CN105002228A - Method for preparing L-ornithine by using arginine as raw material - Google Patents
Method for preparing L-ornithine by using arginine as raw material Download PDFInfo
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Abstract
The present invention relates to a method for preparing L-ornithine by using arginine as a raw material. The method comprises the steps of: (1) acquisition of a substrate: subjecting an arginine ceramic membrane filtration liquid to ammonia 711 resin adsorption, eluting with 2.0N ammonia liquor, so as to obtain an arginine positive column purified liquid; and (2) washing the thalline containing arginase with normal saline once, centrifuging and collecting the thalline; transferring the thalline to a conversion liquid including a 0.3 mol/L carbonate buffer (Na2CO4 and NaHCO4 in the volume ratio of 7:3) and 0.1mg/ml Mn<2+>, 5% of L-arginine acid purification liquid, reacting for 24h (30 DEG C, 200r / min), wherein the conversion rate reaches 98%. The method provided by the invention uses arginine as the starting material for preparing L-ornithine, and has the advantages of high product purity, easy separation and low cost of finished product.
Description
Technical field
The present invention relates to a kind of is the method that L-Orn prepared by raw material with arginine, belongs to biological technical field.
Background technology
Ornithine is 1877, and Jie Fu finds in the hydrolysate of birds urine of feeding M-nitro benzoic acid, hence obtains one's name.It is the moiety of bacterial cell membrane and polypeptide antibiotics, can as amino acid transfusion raw material.The secretion of energy growth promoting effects hormone, to assist organism metabolism excess fat, with arginine with the use of having good fat-reducing effect.Mainly participate in uric acid circulation in animal body, the discharge of ammonia-state nitrogen in body is played an important role.Ornithine be maintain immunity system and liver function normally indispensable, can detoxify ammonia and help liver regeneration, except pharmaceutically as except reagent and injection liquid, also usual together with arginine as protecting the liver, keep fit, the raw material of toxinicide and so on medicament.Ornithine containing high density in skin and reticular tissue is highly profitable for healing and damaged tissue repair.Ornithine is synthesized by the arginine in body, and conversely, it is again the precursor of citrulline, proline(Pro) and L-glutamic acid.Ammonia can be converted into urea and glutaminase by ornithine, therefore supplements ornithine and can be the Substrate that detoxification pathways provides enzyme.When health manufactures urea, arginine just can be fallen by metabolism, and health transfers manufacture ornithine to.The experiment made of animal proves that ornithine and arginine can make health produce hormone that is more how anabolic, growth promoting effects, comprises Regular Insulin and HGH, promotes muscle growth.In addition, ornithine is also for preparing the fizz set up.
In vivo, ornithine can work in ornithine cycle (Krebs.Henseleit circulation).Ornithine cycle is divided into 4 steps, can be urea by the nitrogen cycling of elements in aspartic acid in organism and ammonia, because of but main ammonia excretion mode in organism.The major organs that nitrogen cycling of elements unnecessary in body is urea by animal use metabolism is liver, and the arginine in body is hydrolyzed under the katalysis of arginase, produces urea and ornithine.The ornithine produced after arginine hydrolysis, will react with carbamyl phosphate, and then generation citrulline, citrulline can react with aspartic acid further and generate argininosuccinic acid, and argininosuccinic acid finally can be broken down into the product such as fumaric acid and arginine further under the katalysis of enzyme.The first step due to this reaction is urea by conversion of Arginine, is reacted to and finally generates arginine again, go round and begin again, so this reaction is called ornithine cycle.Due to developing rapidly of current whole world amino acid industry, the scientist of various places has recognized the multiple biological function of L-Orn gradually, wherein Japan is the most outstanding, they just started to possess some special knowledge to L-Orn in the forties in 20th century, just have many articles to deliver afterwards at 10-20, and majority delivers with the form of patent.Produce the fermentation condition optimization etc. of bacterial strain, the seed selection of ornithine superior strain, the quantitative analysis of ornithine and fermentative Production-ornithine comprising ornithine, and deliver with the form of patent documentation.But but seldom hear the research for-ornithine during this at home.Until between 1998 to 2000, just report has the people such as Zhang Kexu, Panver and Chen Ning once to do research in the bacterial screening and fermentation of L-Orn, has several sections of paper publishings.Compared with external research, China's L-Orn industry is also in the starting stage, and the face studied is very narrow, and progress is comparatively slow, and only develop into the shake flask test stage at present, carry out suitability for industrialized production also needs us to carry out a large amount of test and studies.Abroad compare, the research of China to ornithine has very large gap, and research face is narrow, makes little progress, is only limitted to shake flask test, have got long long way to go from suitability for industrialized production.
