CN101348808B - Double enzyme coupling preparation of ornithine hydrochloride - Google Patents
Double enzyme coupling preparation of ornithine hydrochloride Download PDFInfo
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- CN101348808B CN101348808B CN2008101200550A CN200810120055A CN101348808B CN 101348808 B CN101348808 B CN 101348808B CN 2008101200550 A CN2008101200550 A CN 2008101200550A CN 200810120055 A CN200810120055 A CN 200810120055A CN 101348808 B CN101348808 B CN 101348808B
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- ornithine hydrochloride
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- GGTYBZJRPHEQDG-WCCKRBBISA-N (2s)-2,5-diaminopentanoic acid hydrochloride Chemical compound Cl.NCCC[C@H](N)C(O)=O GGTYBZJRPHEQDG-WCCKRBBISA-N 0.000 title claims abstract description 43
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 42
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 42
- 229960003244 ornithine hydrochloride Drugs 0.000 title claims abstract description 40
- 238000010168 coupling process Methods 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims description 35
- 230000008878 coupling Effects 0.000 title claims description 19
- 238000005859 coupling reaction Methods 0.000 title claims description 19
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 23
- 244000068988 Glycine max Species 0.000 claims abstract description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 58
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical group CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 42
- 235000021336 beef liver Nutrition 0.000 claims description 35
- 239000003054 catalyst Substances 0.000 claims description 27
- 239000000047 product Substances 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 238000006243 chemical reaction Methods 0.000 claims description 24
- 239000011572 manganese Substances 0.000 claims description 24
- 239000012043 crude product Substances 0.000 claims description 23
- 239000008367 deionised water Substances 0.000 claims description 23
- 229910021641 deionized water Inorganic materials 0.000 claims description 23
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 21
- 239000013078 crystal Substances 0.000 claims description 21
- 239000004475 Arginine Substances 0.000 claims description 16
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 16
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 16
- 238000001816 cooling Methods 0.000 claims description 16
- 239000000706 filtrate Substances 0.000 claims description 16
- 238000001914 filtration Methods 0.000 claims description 16
- 239000000284 extract Substances 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 15
- 238000002425 crystallisation Methods 0.000 claims description 14
- 238000004042 decolorization Methods 0.000 claims description 14
- 238000000703 high-speed centrifugation Methods 0.000 claims description 14
- 150000002500 ions Chemical class 0.000 claims description 14
- 239000012452 mother liquor Substances 0.000 claims description 14
- 238000010792 warming Methods 0.000 claims description 14
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- 229940071125 manganese acetate Drugs 0.000 claims description 9
- UOGMEBQRZBEZQT-UHFFFAOYSA-L manganese(2+);diacetate Chemical compound [Mn+2].CC([O-])=O.CC([O-])=O UOGMEBQRZBEZQT-UHFFFAOYSA-L 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- CNFDGXZLMLFIJV-UHFFFAOYSA-L manganese(II) chloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Mn+2] CNFDGXZLMLFIJV-UHFFFAOYSA-L 0.000 claims description 8
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical group [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 8
- 239000000758 substrate Substances 0.000 claims description 8
- 210000002421 cell wall Anatomy 0.000 claims description 7
- 238000011161 development Methods 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
- 239000012530 fluid Substances 0.000 claims description 7
- 238000007710 freezing Methods 0.000 claims description 7
- 230000008014 freezing Effects 0.000 claims description 7
- 238000009413 insulation Methods 0.000 claims description 7
- 238000004321 preservation Methods 0.000 claims description 7
- 238000005070 sampling Methods 0.000 claims description 7
- 239000002002 slurry Substances 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 238000000034 method Methods 0.000 abstract description 18
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 241000283690 Bos taurus Species 0.000 abstract 2
- 210000004185 liver Anatomy 0.000 abstract 2
- 108010046334 Urease Proteins 0.000 abstract 1
- 235000009697 arginine Nutrition 0.000 description 14
- 239000003643 water by type Substances 0.000 description 6
- 102000004452 Arginase Human genes 0.000 description 5
- 108700024123 Arginases Proteins 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 238000010298 pulverizing process Methods 0.000 description 5
- 230000009466 transformation Effects 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 2
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 2
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Chemical compound [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 description 2
