CN107916282A - A kind of method that bioanalysis prepares L citrulling and L ornithines - Google Patents
A kind of method that bioanalysis prepares L citrulling and L ornithines Download PDFInfo
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Abstract
The present invention provides a kind of method that bioanalysis prepares L citrulling and L ornithines.The method carries out as follows：1st, the cultivation and fermentation of strain：Total carbon source mass concentration is 1 20g/L in culture medium, and total nitrogen source mass concentration is 1 20g/L, and pH is controlled 7.2；2nd, L citrulling is prepared；3rd, L ornithines are prepared.The present invention prepares two kinds of products of L citrulling and L ornithines respectively using thalline made from one time fermentation and zymotic fluid, resource is made full use of, process optimization is reasonable, and it is gentle with reaction condition that bioanalysis prepares L citrulling and L ornithines, technological process is simple, is adapted to industrialization large-scale production.
Description
Technical field
The present invention relates to bioengineering field, and in particular to a kind of bioanalysis prepares the side of L-citrulline and L-Orn
Method.
Background technology
L-citrulline is a kind of important nonprotein amino acid, is gained the name " melon because first time is isolated from watermelon
Propylhomoserin ", L-citrulline are the important mesostates of urea cycle in human body, important for maintaining human body blood ammonia balance to have
Effect, L-citrulline can absorb and remove harmful free radical, so as to play the role of oxidation resistant, therefore can be used as anti-
Aging, the health products for improving immunity.In field of medicaments, L-citrulline is to preventing prostatic disorders, including prostatitis, forefront
The effect of gland cancer is obvious.It has recently been demonstrated that L-citrulline is in the metabolism of nitric oxide for maintaining cardiovascular normal function
Play an important role.
The preparation method of L-citrulline mainly has chemical synthesis, microbe fermentation method and enzyme transforming process at present
Chemical method synthesis L-citrulline reaction process is complicated, accessory substance is more, and low yield, purity is not high, loses competitiveness substantially.
Production by Microorganism Fermentation L-citrulline is mainly to obtain L-citrulline by the method for mutagenesis or genetic engineering
Superior strain, L-citrulline is synthesized by starting material of carbon sources such as cheap glucose.The culture of consonance fermentation company of Japan can
The arthrobacter paraffineus of carbon source is used as by the use of hydro carbonsArthrobacter paraffineusThe mutant strain fermentation of researches on arginine requirement type
L-citrulline is produced, fermentation 96 accumulates the L-citrulline of 7.1 g/L when small.But Production by Microorganism Fermentation L-citrulline is deposited
The problems such as in low output, fermentation period is grown, often adjoint accessory substance generation, and later separation is difficult, cannot still meet to industrialize
It is required that.
The advantages of enzymatic conversion method production L-citrulline is that enzyme stereoselectivity is strong, and production concentration is high, and purification step is few, production
Cost is low, is the main method of industrial production L-citrulline at present.Beijing University of Chemical Technology's Yao's seapeak etc. is catalyzed with streptococcus fecalis
L-arginine, 25 interior when small obtain 92.72 g/L L-citrullines；Southern Yangtze University's bright grade of will of having mercy on is entirely thin with recombinant corynebacterium crematum
Born of the same parents are catalyst, and L-citrulline is produced by substrate of L-arginine, using the strategy for adding L-arginine stage by stage, conversion 48
Hour, L-citrulline yield reaches 301.4 g/L.
L-Orn is a kind of non-protein amino acid, is mainly present in liver with free state in human body.L-Orn is most
Main physiological function is to participate in urea cycle, there is powerful ammonia function of detoxification, therefore is widely used in treating liver property brain
Medicament research and development in terms of disease, hepatitis and protection liver.In addition, L-Orn also has a lot of other biological functions, life is such as stimulated
Long hormone secretion, improve sleep, fatigue-relieving, moreover it is possible to improves flavour of food products as food additives.
According to the literature, the preparation method of L-Orn mainly has chemical method, fermentation method and enzyme transforming process.