Ornithine is arginic derivative, and have extensive use in fields such as medicine, health care, food and chemical industry, the market requirement increases year by year, there is no the report of scale operation both at home and abroad.Ornithine production method mainly contains chemical method, microbe fermentation method and enzyme transforming process.Chemical process is numerous and diverse, and environmental pollution is serious, and by product is many and not easily separated; Microbe fermentation method, owing to lacking the production bacterial strain of excellent property and the systematic study of production technique, limits development and the application of the method.Enzyme transforming process is that arginine generates ornithine under the single-minded effect of arginase, and product purity is high, is the ornithine production method of most potentiality to be exploited, and the arginine raw material of high-quality cheapness and the acquisition of saccharase are the keys of the method.
The production method of ornithine has 4 kinds: fermentation method: enzyme process; Chemical synthesis and proteolysis extraction method.
(1) fermentation method fermentation method has self amino acid needed ability of synthesis by microorganism, by process such as the matter mutagenesis to bacterial strain, select various auxotroph and Amino acid analogue resistant mutant, to remove feedback inhibition in Metabolism regulation and to check, reach certain amino acid whose object of excessive synthesis.Fermentation method comprises direct fermentation and adds Precurosor fermentation method.Amino Acid-producing Bacteria realizes the amino acid whose prerequisite of fermentative Production, in the foundation and improvement of amino acid fermentation, play very important effect.According to the inherited character of Amino Acid-producing Bacteria, four classes can be divided into: the first kind is directly use wild type strain by sugar and ammonium salt fermentative production amino acid, as L-glutamic acid; Equations of The Second Kind is auxotroph variant; 3rd class is Amino acid analogue resistant mutant; 4th class is double analog anti-the variant of auxotroph (or leakage type).Application of fermentation method produce ornithine because of the lower practical value of acid yield little.Fermentation method mainly utilizes the auxotrophic mutant of arginine (Arg-) or citrulline (Cit-) to accumulate ornithine.Takay asu etc. screens the mutant strain with mycophenolic acid on the basis of auxotrophic strain, and production peak can reach 50g/L; Li Huan etc. utilize fermented by mixed bacterium to produce ornithine, and output reaches about 40g/L.Fermentation method low raw-material cost, but complicated component in fermented liquid, later separation difficulty.
(2) chemical synthesis chemical method is prepared ornithine and can be divided into organic synthesis method and arginine hydrolysis method two kinds according to chemical reaction mechanism.This method adopts basic chemical industry Material synthesis ornithine, and raw material sources are extensive and cost is low.But this method is through multi-step chemical Reactive Synthesis, yield is on the low side; Prussic acid in its raw material is hypertoxic carcinogens, should avoid in the industrial production using as far as possible.The ornithine that chemical method synthesizes is DL type mixture, and the chiral separation of product is also to the process adds difficulty and cost, and these aspects all become the major defect place of this method.Chemical method utilizes weak base to be hydrolyzed arginine to obtain ornithine and a small amount of citrulline.It is DL-type racemic modification that chemical method produces ornithine product, and separation difficulty, production process is wayward, is now eliminated.
(3) proteolysis extraction method is with protein such as hair, blood meal and useless silks for raw material, becomes multiple amino acids mixture by acid, alkali or enzymic hydrolysis, obtains various amino acid whose method be called hydrolysis method through separation and purification.Along with the progress of amino acids production technology.Extract this ancient production method of amino acid by proteolysis method to be severely affected, traditional hydrolysis method is produced L-glutamic acid and has been lost its value existed.