- 239000000920 calcium hydroxide Substances 0.000 description 2
- 235000011116 calcium hydroxide Nutrition 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 229960003104 ornithine Drugs 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- 229930064664 L-arginine Natural products 0.000 description 1
- 235000014852 L-arginine Nutrition 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- RQPZNWPYLFFXCP-UHFFFAOYSA-L barium dihydroxide Chemical class [OH-].[OH-].[Ba+2] RQPZNWPYLFFXCP-UHFFFAOYSA-L 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 1
- 229910001861 calcium hydroxide Inorganic materials 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 150000003983 crown ethers Chemical class 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000011169 microbiological contamination Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003444 phase transfer catalyst Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention relates to a method for preparing ornithine hydrochloride, in particular to a double-enzyme coupling method for preparing the ornithine hydrochloride. The invention provides an economic and environment-friendly double-enzyme coupling method for preparing the ornithine hydrochloride. The method fully utilizes biological enzymes to prepare the product of the ornithine hydrochloride and is distinguished from the most production methods which depend on chemical reagent too much in the prior art. The method adopts urease extracted from fresh soybeans and bovine liver enzyme solution extracted from fresh bovine livers to prepare the ornithine hydrochloride.
Description
Technical field
The present invention relates to a kind of ornithine hydrochloride preparation method, refer in particular to a kind of double enzyme coupling preparation of ornithine hydrochloride.
Background technology
Ornithine (Ornithine), being a seed amino acid, is that the metabolism when arginine (Arginine) produces urea (formation of urine) produces, and is important medicine intermediate and bulk drug and foodstuff additive, be mainly used in the treatment liver cirrhosis, the recovery of disease and enhancing physical efficiency.
Domestic publication number is the preparation method of a kind of L-ornithine hydrochloride of CN1590367, its main contents comprise: the L-arginine is soluble in water, add phase-transfer catalyst crown ether and milk of lime calcium hydroxide, add the promotor choline in stirring and refluxing heating back, pH value is transferred with 6N sulfuric acid in question response mixture cooling back then.Remove calcium sulfate more after filtration, its filtrate is transferred pH value with saturated barium hydroxide solution behind concentrating under reduced pressure, remove barium sulfate precipitate more after filtration, and its filtrate is transferred PH with 3N hydrochloric acid, adds ethanol again behind vacuum concentration, leaches after cooling.The product yield of this method can reach 78～82%, and purity is 〉=99.0%, specific rotatory power [A]
D 20+ 23.0～+ 24.50.
Present most production method too much relies on chemical reagent, and is big to environmental disruption, the cost height, and product yield and purity can not get guaranteeing.
Summary of the invention
The invention provides a kind of double enzyme coupling preparation of ornithine hydrochloride of economic environmental protection.Make full use of biological enzyme and make product, difference is the too much production method that relies on chemical reagent of great majority now.
Above-mentioned technical problem of the present invention is mainly solved by following technical proposals:
The step of the double enzyme coupling preparation of ornithine hydrochloride comprises:
A, the arginine dry product is dissolved in as substrate is made into conversion fluid in the deionized water, regulate between PH to 7～10 with 2N hydrochloric acid, be warming up to 38 ℃～45 ℃, add the beef liver enzyme liquid that from fresh beef liver, extracts, add catalyst, put into 38 ℃～45 ℃ constant temperature waters shaking tables and transform 10～40 hours;
B, the solution sampling that steps A is obtained, the point plate, treat that the arginine spot transforms completely dissolve after, add the urase that extracts in the fresh soya bean of 5～20ml/L, 30 ℃～35 ℃ insulation reaction are after 40～70 minutes, drip the 6N hcl acidifying between PH5～6, decolorization filtering is after being concentrated into small amount of crystal and separating out, the alcohol that in concentrated solution, adds its 2～5 times of volumes, behind the backflow 1h, crystallisation by cooling gets the ornithine hydrochloride crude product after the centrifuging under 60 ℃～90 ℃ conditions;
C, ornithine hydrochloride dissolving crude product that step B is obtained are in deionized water, be warming up to 70 ℃～80 ℃, the gac that adds crude product weight 3～10%, decolorization filtering, after being concentrated into small amount of crystal and separating out, in concentrated solution, add the alcohol of its 2～5 times of volumes, crystallisation by cooling, centrifuging goes to clean the residual mother liquor of plane of crystal with small amount of ethanol behind the mother liquor, gets finished product after the drying.