Ornithine reactions steps are more, yield is low for chemical method synthesis, and what is obtained is raceme, it is necessary to split, and is not suitable for greatly
Large-scale production L-Orn product.George et al. utilizes methacrylaldehyde, hydrogen cyanide, ammonia, CO2For Material synthesis L-Orn,
But effect is undesirable, the DL- ornithines that reactions steps are more, product is racemization are mainly shown as.Rivard et al. utilizes Ba (OH)2
Hydrolyze L-arginine, through hydrolysis, desalination and it is dry after, obtain L-ornithine hydrochloride, there are racemization phenomenon, production in hydrolytic process
Product optical purity is not high.
Fermentation method is favored because raw material is cheap be subject to researcher, has made great progress in recent years, is mainly manifested in production
In terms of the selection and breeding of bacterial strain and the transformation of production technology.Japanese researchers just utilize ultraviolet, cyanide early in the 1950s
Etc. traditional induced-mutation technique, Corynebacterium glutamicum citrulling Auxotrophie mutant is obtained, successfully have accumulated L-Orn.Wan Hong
It is expensive et al. to disclose utilizationCorynebacterium glutamicum3663 fermentations of CGMCC No. prepare the side of L-Orn
Method, L-Ornithine Content in Fermentation Broth accumulation reach 35~45 g/L.But fermentation method also has shortcoming, the unstability of strain, hair
Ferment technique state modulator, all there are urgent problem to be solved for the process such as separation and Extraction of L-Ornithine Content in Fermentation Broth.
Enzymatic conversion method is because with specificity is strong, high catalytic efficiency, reaction condition is gentle, reaction time is short, product is easy to point
From the advantages that and be paid more and more attention, be low cost production L-Orn effective ways.High wisdom et al. utilizes recombination expression
Arginase gene engineering bacteria Enzymatic Resolution DL- arginine prepare D-Arg and L-Orn, product L-Orn hydrochloric acid
Purity salt more than 95%.Jiao's celebrating ability et al. uses immobilized cell enzymatic hydrolysis L-arginine, substrate conversion efficiency more than 95%, L- birds
Propylhomoserin yield more than 80%.
Enzymatic Conversion of L-Citrulline process needs arginine deiminase.At present, fermented and made using wild-type strain
Need to add a small amount of L-arginine induction during standby arginine deiminase, L-arginine, which is decomposed after metabolism, becomes L- birds
Propylhomoserin, accumulates in zymotic fluid.Since the L-arginine added in Induction Process is less, the L- bird ammonia that is accumulated in zymotic fluid
Acid concentration is relatively low, isolates and purifies that the cost is relatively high to it, and zymotic fluid is usually taken as wastewater treatment, has not only polluted environment but also has wasted
Resource.In addition, higher, the arginine deiminase pair that does substrate Enzymatic Conversion of L-Citrulline cost using L-arginine sterling
The purity requirement of substrate L-arginine is not high, therefore produces the zymotic fluid of L-arginine using fermentation method or contain L-arginine
The aqueous solution that ion-exchanging eluent and L-arginine crude product are made into can drop significantly for substrate Enzymatic Conversion of L-Citrulline
Low production cost.
In conclusion Enzymatic Conversion of L-Citrulline and L-Orn have a greater advantage, bottom during enzymatic conversion method
The cost of thing L-arginine and enzyme preparation height is that can this method realize industrialized key factor.Therefore, it is rich to find source
The L-arginine raw material of industry rich, of low cost, the technique for establishing low-cost fermentation preparation enzyme preparation are to realize that enzymatic conversion method is given birth to
Produce the important channel of L-citrulline and L-Orn.
The content of the invention
The object of the present invention is to provide a kind of efficient, low-cost bio method to prepare L-citrulline and the method for L-Orn.