(4) enzyme process Production by Enzymes ornithine is exactly utilize the arginase in animals and plants or microbe to make catalyzer, and hydrolysis arginine, generates ornithine and urea; Production by Enzymes amino acid has that technique is simple, the cycle is short, it is low to consume energy, specificity is strong, yield high.L-arginine enzyme is extensively present in traditional enzyme process in the liver of animal, plant, yeast and bacterium and prepares ornithine method, and adopting with beef liver is enzyme source, extracts L-arginine enzyme from beef liver, with finished product arginine for synthesis substrate conversion ornithine.Production by Enzymes ornithine specificity is strong, and reaction conditions is gentle, and avoid adopting the noxious chemicals such as prussic acid, substrate conversion efficiency is high simultaneously, and product is purer, is conducive to later separation, but the source of arginase not easily, and the arginic cost of substrate is also higher.The synthesis substrate and the enzyme source that how to obtain cheapness are the keys that this method success is applied.
The present invention is with the arginine refined solution of fermentative Production for raw material, and the arginase adopting pseudomonas to produce transforms produces ornithine, and raw material is easy to get, and enzyme is with low cost, in conjunction with enzyme immobilization technology, can save ornithine production cost, produce high-quality product.Applicant is devoted to the research of fermentative Production arginine and derivative gordian technique thereof for many years, has the arginine production line of annual output 500 tons.In view of the present situation that domestic and international market is sought-after, applicant studies ornithine high-performance bio manufacturing technology and industrialization, and the creationary microbial enzyme method that proposes produces bird ammonia.Rely on existing arginine technology and raw material advantage, the high efficient strain of the arginase of ornithine is produced in seed selection, is raw material obtains ornithine by enzymatic conversion with arginine.Product purity height of the present invention is easily separated, overcomes raw material and the expensive inferior position not easily obtained of enzyme simultaneously, has the prospect of large-scale production.
Summary of the invention
A strain is the object of the present invention is to provide to produce the bacterial strain MQO-154 of arginase, the present invention is through carrying out ultraviolet mutagenesis and chemomorphosis to bacillus thuringiensis (soil separation), screen the bacterial strain that a strain arginase activities is higher, after separation and purification, obtain bacterial strain MQO-154, its deposit number is: CGMCCNo.10728; Depositary institution is: China Committee for Culture Collection of Microorganisms's common micro-organisms center; Preservation date is: on April 21st, 2015; Preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
The biological nature of bacterial strain MQO-154 is: this bacterial strain cultivates 24h on slant medium, become oyster white bacterium colony, moistening, mattness, lawn is thick and opaque, bacterium colony is comparatively large, and the irregular thalline in edge is shaft-like, and cell is about 3-4 μm, diameter 1-2 μm, there is endogenous spore, can not move, aerobic or amphimicrobian.The Classification And Nomenclature of bacterial strain MQO-154 is bacillus thuringiensis (Bacillusthuringensis).
Utilize bacterial strain MQO-154 to prepare the method for arginase, step is as follows:
(1) slant culture: be aseptically inoculated on slant medium by bacterial strain MQO-154 and cultivate, culture temperature is 33 DEG C, incubation time is 24h;
Slant medium: glucose 25g, fish peptone 15g, KH
2pO
40.6g, MgSO
47H
2o 0.5g, K
2hPO
41.5g, agar 16g, add pure water to 1L, adjusts pH most 7.0, sterilising temp 115 DEG C, 15min with NaOH.
(2) seed enlarged culturing: picking colony from the slant medium of step (1), thin up obtains phage solution, and the concentration after dilution is 3 × 10
5-5 × 10
5individual/mL, phage solution is inoculated on seed culture medium and carries out enlarged culturing, inoculum size is 10% (v/v), and the temperature of enlarged culturing is 30 DEG C, and incubation time is 24h, 0-10h, shaking speed 100r/min, 10-20h, shaking speed 120r/min, 20-24h, shaking speed 100r/min.
Seed culture medium: glucose 25g, ammonium acetate 9g, KH
2p0
40.59g, K
2hPO
41.5g, MgSO
47H
20 0.5g, FeSO
40.05g, MnSO
40.05g, VB
1hCl 0.005g, agar 16g, add pure water to 1L, adjusts pH to 7.0, sterilising temp 115 DEG C, 15min with NaOH.