The double enzyme coupling preparation of above-mentioned ornithine hydrochloride, as preferably, in the described steps A, the amount that adds beef liver enzyme liquid in the solution is with in every 1L conversion fluid, adds the beef liver enzyme liquid of 20ml～120ml.
Requirement according to technologies such as present method transformation efficiency and subsequent extractions, refining processing, effectively control the concentration of enzyme in conversion fluid, not only can improve transformation efficiency, also can make full use of enzyme activity, make reaction system reach the balance environment an of the best, guarantee the subsequent extractions of product, the requirement of refining processing.
The double enzyme coupling preparation of above-mentioned ornithine hydrochloride, as preferably, in the described steps A, the conversion fluid concentration of substrate is controlled at 50～300g/L.The control decision reaction substrate of concentration of substrate is to the transformation efficiency of target product, and meet the requirement of postorder complete processing, therefore effectively control the concentration of reaction substrate, not only can improve the reaction transformation efficiency, easily can utilize enzyme activity fully, make reaction environment meet the requirement of reaction kinetics, thereby reach stabilization process, enhance productivity the purpose of save energy.
The double enzyme coupling preparation of above-mentioned ornithine hydrochloride, as preferably, in the described steps A, catalyst is Mn
2+Ion, Mn
2+Ionic concn is controlled at 0.2～0.6mmol/L.Mn
2+Catalyst can be: manganous sulfate, manganese acetate, Manganous chloride tetrahydrate etc.
Mn
2+The release of living of the concentration decision enzyme in reaction system, and reaction is to the transfer of product direction, concentration is low excessively, too high all can live by inhibitory enzyme, impels the prolongation in reaction times.
The double enzyme coupling preparation of above-mentioned ornithine hydrochloride, as preferably, among the described step B, the agent of some plate development is n-propyl alcohol and ammoniacal liquor, its mass percent is a n-propyl alcohol: ammoniacal liquor=65～75: 35～25.The mass percent of above-mentioned solution is the mass percent of effective constituent.
The decision of the selection of developping agent and proportioning is to the dissolving of composition and the separating effect between the component, and and between component do not react, and the component of this developping agent is selected and proportioning can make other related impuritieses of product and substrate effectively separate, and presents independent clearly spot.
The most preferred, the mass percent of described n-propyl alcohol and ammoniacal liquor is a n-propyl alcohol: ammoniacal liquor=72: 28.
The double enzyme coupling preparation of above-mentioned ornithine hydrochloride, as preferably, among the described step C, the ornithine hydrochloride crude product is dissolved in the deionized water by weight 10%～30% concentration.
This step is a treating process, and in the production process, disposable products come out to be difficult to directly to reach qualified, therefore needs a treating process.
The double enzyme coupling preparation of above-mentioned ornithine hydrochloride, as preferably, in the described steps A, the preparation method of beef liver enzyme liquid is: get that fresh beef liver is pulverized in freezing pulverizer, cell wall breaking, stand-by 4 ℃ of preservations then, the beef liver slurry is dissolved in the deionized water of 1～4 times of mass ratio, adds Mn
2+Ion, and concentration is controlled at 0.2～0.6mmol/L, heat be incubated 0.5～2 hour between 30～70 ℃ altogether, filters, collection filtrate, and filtrate is deposited in 4 ℃ of refrigerators stand-by behind high speed centrifugation immediately.Mn
2+Catalyst can be: manganous sulfate, manganese acetate, Manganous chloride tetrahydrate etc.