The design philosophy of the present invention is stream plus L-arginine during Enterococcus faecalis fermentation, and induction produces arginine and takes off Asia
Amine enzyme, while by-product L-Orn, the somatic cells with arginine deiminase activity obtained after zymotic fluid centrifugation are used for
Conversion of substrate L-arginine prepares L-citrulline, and fermented liquid supernatant utilizes the isolated L-Orn of ion exchange resin.
The present invention improves stream plus L-arginine concentration during wild type strain fermentation prepares arginine deiminase,
While induction produces arginine deiminase, realize that L-Ornithine Content in Fermentation Broth high concentration accumulates, finally handed over using ion
Change the isolated L-Orn of resin；The arginine deiminase that induction produces with the zymotic fluid of L-arginine or contains L- essence ammonia
The aqueous solution that the ion-exchanging eluent or L-arginine crude product of acid are made into is substrate Enzymatic Conversion of L-Citrulline, is dropped significantly
Low production cost.One time fermentation of the present invention and conversion can obtain two kinds of high value added products of L-Orn and L-citrulline, make
Production cost is greatly lowered, and has great prominent effect and industrial value.
To achieve the above object, the present invention reaches by the following technical programs：
A kind of bioanalysis prepares L-citrulline and the method for L-Orn, it is characterised in that：The method is made of following steps：
A kind of bioanalysis prepares L-citrulline and the method for L-Orn, it is characterised in that：The method carries out as follows：
1st, the cultivation and fermentation of strain：Enterococcus faecalis is cultivated in the medium, is fermented, culture medium includes following component：Corn
Starch 0.5-2.0g/ml, protein hydrolysate 0.5g/ml, beef extract 1.0-2.0 g/ml, maltose 0.5-1.0 g/ml, lactose
0.5-1.0 g/ml 、NaCl 0.5 g/ml、KH2PO4 0.04-0.5 g/ml、MgSO4•7H2O 0.02-0.05 g/ml, Portugal
1.0 g/ml of grape sugar, peptone 0.5-1.0 g/ml, 0.5 g/ml of sucrose, 1.0 g/ml of fructose, 3 g/ml of soya-bean cake hydrolyzate,
Yeast extract 0.5-1.0 g/ml, pH controls in culture medium are 7.2, and total carbon source mass concentration is 1-20g/L in the medium,
Total nitrogen source mass concentration be 1-20g/L, by the inoculum concentration kind of seed liquor 1-2% by volume in 300L fermentation tanks, is fermented canned
Liquid measure is 200L, and throughput 12m3/h, tank pressure is 0.05MPa, and rotating speed 100r/min, cultivation temperature is 35 DEG C, is fermented
Cheng Zhongliu adds L-arginine solution, during fermentation pH controls in 6.8-7.2, fermentation time for 15-24 it is small when, centrifuged after the completion of fermentation
Zymotic fluid, obtains the somatic cells with arginine depickling imines enzymatic activity and the fermented supernatant fluid containing L-Orn；
2nd, L-citrulline is prepared：Substrate solution is prepared with the zymotic fluid of L-arginine, the pH value of substrate solution is 6.0-8.0, is added
Enter surfactant, somatic cells or enzyme extract with arginine deiminase activity are added, under the conditions of 30-50 DEG C
Enzymatic reaction is carried out, it is isolated using the method for isoelectric point crystallizing after conversion reaction when the reaction time is 16-30 small
Product L-citrulline；
3rd, L-Orn is prepared：By the fermented supernatant fluid containing L-Orn after decolorization, upper cation exchange column, L- birds
Propylhomoserin is adsorbed, and the hydrochloric acid for being 1mol/L with concentration desorbs, and efflux is neutralized to pH value to 6.0 with sodium hydroxide, and activated carbon takes off
Color, be concentrated in vacuo to crystal precipitation, adds the absolute ethyl alcohol of 3 times of volumes, crystallisation by cooling, suction filtration, crystalline substance is washed with 10 liters of ethanol
Body, L-Orn is obtained after dry.
The concentration that institute's stream adds to the L-arginine solution in zymotic fluid in the step 1 is 10-60g/L.