(3) fermentation culture: be inoculated in fermentation broth by the thalline after enlarged culturing, inoculum size is 10% (v/v), and during inoculation, the concentration of phage solution is 3 × 10
5-5 × 10
5individual/mL, in 30 DEG C, the shaking table top fermentation 24h of 170r/min, obtains the fermentation culture containing arginine kinase;
Fermention medium: glucose 25g, fish peptone 15g, corn steep liquor 10g, yeast extract paste 1g, KH
2pO
40.5g, MgSO
47H
2o 0.5g, FeSO
40.05g, MnSO
40.05g, VB
1hCl 0.005g, is made into 1L solution with tap water, adjusts pH to 7.0, sterilising temp 121 DEG C, sterilization time 20min with NaOH.
(4) 30L fermentor cultivation: be inoculated in the substratum of 30L fermentor tank by the fermented liquid after fermentation culture in step (3), inoculum size is 10% (v/v), and during inoculation, the concentration of phage solution is 3 × 10
5-5 × 10
5individual/mL, controlling tank pressure is by force 0.05MPa, cultivate 28 hours under 33 DEG C of conditions, mixing speed is 600-800rpm, and air quantity is 1:0.8, and control dissolved oxygen amount is 30-40%, omnidistance is 6.7-7.0 with aseptic saturated ammonia water management pH, it is 1% that sugar control residual sugar is mended in midway, and putting tank residual sugar is 0%, must contain the thalline of arginase after fermentation.
The present invention utilizes arginase, and be that L-Orn prepared by raw material with arginine, step is as follows:
(1) acquisition of conversion of substrate: by arginine ceramic membrane filter liquid through 711 ammonia type resin absorption, with 2.0N ammoniacal liquor wash-out, obtains arginine sun column purification liquid
(2) by the thalline containing arginase, with brine 1 time, collected after centrifugation thalline.Transferred to by thalline in conversion fluid, conversion fluid comprises 0.3mol/L carbonate buffer solution (Na
2cO
4with NaHCO
4volume ratio is 7:3) and the Mn of 0.1mg/ml
2+, add the L-arginine refined solution of 5%, reaction 24h (30 DEG C, 200r/min); Transformation efficiency can reach 98%.
(3) separation and purification:
Ion-exchange: conversion fluid is limpid to effluent liquid with water backwash resin after ammonia type 732 resin absorption, with 1.0N ammoniacal liquor wash-out, elutriant obtains L-Orn refined solution after stream D335-OH type resin removing anionic impurity, and yield reaches 95%;
Nanofiltration: adopt 800 molecular weight nanofiltration membrane L-Orn refined solutions to obtain L-Orn nanofiltration clear liquid, after deionization moisture 3 cleaning concentrates (nanofiltration clear liquid) of 3 times of concentrated solution volumes, discard concentrated solution, collect the nanofiltration dialyzate containing L-Orn product,, yield reaches 98%;
Adjust pH, decolouring: the pH to 4.5 regulating L-Orn nanofiltration clear liquid with hydrochloric acid, decolour with gac, the consumption of gac is 0.5% (massfraction) of L-Orn nanofiltration clear liquid, bleaching temperature is 50 DEG C, bleaching time is 30min, obtain destainer with 0.22 μm of membrane filtration, destainer transmittance >=99%, yield reaches 99%;
Reverse osmosis concentration: L-Orn destainer is concentrated into original volume half through reverse osmosis membrane and obtains the pre-concentration liquid after L-Orn deamination, yield reaches 99%;
Condensing crystal: by pre-concentration liquid in vacuum tightness >=0.095, is concentrated into the concentrated solution of degree of enrichment content >=80% under vaporization temperature 50 DEG C of conditions, put by concentrated solution to 5 DEG C of refrigerators insulation 2h and obtain L-Orn crystallization;
The acquisition of L-ornithine hydrochloride: the ornithine of crystallization is obtained ornithine hydrochloride wet product 3 times with absolute ethanol washing after suction filtration, and yield reaches 85%;
Dry: ornithine hydrochloride wet product to be put into 55 DEG C of vacuum drying ovens dry 6h under >=-0.098 vacuum tightness and obtain ornithine hydrochloride finished product.