The double enzyme coupling preparation of above-mentioned ornithine hydrochloride, as preferably, the preparation method of urase is among the described step B: after fresh soya bean is pulverized, mix with the ethanol of 30% volume percent, soya bean and ethanol mass percent are 1: 2～3, shake 10 minutes down at 0～10 ℃ then, get supernatant liquor behind the high speed centrifugation, 30～35 ℃ of uses are incubated 40～70 minutes.
The present invention has following advantage:
The yield of the inventive method can reach 80-85%, and product purity can reach more than 99%.
Enzyme of the present invention all comes from resource cheap and easy to get, and animal ferment extracts from commercially available beef liver, and plant enzyme comes from the extraction liquid of soya bean.
The inventive method compares with chemical method that to have technology controlling and process stable, and reactions steps is few, production efficiency height, low cost and other advantages.
The inventive method is compared with fermentation method, adopt zymotechnic to prepare the ornithine hydrochloride product, overcome the difficult problem of fermentation method culture presevation and fermenting process control microbiological contamination, and the fermentation method environmental pollution is big, the production waste amount is big, because the output of every liter of fermented liquid of strain fermentation is extremely low, problems such as technology instability become the difficult problem that fermentation method prepares this product.
Characteristics of the present invention are that technology controlling and process is stable, and reactions steps is few, and production cost is low, and environmental pollution is little, and energy consumption is low, and products obtained therefrom purity advantages of higher has the prospect of industrialization.
Embodiment
Below by embodiment, technical scheme of the present invention is described in further detail.
Embodiment 1:
The preparation of arginase:
Get fresh beef liver 100g, in freezing pulverizer, pulverize, cell wall breaking, stand-by 4 ℃ of preservations then, get 100g beef liver slurry and be dissolved in the 400ml deionized water, add catalyzer, catalyzer is a manganous sulfate, effectively catalyst component is Mn
2+Ion, and concentration is controlled at 0.2mmol/L, heat be incubated 0.5 hour to 30 ℃ altogether, filters, collection filtrate, and filtrate is deposited in 4 ℃ of refrigerators stand-by behind high speed centrifugation immediately.
The preparation of urase:
Get fresh soya bean 100g, after the pulverizing, mix with the ethanol of 30% volume percent, soya bean and ethanol mass percent are 1: 3, shake 10 minutes down at 10 ℃ then, get supernatant liquor behind the high speed centrifugation, and 30 ℃ of uses are incubated 40 minutes, are kept at below 4 ℃.
Conversion reaction:
100g arginine dry product is dissolved in the 2000ml deionized water, regulates PH to 10 with 2N hydrochloric acid, be warming up to 45 ℃, add the beef liver enzyme liquid that 120ml extracts from fresh beef liver, add catalyst, catalyst is a manganese acetate, and effectively catalyst component is Mn
2+Ion, Mn
2+Ionic concn is controlled at 0.6mmol/L, puts into 45 ℃ of constant temperature waters shaking tables and transforms 40 hours;
With above-mentioned solution sampling, the point plate, the agent of some plate development is n-propyl alcohol and ammoniacal liquor, n-propyl alcohol and ammoniacal liquor mass percent are n-propyl alcohol: ammoniacal liquor=65: 35, after treating that the arginine spot transforms completely dissolve, add the urase that extracts in the fresh soya bean of 5ml/L, 30 ℃ of insulation reaction are after 40 minutes, drip the 6N hcl acidifying to PH5, decolorization filtering is after being concentrated into small amount of crystal and separating out, the alcohol that in concentrated solution, adds its 2 times of volumes, behind the backflow 1h, crystallisation by cooling gets the ornithine hydrochloride crude product after the centrifuging under 60 ℃ of conditions;
The ornithine hydrochloride crude product that obtains is got the concentration of 100g by weight 10% to be dissolved in the deionized water, be warming up to 70 ℃, the gac that adds crude product weight 3%, decolorization filtering, after being concentrated into small amount of crystal and separating out, in concentrated solution, add the alcohol of its 2 times of volumes, crystallisation by cooling, centrifuging goes to clean the residual mother liquor of plane of crystal with small amount of ethanol behind the mother liquor, gets finished product after the drying.
