by enzyme work is (purchased from the glad life in Shanghai hundred millionThing Co., Ltd) 0.15 gram add buffer solution, stir, then stir 15 points with 90 revs/minClock, makes lipase mixed liquor. (2) at 130ml chemical pure isopropanol (purchased from the two woodsizations in HangzhouWork chemical reagent work) in add 1.8 grams of meso benzene glycolylureas (limited purchased from Macheng, hubei Province rapid development biotechnologyCompany), be uniformly dissolved, make benzene glycolylurea solution. (3) by benzene glycolylurea solution and step (1) preparationLipase mixed liquor mix, regulate pH value with the sodium hydrate aqueous solution of 1.0mol/L concentrationTo 6.6, form the reaction system of cumulative volume 232ml, in water-bath, keep 45 DEG C, 300 turning/Divide mechanical agitation, react termination in 56 hours. With liquid chromatography (chromatographic condition: C18 post, bodyLong-pending than the pH5.2 acetic acid of 99:1: sodium-acetate buffer and methyl alcohol are mobile phase, 0.5 milliliter of flow velocity/Minute, 30 DEG C of column temperatures, sample size 5 microlitres, UV-detector, wavelength 254nm) detect insteadAnswer liquid, benzene glycolylurea is that standard items are quantitative, has 0.42 gram of N-carbamyl phenylglycine to generate, anti-Product yield 23.3%.
Comparative example 2:
(1) get 100ml distilled water, allocate 0.36 gram of disodium-hydrogen and 0.30 gram of biphosphate intoSodium, stirring and dissolving is even, and regulating pH value with the sodium hydrate aqueous solution of 1.0mol/L concentration is 5.9,Make buffer solution. Be that (the safe new century is opened in Beijing for the lipase from candida sp of 5000 units/gram by enzyme workBiological Co., Ltd) 0.10 gram allocate buffer solution into, stir, then stir 20 with 90 revs/minMinute, make lipase mixed liquor. (2) at 130ml chemical pure isopropanol (the two woods chemical industry in HangzhouChemical reagent work) in allocate 2.0 grams of meso benzene glycolylureas (Macheng, hubei Province rapid development bio tech ltd) into,Be uniformly dissolved, make benzene glycolylurea solution. (3) fat of being prepared by benzene glycolylurea solution and step (1)Enzyme mixation mixes, by the sodium hydrate aqueous solution adjusting pH value to 6.8 of 1.0mol/L concentration,The reaction system that forms cumulative volume 232ml keeps 45 DEG C in water-bath, stirs with 300 revs/min of machineriesMix, react termination in 60 hours. With liquid chromatography (chromatographic condition: C18 post, volume ratio 99:1PH5.2 acetic acid: sodium-acetate buffer and methyl alcohol are mobile phase, flow velocity 0.5 ml/min, column temperature30 DEG C, sample size 5 microlitres, UV-detector, wavelength 254nm) detection reaction liquid, benzene seaBecause standard items are quantitative, generate productive rate 19.5% in respect of 0.39 gram of N-carbamyl phenylglycine.
Comparative example 3:
(1) get 99ml distilled water, add the chemical pure acetic acid of 1.0ml, be made into 1.0% volume denseThe aqueous acetic acid of degree. In aqueous acetic acid, add the shitosan of 0.3 gram of deacetylation 92.1% (to purchaseFrom Zhejiang Province gold shell Biochemie Co., Ltd), be stirred to shitosan and dissolve completely, allocate 0.36 gram of phosphorus intoAcid one hydrogen sodium and 0.30 gram of sodium dihydrogen phosphate, stirring and dissolving is even, with the hydrogen-oxygen of 1.0mol/L concentrationThe pH value of changing sodium water solution adjusting chitosan solution is 5.9, makes the buffer solution of chitosan-containing. Will0.15 gram of the Penicillium lipase (biological Co., Ltd of safe new century is opened in Beijing) that enzyme work is 3000U/ gramAdd shitosan buffer solution, stir, then with 90 revs/min of stirrings 15 minutes, make fatEnzyme mixation. (2) in 130ml chemical pure isopropanol (purchased from Hangzhou Shuan Lin chemical reagent factory)Add 1.8 grams of meso benzene glycolylureas (purchased from Macheng, hubei Province rapid development bio tech ltd), dissolveEvenly, make benzene glycolylurea solution. (3) lipase of being prepared by benzene glycolylurea solution and step (1) is mixedClose liquid and mix, with the sodium hydrate aqueous solution adjusting pH value to 6.6 of 1.0mol/L concentration, structureThe reaction system that becomes cumulative volume 233ml keeps 45 DEG C in water-bath, with 300 revs/min of mechanical agitation,React termination in 56 hours. With liquid chromatography (chromatographic condition: C18 post, the pH5.2 of volume ratio 99:1Acetic acid: sodium-acetate buffer and methyl alcohol are mobile phase, flow velocity 0.5 ml/min, 30 DEG C of column temperatures,Sample size 5 microlitres, UV-detector, wavelength 254nm) detection reaction liquid, benzene glycolylurea is markAccurate product are quantitative, have 0.19 gram of N-carbamyl phenylglycine to generate, reaction yield 10.6%.