The present invention compared with prior art has the following advantages:
(1) screen a strain MQO-154, solve enzymatic translation technics enzyme source and the high restriction of enzyme cost;
(2) utilize arginase, with arginine refined solution for L-Orn prepared by raw material, product purity is high, easily separated, and cost of goods manufactured is low;
(3) adopt the advanced treatment process such as membrane sepn, nanofiltration decolouring, membrane concentration to save energy consumption, save the consumption of gac, decrease the pollution to environment, reduce production cost, improve quality product.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The bacterial strain MQO-154 of arginase is produced in embodiment 1 one strain,
The present invention is through carrying out ultraviolet mutagenesis and chemomorphosis to bacillus thuringiensis, and screen the bacterial strain that a strain enzymic activity is higher, obtain bacterial strain MQO-154 after separation and purification, its deposit number is: CGMCCNo.10728; Depositary institution is: China Committee for Culture Collection of Microorganisms's common micro-organisms center; Preservation date is: on April 21st, 2015; Preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
The biological nature of bacterial strain MQO-154 is: bacterial strain MQO-154 is inoculated on slant medium and carries out cultivation 24h, become oyster white bacterium colony, moistening, mattness, lawn is thick and opaque, bacterium colony is comparatively large, and the irregular thalline in edge is shaft-like, and cell is about 3-4 μm, diameter 1-2 μm, there is endogenous spore, can not move, aerobic or amphimicrobian.
Embodiment 2 utilizes bacterial strain MQO-154 to prepare the method for arginase, and step is as follows:
(1) slant culture: be aseptically inoculated on slant medium by bacterial strain MQO-154 and cultivate, culture temperature is 33 DEG C, incubation time is 24h;
Slant medium: glucose 25g, fish peptone 15g, KH
2pO
40.6g, MgSO
47H
20 0.5g, K
2hPO
41.5g, agar 16g, add pure water to 1L, adjusts pH most 7.0, sterilising temp 115 DEG C, 15min with NaOH.
(2) seed enlarged culturing: picking colony from the slant medium of step (1), thin up obtains phage solution, and the concentration after dilution is 3 × 10
5-5 × 10
5individual/mL, phage solution is inoculated on seed culture medium and carries out enlarged culturing, inoculum size is 10% (v/v), and the temperature of enlarged culturing is 30 DEG C, and incubation time is 24h, 0-10h, shaking speed 100r/min, 10-20h, shaking speed 120r/min, 20-24h, shaking speed 100r/min.
Seed culture medium: glucose 25g, ammonium acetate 9g, KH
2pO
40.59g, K
2hPO
41.5g, MgSO
47H
20 0.5g, FeSO
40.05g, MnSO
40.05g, VB
1hCl 0.005g, agar 16g, add pure water to 1L, adjusts pH to 7.0, sterilising temp 115 DEG C, 15min with NaOH.
(3) fermentation culture: be inoculated in fermentation broth by the thalline after enlarged culturing, inoculum size is 10% (v/v), and during inoculation, the concentration of phage solution is 3 × 10
5-5 × 10
5individual/mL, in 30 DEG C, the shaking table top fermentation 24h of 170r/min, obtains the fermentation culture containing arginine kinase;
Fermention medium: glucose 25g, fish peptone 15g, corn steep liquor 10g, yeast extract paste 1g, KH
2pO
40.5g, MgSO
47H
2o 0.5g, FeSO
40.05g, MnSO
40.05g, VB
1hCl 0.005g, is made into 1L solution with tap water, adjusts pH to 7.0, sterilising temp 121 DEG C, sterilization time 20min with NaOH.
(4) 30L fermentor cultivation: be inoculated in the substratum of 30L fermentor tank by the fermented liquid after fermentation culture in step (3), inoculum size is 10% (v/v), and during inoculation, the concentration of phage solution is 3 × 10
5-5 × 10
5individual/mL, controlling tank pressure is by force 0.05MPa, cultivate 28 hours under 35 DEG C of conditions, mixing speed is 600-800rpm, and air quantity is 1:0.8, and control dissolved oxygen amount is 30-40%, omnidistance is 6.7-7.0 with aseptic saturated ammonia water management pH, it is 1% that sugar control residual sugar is mended in midway, and putting tank residual sugar is 0%, must contain the thalline of arginase after fermentation.