Embodiment 2:
The preparation of arginase:
Get fresh beef liver 100g, in freezing pulverizer, pulverize, cell wall breaking, stand-by 4 ℃ of preservations then, get 100g beef liver slurry and be dissolved in the 100ml deionized water, add catalyzer, catalyzer is a manganese acetate, effectively catalyst component is Mn
2+Ion, and concentration is controlled at 0.6mmol/L, heat be incubated 2 hours to 70 ℃ altogether, filters, collection filtrate, and filtrate is deposited in 4 ℃ of refrigerators stand-by behind high speed centrifugation immediately.
The preparation of urase:
Get fresh soya bean 100g, after the pulverizing, mix with the ethanol of 30% volume percent, soya bean and ethanol mass percent are 1: 2, shake 10 minutes down at 0 ℃ then, get supernatant liquor behind the high speed centrifugation, and 35 ℃ of uses are incubated 70 minutes, are kept at below 4 ℃.
Conversion reaction:
100g arginine dry product is dissolved in the 300ml deionized water, regulates PH to 7 with 2N hydrochloric acid, be warming up to 38 ℃, add the beef liver enzyme liquid that 20ml extracts from fresh beef liver, add catalyst, catalyst is a Manganous chloride tetrahydrate, and effectively catalyst component is Mn
2+Ion, Mn
2+Ionic concn is controlled at 0.2mmol/L, puts into 38 ℃ of constant temperature waters shaking tables and transforms 10 hours;
With above-mentioned solution sampling, the point plate, the agent of some plate development is n-propyl alcohol and ammoniacal liquor, n-propyl alcohol and ammoniacal liquor mass percent are n-propyl alcohol: ammoniacal liquor=75: 25, after treating that the arginine spot transforms completely dissolve, add the urase that extracts in the fresh soya bean of 20ml/L, 35 ℃ of insulation reaction are after 70 minutes, drip the 6N hcl acidifying to PH6, decolorization filtering is after being concentrated into small amount of crystal and separating out, the alcohol that in concentrated solution, adds its 5 times of volumes, behind the backflow 1h, crystallisation by cooling gets the ornithine hydrochloride crude product after the centrifuging under 90 ℃ of conditions;
The ornithine hydrochloride crude product that obtains is got the concentration of 100g by weight 30% to be dissolved in the deionized water, be warming up to 80 ℃, the gac that adds crude product weight 4%, decolorization filtering, after being concentrated into small amount of crystal and separating out, in concentrated solution, add the alcohol of its 5 times of volumes, crystallisation by cooling, centrifuging goes to clean the residual mother liquor of plane of crystal with small amount of ethanol behind the mother liquor, gets finished product after the drying.
Embodiment 3:
The preparation of arginase:
Get fresh beef liver 100g, in freezing pulverizer, pulverize, cell wall breaking, stand-by 4 ℃ of preservations then, get 100g beef liver slurry and be dissolved in the 200ml deionized water, add catalyzer, catalyzer is a Manganous chloride tetrahydrate, effectively catalyst component is Mn
2+Ion, and concentration is controlled at 0.4mmol/L, heat be incubated 1 hour to 35 ℃ altogether, filters, collection filtrate, and filtrate is deposited in 4 ℃ of refrigerators stand-by behind high speed centrifugation immediately.
The preparation of urase:
Get fresh soya bean 100g, after the pulverizing, mix with the ethanol of 30% volume percent, soya bean and ethanol mass percent are 1: 2.9, shake 10 minutes down at 2 ℃ then, get supernatant liquor behind the high speed centrifugation, and 35 ℃ of uses are incubated 60 minutes, are kept at below 4 ℃.