The mass concentration of L-arginine in the method step 2 in substrate solution is 150-250 g/L, surfactant
For：Tween 80, cetyl trimethylammonium bromide, TritonX-100, its concentration are 0.1~1.0 g/L.
Compared with prior art, the positive effect of the present invention is：
1st, the present invention during fermentation prepares arginine deiminase, realizes L- by improving stream plus L-arginine concentration
Ornithine high concentration accumulates, and L-Ornithine Content in Fermentation Broth concentration reaches 45 g/L；
2nd, the present invention prepares two kinds of products of L-citrulline and L-Orn respectively using thalline made from one time fermentation and zymotic fluid,
Resource is made full use of, process optimization is reasonable；
3rd, during Enzymatic Conversion of L-Citrulline, employ the zymotic fluid of cheap L-arginine or contain L-arginine
Ion-exchanging eluent or L-arginine crude product be substrate, take full advantage of industrial processes material, eliminate L- essence ammonia
The separation of acid, refining step, reduce production cost, and implementation result protrudes, with good economic efficiency and social benefit；
4th, bioanalysis preparation L-citrulline and L-Orn are gentle with reaction condition, and technological process is simple, are adapted to industrialization big
Large-scale production.
Embodiment
Clear, complete description is further carried out to technical scheme with reference to embodiment.
Embodiment one
1st, the cultivation and fermentation of strain：Enterococcus faecalis is cultivated in the medium, is activated, culture medium prescription is（g/mL）：Corn
Slurry 2.0%, protein hydrolysate 0.5%, beef extract 1%, maltose 1.0%, lactose 0.5%, NaCl 0.5%, KH2PO40.5%,
MgSO4•7H2O 0.03%, pH 7.2, seed liquor is inoculated in 300 L fermentation tanks according to the inoculum concentration of volume ratio 1%, fermentation tank
200 L of liquid amount, 12 m of throughput3/ h, tank press 0.05 MPa, and 100 r/min of rotating speed, 35 DEG C are cultivated, and stream adds in fermentation process
The L-arginine of final concentration of 10 g/L, pH controls are 6.8 or so during fermentation, and the control of fermentation later stage pH is 7.2 or so, fermentation 15
Hour terminates, and zymotic fluid obtains 4 kg of wet cell, 200 L of fermented liquid supernatant with arginine deiminase activity after centrifugation,
7.2 g/L of L-Orn content in fermented liquid supernatant.
2nd, L-citrulline is prepared：Substrate solution is prepared with the zymotic fluid of L-arginine, wherein L-arginine content is 150
G/L, pH 6.0, adds 1.0 g/L TritonX-100, adds 4 kg arginine deiminase wet thallus, cumulative volume 400
L, 30 DEG C, when 50 r/min enzymatic reactions 20 are small, L-citrulline concentration is 149.4 in sampling detection conversion fluid after reaction
G/L, is 99% to L-arginine molar yield, conversion fluid is centrifuged and removes thalline, supernatant is warming up to 80 DEG C of activity after desalting
Carbon decoloring, plate compression, filtrate have been concentrated into a small amount of crystal and have separated out, and the absolute ethyl alcohol of 3 times of volumes, cooling are added into concentrate
Crystallization, filters, crystal is washed with 10 liters of ethanol, 50.8 kg of L-citrulline, yield 85%, specific rotation are obtained after dry=
24.9o（c=8,6 mol/L hydrochloric acid）, crystalline mother solution recycling ethanol Posterior circle apply mechanically.
3rd, L-Orn is prepared：Fermented liquid supernatant containing L-Orn is after decolorization, upper cation exchange column, L- birds
Propylhomoserin is adsorbed, and is desorbed with 1 mol/L hydrochloric acid, and efflux is neutralized to pH 6.0, activated carbon decolorizing, vacuum concentration with sodium hydroxide
To there is crystal precipitation, the absolute ethyl alcohol of 3 times of volumes is added, crystallisation by cooling, filters, crystal is washed with a small amount of ethanol, obtained after dry
1.47 kg of L-ornithine hydrochloride, specific rotation= 23.6o（c=4,6 mol/L hydrochloric acid）.