Embodiment 3 the present invention utilizes arginase, and with arginine refined solution for L-Orn prepared by raw material, step is as follows:
(1) acquisition of substrate: by arginine ceramic membrane filter liquid through 711 ammonia type resin absorption, with 2.0N ammoniacal liquor wash-out, obtains arginine sun column purification liquid;
(2) transform and cultivate: by the thalline containing arginase, with brine 1 time, collected after centrifugation thalline.Transferred to by thalline in conversion fluid, conversion fluid comprises 0.3mol/L carbonate buffer solution (Na
2cO
4with NaHCO
4volume ratio is 7:3) and the Mn of 0.1mg/ml
2+, add the L-arginine refined solution of 5%, reaction 24h (30 DEG C, 200r/min); Transformation efficiency can reach 98%.
The separation and purification of L-Orn prepared by embodiment 4 the present invention
(1) ion-exchange: L-Orn conversion fluid embodiment 3 obtained is limpid to effluent liquid with water backwash resin after ammonia type 732 resin absorption, with 1.0N ammoniacal liquor wash-out, elutriant obtains ornithine refined solution after stream D335-OH type resin removing anionic impurity, and yield reaches 95%;
(2) nanofiltration: adopt 800 molecular weight nanofiltration membrane ornithine refined solutions to obtain ornithine nanofiltration clear liquid, after deionization moisture 3 cleaning concentrates (nanofiltration clear liquid) of 3 times of concentrated solution volumes, discard concentrated solution, collect the nanofiltration dialyzate containing L-Orn product,, yield reaches 98%;
(3) pH, decolouring is adjusted: the pH to 4.5 regulating ornithine nanofiltration clear liquid with hydrochloric acid, decolour with gac, the consumption of gac is 0.5% (massfraction) of ornithine nanofiltration clear liquid, bleaching temperature is 50 DEG C, bleaching time is 30min, obtain destainer with 0.22 μm of membrane filtration, destainer transmittance >=99%, yield reaches 99%;
(4) reverse osmosis concentration: ornithine destainer is concentrated into original volume half through reverse osmosis membrane and obtains the pre-concentration liquid after ornithine deamination, yield reaches 99%;
(5) condensing crystal: by pre-concentration liquid in vacuum tightness >=0.095, is concentrated into the concentrated solution of degree of enrichment content >=80% under vaporization temperature 50 DEG C of conditions, put by concentrated solution to 5 DEG C of refrigerators insulation 2h and obtain ornithine crystallization.
The determination of conversion condition in test example 1 the present invention
(1) determination of pH value, when other condition is constant, when reaction pH is 9.0-10.0, arginine complete reaction, when pH is 10.0, arginine molar yield significantly improves.
(2) determination of transformation time, when other condition is constant, react that to test color for slope cause for gossip during 10h be dark rose, show still there is a large amount of L-arginine hydrochlorides, reaction is not exclusively; Along with the prolongation in reaction times, slope cause for gossip is tested color and is being shoaled, and during 15h, L-arginine hydrochloride reacts completely, but is not also converted into L-Orn completely, and productive rate is still very low; During 24h, the existence can not checking L-arginine hydrochloride is tested in slope cause for gossip, and productive rate is very high.But along with the further prolongation of time, the productive rate of L-Orn considerable change does not occur, when 24h is described, reaction completely, and therefore 24h is the better reaction times.
(3) determination of temperature of reaction, when other condition is constant, when temperature of reaction is 36 DEG C, productive rate is the highest, illustrates that 36 DEG C can make the catalytic activity of L-arginine enzyme play best.
(4) determination of different concentration of substrate, when initial substrate arginine concentrations is 5%, still can remain on a higher level through the conversion arginine molar yield of about 24h; But when the arginic concentration of initial substrate reaches 7% and 10% time, initial arginine concentrations is the conversion fluid of 5%, and in its conversion fluid, the concentration of product ornithine only has very little raising.
Show that arginase transforms the best enzymatic conversion condition of ornithine by above-mentioned serial experiment as follows: reaction pH is 9.5, temperature of reaction 36 DEG C, reaction times 24h, substrate arginine concentrations 5%.
The mensuration of ornithine concentration, arginine molar yield in test example 2 the present invention
(1) mensuration of ornithine concentration
Ornithine and triketohydrindene hydrate can react and generate red material under pH is the condition of 11, and this material has maximum light absorption at 515nm place, is reacted by the ornithine and acid ninhydrine preparing different concentration known, measures the light absorption value drawing standard curve at its 515nm place.Ornithine concentration (mol/mL) can be calculated by typical curve.