Conversion reaction:
100g arginine dry product is dissolved in the 1800ml deionized water, regulates PH to 8 with 2N hydrochloric acid, be warming up to 44 ℃, add the beef liver enzyme liquid that 70ml extracts from fresh beef liver, add catalyst, catalyst is a manganous sulfate, and effectively catalyst component is Mn
2+Ion, Mn
2+Ionic concn is controlled at 0.25mmol/L, puts into 43 ℃ of constant temperature waters shaking tables and transforms 35 hours;
With above-mentioned solution sampling, the point plate, the agent of some plate development is n-propyl alcohol and ammoniacal liquor, n-propyl alcohol and ammoniacal liquor mass percent are n-propyl alcohol: ammoniacal liquor=68: 32, after treating that the arginine spot transforms completely dissolve, add the urase that extracts in the fresh soya bean of 18ml/L, 31 ℃ of insulation reaction are after 45 minutes, drip the 6N hcl acidifying to PH6, decolorization filtering is after being concentrated into small amount of crystal and separating out, the alcohol that in concentrated solution, adds its 4.5 times of volumes, behind the backflow 1h, crystallisation by cooling gets the ornithine hydrochloride crude product after the centrifuging under 65 ℃ of conditions;
The ornithine hydrochloride crude product that obtains is got the concentration of 100g by weight 15% to be dissolved in the deionized water, be warming up to 77 ℃, the gac that adds crude product weight 10%, decolorization filtering, after being concentrated into small amount of crystal and separating out, in concentrated solution, add the alcohol of its 2 times of volumes, crystallisation by cooling, centrifuging goes to clean the residual mother liquor of plane of crystal with small amount of ethanol behind the mother liquor, gets finished product after the drying.
Embodiment 4:
The preparation of arginase:
Get fresh beef liver 100g, in freezing pulverizer, pulverize, cell wall breaking, stand-by 4 ℃ of preservations then, get 100g beef liver slurry and be dissolved in the 300ml deionized water, add catalyzer, catalyzer is manganous sulfate and manganese acetate and Manganous chloride tetrahydrate, effectively catalyst component is Mn
2+Ion, and concentration is controlled at 0.5mmol/L, heat be incubated 1.5 hours to 50 ℃ altogether, filters, collection filtrate, and filtrate is deposited in 4 ℃ of refrigerators stand-by behind high speed centrifugation immediately.
The preparation of urase:
Get fresh soya bean 100g, after the pulverizing, mix with the ethanol of 30% volume percent, soya bean and ethanol mass percent are 1: 2, shake 10 minutes down at 8 ℃ then, get supernatant liquor behind the high speed centrifugation, and 32 ℃ of uses are incubated 45 minutes, are kept at below 4 ℃.
Conversion reaction:
100g arginine dry product is dissolved in the 1500ml deionized water, regulates PH to 7 with 2N hydrochloric acid, be warming up to 39 ℃, add the beef liver enzyme liquid that 35ml extracts from fresh beef liver, add catalyst, catalyst is manganous sulfate and manganese acetate and Manganous chloride tetrahydrate, and effectively catalyst component is Mn
2+Ion, Mn
2+Ionic concn is controlled at 0.2mmol/L, puts into 44 ℃ of constant temperature waters shaking tables and transforms 15 hours;
With above-mentioned solution sampling, the point plate, the agent of some plate development is n-propyl alcohol and ammoniacal liquor, n-propyl alcohol and ammoniacal liquor mass percent are n-propyl alcohol: ammoniacal liquor=70: 30, after treating that the arginine spot transforms completely dissolve, add the urase that extracts in the fresh soya bean of 18ml/L, 31 ℃ of insulation reaction are after 65 minutes, drip the 6N hcl acidifying to PH5, decolorization filtering is after being concentrated into small amount of crystal and separating out, the alcohol that in concentrated solution, adds its 2～5 times of volumes, behind the backflow 1h, crystallisation by cooling gets the ornithine hydrochloride crude product after the centrifuging under 85 ℃ of conditions;
The ornithine hydrochloride crude product that obtains is got the concentration of 100g by weight 15% to be dissolved in the deionized water, be warming up to 80 ℃, the gac that adds crude product weight 5%, decolorization filtering, after being concentrated into small amount of crystal and separating out, in concentrated solution, add the alcohol of its 4 times of volumes, crystallisation by cooling, centrifuging goes to clean the residual mother liquor of plane of crystal with small amount of ethanol behind the mother liquor, gets finished product after the drying.