Embodiment two
1st, the cultivation and fermentation of strain：Enterococcus faecalis is cultivated in the medium, is activated, culture medium prescription is（g/mL）：Corn
Starch 0.5%, NaCl 0.5%, KH2PO40.05%, MgSO4•7H2O 0.02%, beef extract 2%, glucose 1%, maltose 0.5%, pH
7.2, seed liquor is inoculated in 300 L fermentation tanks according to the inoculum concentration of volume ratio 2%, 200 L of fermentation tank liquid amount, throughput
12 m3/ h, tank press 0.05 MPa, and 100 r/min of rotating speed, 37 DEG C are cultivated, the L- of stream plus final concentration of 20 g/L in fermentation process
Arginine, pH controls are 6.8 or so during fermentation, and the control of fermentation later stage pH is 7.2 or so, and fermentation 15 terminates when small, zymotic fluid warp
4.4 kg of wet cell with arginine deiminase activity, 200 L of fermented liquid supernatant are obtained after centrifugation, L- birds in fermented liquid supernatant
14.5 g/L of histidine content.
2nd, L-citrulline is prepared：Substrate solution is prepared with the zymotic fluid of L-arginine, wherein L-arginine content is 200
G/L, pH 6.5, adds 1.0 g/L Tween-80s, adds 4 kg arginine deiminase wet thallus, cumulative volume 400 L, and 40
DEG C, when 50 r/min enzymatic reactions 16 are small, L-citrulline concentration is 199.1 g/L in sampling detection conversion fluid after reaction,
It is 99% to L-arginine molar yield, conversion fluid is centrifuged and removes thalline, supernatant is warming up to 80 DEG C of activated carbons and takes off after desalts
Color, plate compression, filtrate have been concentrated into a small amount of crystal and have separated out, and the absolute ethyl alcohol of 3 times of volumes, cooling knot are added into concentrate
Crystalline substance, filters, crystal is washed with 10 liters of ethanol, 67.8 kg of L-citrulline, yield 85%, specific rotation are obtained after dry= 25.1o
（c=8,6 mol/L hydrochloric acid）, crystalline mother solution recycling ethanol Posterior circle apply mechanically.
3rd, L-Orn is prepared：Fermented liquid supernatant containing L-Orn is after decolorization, upper cation exchange column, L- birds
Propylhomoserin is adsorbed, and is desorbed with 1 mol/L hydrochloric acid, and efflux is neutralized to pH 6.0, activated carbon decolorizing, vacuum concentration with sodium hydroxide
To there is crystal precipitation, the absolute ethyl alcohol of 3 times of volumes is added, crystallisation by cooling, filters, crystal is washed with a small amount of ethanol, obtained after dry
3.0 kg of L-ornithine hydrochloride, specific rotation= 23.2o（c=4,6 mol/L hydrochloric acid）.
Embodiment three
1st, the cultivation and fermentation of strain：Enterococcus faecalis is cultivated in the medium, is activated, culture medium prescription is（g/mL）：Corn
Slurry 1.0%, protein hydrolysate 0.5%, beef extract 2%, maltose 1.0%, lactose 0.5%, NaCl 0.5%, KH2PO40.5%,
MgSO4•7H2O 0.03%, pH 7.2, seed liquor is inoculated in 300 L fermentation tanks according to the inoculum concentration of volume ratio 1.5%, fermentation
Canned 200 L of liquid measure, 12 m of throughput3/ h, tank press 0.05 MPa, and 100 r/min of rotating speed, 35 DEG C are cultivated, and are flowed in fermentation process
Add the L-arginine of final concentration of 30 g/L, pH controls are 6.8 or so during fermentation, and the control of fermentation later stage pH is 7.2 or so, fermentation
17 terminate when small, and zymotic fluid must have 4.5 kg of wet cell of arginine deiminase activity, fermented liquid supernatant after centrifugation