(2) mensuration of arginine molar yield
Thalline is inoculated in 50mL liquid nutrient medium and cultivates 24h in 33 DEG C.The centrifugal 30min of 4000r/min at cultured fermented liquid 4 DEG C, abandoning supernatant.Thalline is transferred in 10mL carbonate buffer solution, adds 0.15g arginine, at 30 DEG C, transform 24h.The centrifugal 10min of 6000r/min, measures the ornithine concentration in supernatant liquor, and calculates the arginic molar yield y of substrate, represent the vigor of arginase with this.It is 98% that the present invention records arginine molar yield.
y＝nA/nB
Wherein nA is the amount of substance (mol) of ornithine in conversion fluid; NB is arginic amount of substance (mol) original in conversion fluid.
The inspection of the ornithine hydrochloride obtained in test example 4 the present invention
Pilot product is through the third party inspection of Pony Testing International Group, and indices meets US Federal Communication Committee, L-ornithine hydrochloride USP standard, and main performance index is shown in Table 1:
Table 1
Claims (9)
1. be the method that L-Orn prepared by raw material with arginine, it is characterized in that, said method comprising the steps of:
(1) acquisition of substrate: by arginine ceramic membrane filter liquid through 711 ammonia type resin absorption, with 2.0N ammoniacal liquor wash-out, obtains arginine sun column purification liquid;
(2) transform and cultivate: by the thalline containing arginase, with brine 1 time, collected after centrifugation thalline.Transferred to by thalline in conversion fluid, conversion fluid comprises 0.3mol/L carbonate buffer solution (Na
2cO
4with NaHCO
4volume ratio is 7:3) and the Mn of 0.1mg/ml
2+, add the L-arginine refined solution of 5%, reaction 24h (30 DEG C, 200r/min); L-Orn can be obtained.
2. method according to claim 1, is characterized in that, described in described step (2), the preparation method of arginase is as follows:
(1) slant culture: be aseptically inoculated on slant medium by bacterial strain MQO-154 and cultivate, culture temperature is 33 DEG C, incubation time is 24h;
Slant medium: glucose 25g, fish peptone 15g, KH
2p0
40.6g, MgS0
47H
20 0.5g, K
2hP0
41.5g, agar 16g, add pure water to 1L, adjusts pH most 7.0, sterilising temp 115 DEG C, 15 minutes with NaOH.
(2) seed enlarged culturing: picking colony from the slant medium of step (1), thin up obtains phage solution, and the concentration after dilution is 3 × 10
5-5 × 10
5individual/mL, phage solution is inoculated on seed culture medium and carries out enlarged culturing, inoculum size is 10% (v/v), and the temperature of enlarged culturing is 30 DEG C, and incubation time is 24h, 0-10h, shaking speed 100r/min, 10-20h, shaking speed 120r/min, 20-24h, shaking speed 100r/min.
Seed culture medium: glucose 25g, ammonium acetate 9g, KH
2p0
40.59g, K
2hP0
41.5g, MgS0
47H
20 O.5g, ferrous sulfate 0.05g, iron manganous sulfate 0.05g, VB
1hCl 0.005g, agar 16g, add pure water to 1L, adjusts pH to 7.0, sterilising temp 115 DEG C, 15 minutes with NaOH.
(3) fermentation culture: be inoculated in fermentation broth by the thalline after enlarged culturing, inoculum size is 10% (v/v), and during inoculation, the concentration of phage solution is 3 × 10
5-5 × 10
5individual/mL, in 30 DEG C, the shaking table top fermentation 24h of 170r/min, obtains the fermentation culture containing arginine kinase;
Fermention medium: glucose 25g, fish peptone 15g, corn steep liquor 10g, yeast extract paste 1g, KH
2p0
40.5g, MgS0
47H
2o 0.5g, FeSO
40.05g, MnSO
40.05g, VB
1hCl 0.005g, is made into 1L solution with tap water, adjusts pH to 7.0, sterilising temp 121 DEG C, sterilization time 20 minutes with NaOH.