Embodiment 5:
The preparation of arginase:
Get fresh beef liver 100g, in freezing pulverizer, pulverize, cell wall breaking, stand-by 4 ℃ of preservations then, get 100g beef liver slurry and be dissolved in the 250ml deionized water, add catalyzer, catalyzer is manganese acetate and Manganous chloride tetrahydrate, effectively catalyst component is Mn
2+Ion, and concentration is controlled at 0.3mmol/L, heat be incubated 1.5 hours to 55 ℃ altogether, filters, collection filtrate, and filtrate is deposited in 4 ℃ of refrigerators stand-by behind high speed centrifugation immediately.
The preparation of urase:
Get fresh soya bean 100g, after the pulverizing, mix with the ethanol of 30% volume percent, soya bean and ethanol mass percent are 1: 2.5, shake 10 minutes down at 5 ℃ then, get supernatant liquor behind the high speed centrifugation, and 33 ℃ of uses are incubated 30 minutes, are kept at below 4 ℃.
Conversion reaction:
100g arginine dry product is dissolved in the 700ml deionized water, regulates PH to 8 with 2N hydrochloric acid, be warming up to 41 ℃, add the beef liver enzyme liquid that 60ml extracts from fresh beef liver, add catalyst, catalyst is manganous sulfate and manganese acetate, and effectively catalyst component is Mn
2+Ion, Mn
2+Ionic concn is controlled at 0.5mmol/L, puts into 40 ℃ of constant temperature waters shaking tables and transforms 30 hours;
With above-mentioned solution sampling, the point plate, the agent of some plate development is n-propyl alcohol and ammoniacal liquor, n-propyl alcohol and ammoniacal liquor mass percent are n-propyl alcohol: ammoniacal liquor=72: 28, after treating that the arginine spot transforms completely dissolve, add the urase that extracts in the fresh soya bean of 10ml/L, 33 ℃ of insulation reaction are after 50 minutes, drip the 6N hcl acidifying to PH5, decolorization filtering is after being concentrated into small amount of crystal and separating out, the alcohol that in concentrated solution, adds its 3 times of volumes, behind the backflow 1h, crystallisation by cooling gets the ornithine hydrochloride crude product after the centrifuging under 80 ℃ of conditions;
The ornithine hydrochloride crude product that obtains is got the concentration of 100g by weight 20% to be dissolved in the deionized water, be warming up to 75 ℃, the gac that adds crude product weight 9%, decolorization filtering, after being concentrated into small amount of crystal and separating out, in concentrated solution, add the alcohol of its 3 times of volumes, crystallisation by cooling, centrifuging goes to clean the residual mother liquor of plane of crystal with small amount of ethanol behind the mother liquor, gets finished product after the drying.
At specific embodiment described herein only is that the present invention's spirit and part experiment are illustrated.Those skilled in the art in the invention can make various modifications or replenish or adopt similar mode to substitute described specific embodiment, but can't depart from spirit of the present invention or surmount the defined scope of appended claims.