220 L, 20.3 g/L of L-Orn content in fermented liquid supernatant.
2nd, L-citrulline is prepared：Substrate solution is prepared with the zymotic fluid of L-arginine, wherein L-arginine content is 220
G/L, pH 7.0, adds 1.0 g/L cetyl trimethylammonium bromides, adds 4 kg arginine deiminase wet thallus, always
Volume 400 L, 45 DEG C, when 50 r/min enzymatic reactions 20 are small, L-citrulline concentration in sampling detection conversion fluid after reaction
For 219 g/L, it is 99% to L-arginine molar yield, conversion fluid is centrifuged and removes thalline, supernatant is warming up to 80 after desalting
DEG C activated carbon decolorizing, plate compression, filtrate have been concentrated into a small amount of crystal and have separated out, and the anhydrous second of 3 times of volumes is added into concentrate
Alcohol, crystallisation by cooling, filters, crystal is washed with 10 liters of ethanol, 75.3 kg of L-citrulline, yield 86%, specific rotation are obtained after dry= 24.7o（c=8,6 mol/L hydrochloric acid）, crystalline mother solution recycling ethanol Posterior circle apply mechanically.
3rd, L-Orn is prepared：Fermented liquid supernatant containing L-Orn is after decolorization, upper cation exchange column, L- birds
Propylhomoserin is adsorbed, and is desorbed with 1 mol/L hydrochloric acid, and efflux is neutralized to pH 6.0, activated carbon decolorizing, vacuum concentration with sodium hydroxide
To there is crystal precipitation, the absolute ethyl alcohol of 3 times of volumes is added, crystallisation by cooling, filters, crystal is washed with a small amount of ethanol, obtained after dry
4.7 kg of L-ornithine hydrochloride, specific rotation= 23.3o（c=4,6 mol/L hydrochloric acid）.
Example IV
1st, the cultivation and fermentation of strain：Enterococcus faecalis is cultivated in the medium, is activated, culture medium prescription is（g/mL）：Albumen
Peptone 1.0%, beef extract 1.0%, sucrose 0.5%, fructose 1.0%, NaCl 0.5%, KH2PO40.3%, K2HPO40.1%, MgSO4•
7H2O 0.05%, pH 7.2, seed liquor is inoculated in 300 L fermentation tanks according to the inoculum concentration of volume ratio 1%, ferment canned liquid
Measure 200 L, 12 m of throughput3/ h, tank press 0.05 MPa, and 100 r/min of rotating speed, 35 DEG C are cultivated, and stream adds dense eventually in fermentation process
Spend for the L-arginine of 40 g/L, pH controls are 6.8 or so during fermentation, and for the control of fermentation later stage pH 7.2 or so, fermentation 19 is small
When terminate, zymotic fluid after centrifugation obtain have arginine deiminase activity 4.8 kg of wet cell, 210 L of fermented liquid supernatant,
29 g/L of L-Orn content in fermented liquid supernatant.
2nd, L-citrulline is prepared：Substrate solution is prepared with the zymotic fluid of L-arginine, wherein L-arginine content is 240
G/L, pH 7.0, adds 1.0 g/L TritonX-100, adds 4 kg arginine deiminase wet thallus, cumulative volume 400
L, 50 DEG C, when 50 r/min enzymatic reactions 24 are small, L-citrulline concentration is 239 g/ in sampling detection conversion fluid after reaction
L, is 99% to L-arginine molar yield, conversion fluid is centrifuged and removes thalline, supernatant is warming up to 80 DEG C of activated carbons after desalting
Decoloration, plate compression, filtrate have been concentrated into a small amount of crystal and have separated out, and the absolute ethyl alcohol of 3 times of volumes, cooling knot are added into concentrate
Crystalline substance, filters, crystal is washed with 10 liters of ethanol, 82.2 kg of L-citrulline, yield 86%, specific rotation are obtained after dry= 24.9o
（c=8,6 mol/L hydrochloric acid）, crystalline mother solution recycling ethanol Posterior circle apply mechanically.