Claims (7)
1. lipase-catalyzed benzene glycolylurea is prepared a method for N-carbamyl phenylglycine, and its feature existsIn described method be: taking meso benzene glycolylurea as raw material, taking isopropyl alcohol as solvent, taking lipase as catalysisAgent forms pH value and is 6.4～6.8 reaction system, at 40～50 DEG C of bars in the buffer solution of chitosan-containingUnder part, carry out stirring reaction, after reacting completely, by reactant liquor separation and purification, obtain N-carbamyl benzene sweetPropylhomoserin; The buffer solution of described chitosan-containing is that the acetic acid aqueous solution of shitosan and volumetric concentration 1% is mixed intoAfter the chitosan solution of chitosan mass concentration 2.5～3.6mg/ml, regulate pH value to 5.6～6.2, thenAdd again final concentration to be disodium-hydrogen and the sodium dihydrogen phosphate of 0.025mol/L, stir, obtainThe buffer solution of chitosan-containing; The consumption of described lipase is 1200～2150U/L reaction system, disappears in describedThe initial concentration that revolves benzene glycolylurea is 7.0～8.8g/L reaction system, and the volume final concentration of described isopropyl alcohol is49～62%, the consumption of the buffer solution of described chitosan-containing is in the quality of shitosan, and the consumption of shitosan is0.8～2.0g/L reaction system.
2. the side that lipase-catalyzed benzene glycolylurea is prepared N-carbamyl phenylglycine as claimed in claim 1Method, is characterized in that described lipase is porcine pancreatic lipase or lipase from candida sp.
3. the side that lipase-catalyzed benzene glycolylurea is prepared N-carbamyl phenylglycine as claimed in claim 1Method, is characterized in that the deacetylation of described shitosan is greater than 90%.
4. the side that lipase-catalyzed benzene glycolylurea is prepared N-carbamyl phenylglycine as claimed in claim 1Method, is characterized in that the described catalytic reaction time is 52～60 hours.
5. the side that lipase-catalyzed benzene glycolylurea is prepared N-carbamyl phenylglycine as claimed in claim 1Method, the consumption that it is characterized in that described lipase is 1500～1700U/L reaction system, described meso benzeneThe initial concentration of glycolylurea is 7.7～7.9g/L reaction system, and the volume final concentration of described isopropyl alcohol is 55-57%,The consumption of the buffer solution of described chitosan-containing is in the quality of shitosan, and the consumption of shitosan is 1.0～1.8g/LReaction system.
6. the side that lipase-catalyzed benzene glycolylurea is prepared N-carbamyl phenylglycine as claimed in claim 1Method, is characterized in that described method carries out as follows: meso benzene glycolylurea is dissolved in isopropyl alcohol by (1)In, make meso benzene glycolylurea solution; (2) shitosan is mixed with the acetic acid aqueous solution of volumetric concentration 1%After the chitosan solution of the mass concentration 2.5～3.6mg/ml of one-tenth shitosan, use the NaOH of 1.0M concentrationThe aqueous solution regulates pH value to 5.6～6.2, and then adds final concentration to be phosphoric acid one hydrogen of 0.025mol/LSodium and sodium dihydrogen phosphate, stir, and makes the buffer solution of chitosan-containing, then by lipase and poly-containing shellThe buffer solution of sugar is mixed and made into lipase mixed liquor; (3) the meso benzene sea of then being prepared by step (1)The lipase mixed liquor of preparing with step (2) because of solution mixes the rear NaOH by 1.0M concentrationThe aqueous solution regulates pH value to 6.4～6.8, forms reaction system, 40～50 DEG C, 200～300rpm conditionUnder carry out stirring reaction, after reacting completely, by reactant liquor separation and purification, obtain N-carbamyl benzene sweet ammoniaAcid; The deacetylation of described shitosan is greater than 90%; The consumption of described lipase is 1500～1700U/LReaction system, the initial concentration of described meso benzene glycolylurea is 7.7～7.9g/L reaction system, described isopropylThe volume final concentration of alcohol is 55～57%, and the consumption of the buffer solution of described chitosan-containing is with the quality of shitosanMeter, the consumption of shitosan is 1.0～1.8g/L reaction system; Described lipase is porcine pancreatic lipase or false silkYeast-lipase.
7. as described in claim 1 or 6, lipase-catalyzed benzene glycolylurea is prepared N-carbamyl phenylglycineMethod, it is characterized in that the method for described reactant liquor separation and purification is: reaction finish after, by reactant liquorUnder 0.1 atmospheric pressure, 50 DEG C of vacuum reclaim isopropyl alcohol, obtain concentrate, in concentrate, add benzinumExtract, after stratification, get petroleum ether layer and extract with the ethanol water of volumetric concentration 30%,Get ethanol water phase, under 0.1 atmospheric pressure, 50 DEG C of vacuum reclaim ethanol dry, obtain N-carbamylBase phenylglycine.
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