(4) 30L fermentor cultivation: be inoculated in the substratum of 30L fermentor tank by the fermented liquid after fermentation culture in step (3), inoculum size is 10% (v/v), and during inoculation, the concentration of phage solution is 3 × 10
5-5 × 10
5individual/mL, controlling tank pressure is by force 0.05MPa, cultivate 28 hours under 33 DEG C of conditions, mixing speed is 600-800rpm, and air quantity is 1:0.8, and control dissolved oxygen amount is 30-40%, omnidistance is 6.7-7.0 with aseptic saturated ammonia water management pH, it is 1% that sugar control residual sugar is mended in midway, and putting tank residual sugar is 0%, must contain the thalline of arginase after fermentation.
3. method according to claim 2, is characterized in that, consisting of of slant medium described in described step (1):
Glucose 25g, fish peptone 15g, KH
2pO
40.6g, MgSO
47H
200.5g, K
2hPO
41.5g, agar 16g, add pure water to 1L.
4. method according to claim 3, is characterized in that, slant medium NaOH described in described step (1) adjusts pH most 7.0, sterilising temp 115 DEG C, 15min.
5. method according to claim 2, is characterized in that, consisting of of seed culture medium described in described step (2):
Glucose 25g, ammonium acetate 9g, KH
2p0
40.59g, K
2hPO
41.5g, MgSO
47H
200.5g, FeSO
40.05g, MnSO
40.05g, VB
1hCl 0.005g, agar 16g, add pure water to 1L, adjusts pH to 7.0, sterilising temp 115 DEG C, 15min with NaOH.
6. method according to claim 5, is characterized in that, seed culture medium NaOH described in described step (2) adjusts pH to 7.0, sterilising temp 115 DEG C, 15min.
7. method according to claim 2, is characterized in that, consisting of of fermention medium described in described step (3):
Glucose 25g, fish peptone 15g, corn steep liquor 10g, yeast extract paste 1g, KH
2pO
40.5g, MgSO
47H
2o 0.5g, FeSO
40.05g, MnSO
40.05g, VB
1hCl 0.005g, is made into 1L solution with tap water, adjusts pH to 7.0, sterilising temp 121 DEG C, sterilization time 20min with NaOH.
8. method according to claim 7, is characterized in that, fermention medium NaOH described in described step (3) adjusts pH to 7.0, sterilising temp 121 DEG C, sterilization time 20min.
9. method according to claim 2, is characterized in that, the deposit number of described bacterial strain MQO-154 is: CGMCC No.10728; Depositary institution is: China Committee for Culture Collection of Microorganisms's common micro-organisms center; Preservation date is: on April 21st, 2015; Preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105886566A (en) * | 2016-05-10 | 2016-08-24 | 宁波市镇海海德生化科技有限责任公司 | Production method for L-ornithine through microorganism enzymatic conversion |
| CN106318888A (en) * | 2016-08-27 | 2017-01-11 | 山东民强生物科技股份有限公司 | Strain MQO-160 for generating L-glutamate oxidase and application of strain |
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| CN103667381A (en) * | 2013-12-24 | 2014-03-26 | 山东民强生物科技股份有限公司 | Method for improving yield of arginine |
| CN103695487A (en) * | 2013-12-24 | 2014-04-02 | 山东民强生物科技股份有限公司 | Process for producing arginine by microbial fermentation |
| CN103695489A (en) * | 2013-12-24 | 2014-04-02 | 山东民强生物科技股份有限公司 | Arginine refining technology |
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| CN103667381A (en) * | 2013-12-24 | 2014-03-26 | 山东民强生物科技股份有限公司 | Method for improving yield of arginine |
| CN103695487A (en) * | 2013-12-24 | 2014-04-02 | 山东民强生物科技股份有限公司 | Process for producing arginine by microbial fermentation |
| CN103695489A (en) * | 2013-12-24 | 2014-04-02 | 山东民强生物科技股份有限公司 | Arginine refining technology |
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Cited By (2)
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| CN105886566A (en) * | 2016-05-10 | 2016-08-24 | 宁波市镇海海德生化科技有限责任公司 | Production method for L-ornithine through microorganism enzymatic conversion |
| CN106318888A (en) * | 2016-08-27 | 2017-01-11 | 山东民强生物科技股份有限公司 | Strain MQO-160 for generating L-glutamate oxidase and application of strain |
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