Claims (7)
1. the double enzyme coupling preparation of an ornithine hydrochloride, its step comprises:
A, the arginine dry product is dissolved in as substrate is made into conversion fluid in the deionized water, regulate between PH to 7～10 with 2N hydrochloric acid, be warming up to 38 ℃～45 ℃, add the beef liver enzyme liquid that from fresh beef liver, extracts, add catalyst, put into 38 ℃～45 ℃ water bath with thermostatic control shaking tables and transform 10～40 hours; Described catalyst is Mn
2+Ion, Mn
2+Ionic concn is controlled at 0.2～0.6mmol/L;
B, the solution sampling that steps A is obtained, the point plate, treat that the arginine spot transforms completely dissolve after, add the urase that extracts in the fresh soya bean of 5～20ml/L, 30 ℃～35 ℃ insulation reaction are after 40～70 minutes, drip the 6N hcl acidifying between PH5～6, decolorization filtering is after being concentrated into small amount of crystal and separating out, the alcohol that in concentrated solution, adds its 2～5 times of volumes, behind the backflow 1h, crystallisation by cooling gets the ornithine hydrochloride crude product after the centrifuging under 60 ℃～90 ℃ conditions; The agent of some plate development is n-propyl alcohol and ammoniacal liquor, and its mass percent is a n-propyl alcohol: ammoniacal liquor=65～75: 35～25;
C, ornithine hydrochloride dissolving crude product that step B is obtained are in deionized water, be warming up to 70 ℃～80 ℃, the gac that adds crude product weight 3～10%, decolorization filtering, after being concentrated into small amount of crystal and separating out, in concentrated solution, add the alcohol of its 2～5 times of volumes, crystallisation by cooling, centrifuging goes to clean the residual mother liquor of plane of crystal with small amount of ethanol behind the mother liquor, gets finished product after the drying.
2. the double enzyme coupling preparation of ornithine hydrochloride according to claim 1 is characterized in that, in the described steps A, the amount that adds beef liver enzyme liquid in the solution is with in every 1L conversion fluid, adds the beef liver enzyme liquid of 20ml～120ml.
3. the double enzyme coupling preparation of ornithine hydrochloride according to claim 1 is characterized in that, in the described steps A, the conversion fluid concentration of substrate is controlled at 50～300g/L.
4. the double enzyme coupling preparation of ornithine hydrochloride according to claim 1 is characterized in that, described catalyst is manganous sulfate or manganese acetate or Manganous chloride tetrahydrate.
5. the double enzyme coupling preparation of ornithine hydrochloride according to claim 1 is characterized in that, among the described step C, the ornithine hydrochloride crude product is dissolved in the deionized water by weight 10%～30% concentration.
6. according to the double enzyme coupling preparation of claim 1 or 2 or 3 or 4 or 5 described ornithine hydrochlorides, it is characterized in that, in the described steps A, the preparation method of beef liver enzyme liquid is: get that fresh beef liver is pulverized in freezing pulverizer, cell wall breaking, stand-by 4 ℃ of preservations then, the beef liver slurry is dissolved in the deionized water of 1～4 times of mass ratio, adds Mn
2+Ion, and concentration is controlled at 0.2～0.6mmol/L, heat be incubated 0.5～2 hour between 30～70 ℃ altogether, filters, collection filtrate, and filtrate is deposited in 4 ℃ of refrigerators stand-by behind high speed centrifugation immediately.
7. according to the double enzyme coupling preparation of claim 1 or 2 or 3 or 4 or 5 described ornithine hydrochlorides, it is characterized in that, the preparation method of urase is among the described step B: after fresh soya bean is pulverized, mix with the ethanol of 30% volume percent, soya bean and ethanol mass percent are 1: 2～3, shake 10 minutes down at 0～10 ℃ then, get supernatant liquor behind the high speed centrifugation, 30～35 ℃ of uses are incubated 40～70 minutes.
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| CN1590367A (en) * | 2003-09-04 | 2005-03-09 | 上海依福瑞实业有限公司 | Preparation method of L-ornithine hydrochloride |
| CN1594282A (en) * | 2004-07-15 | 2005-03-16 | 武汉武大弘元股份有限公司 | Method for producing L-ornithine hydrochloride |
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| CN1590367A (en) * | 2003-09-04 | 2005-03-09 | 上海依福瑞实业有限公司 | Preparation method of L-ornithine hydrochloride |
| CN1594282A (en) * | 2004-07-15 | 2005-03-16 | 武汉武大弘元股份有限公司 | Method for producing L-ornithine hydrochloride |
Non-Patent Citations (2)
| Title |
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| 万红贵等.L_鸟氨酸的制备及应用.《生物加工过程》.2008,第6卷(第1期),全文. * |
| 李海燕.化学法和酶法制备L_鸟氨酸工艺条件的探讨.《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》.2007,(第01期),全文. * |
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