3rd, L-Orn is prepared：Fermented liquid supernatant containing L-Orn is after decolorization, upper cation exchange column, L- birds
Propylhomoserin is adsorbed, and is desorbed with 1 mol/L hydrochloric acid, and efflux is neutralized to pH 6.0, activated carbon decolorizing, vacuum concentration with sodium hydroxide
To there is crystal precipitation, the absolute ethyl alcohol of 3 times of volumes is added, crystallisation by cooling, filters, crystal is washed with a small amount of ethanol, obtained after dry
6.4 kg of L-ornithine hydrochloride, specific rotation= 23.6o（c=4,6 mol/L hydrochloric acid）.
Embodiment five
1st, the cultivation and fermentation of strain：Enterococcus faecalis is cultivated in the medium, is activated, culture medium prescription is（g/mL）：Soya-bean cake
Hydrolyzate 3%, peptone 0.5%, yeast extract 0.5%, lactose 1.0%, sucrose 0.5%, maltose 0.5%, NaCl 0.5%, KH2PO4
0.1%, MgSO4•7H2O 0.03%, pH 7.2, seed liquor is inoculated in 300 L fermentation tanks according to the inoculum concentration of volume ratio 1%,
200 L of fermentation tank liquid amount, 12 m of throughput3/ h, tank press 0.05 MPa, 100 r/min of rotating speed, 35 DEG C of cultures, fermentation process
The L-arginine of middle stream plus final concentration of 50 g/L, during fermentation pH controls 6.8 or so, the control of fermentation later stage pH 7.2 or so,
Ferment 22 it is small when terminate, zymotic fluid obtains 5.1 kg of wet cell with arginine deiminase activity after centrifugation, on zymotic fluid
Clear 230 L, 36.2 g/L of L-Orn content in fermented liquid supernatant.
2nd, L-citrulline：The zymotic fluid of preparation L-arginine prepares substrate solution, and wherein L-arginine content is 250
G/L, pH 8.0, adds 1.0 g/L TritonX-100, adds 4 kg arginine deiminase wet thallus, cumulative volume 400
L, 42 DEG C, when 50 r/min enzymatic reactions 30 are small, L-citrulline concentration is 249 g/ in sampling detection conversion fluid after reaction
L, is 99% to L-arginine molar yield.Conversion fluid is centrifuged and removes thalline, supernatant is warming up to 80 DEG C of activated carbons after desalting
Decoloration, plate compression, filtrate have been concentrated into a small amount of crystal and have separated out, and the absolute ethyl alcohol of 3 times of volumes, cooling knot are added into concentrate
Crystalline substance, filters, crystal is washed with 10 liters of ethanol, 85.6 kg of L-citrulline, yield 86%, specific rotation are obtained after dry= 24.4o
（c=8,6 mol/L hydrochloric acid）, crystalline mother solution recycling ethanol Posterior circle apply mechanically.
3rd, L-Orn is prepared：Fermented liquid supernatant containing L-Orn is after decolorization, upper cation exchange column, L- birds
Propylhomoserin is adsorbed, and is desorbed with 1 mol/L hydrochloric acid, and efflux is neutralized to pH 6.0, activated carbon decolorizing, vacuum concentration with sodium hydroxide
To there is crystal precipitation, the absolute ethyl alcohol of 3 times of volumes is added, crystallisation by cooling, filters, crystal is washed with a small amount of ethanol, obtained after dry
8.8 kg of L-ornithine hydrochloride, specific rotation= 23.1o（c=4,6 mol/L hydrochloric acid）.
Embodiment six
1st, the cultivation and fermentation of strain：Enterococcus faecalis is cultivated in the medium, is activated, culture medium prescription is（g/mL）：Albumen
Peptone 1%, yeast extract 1%, glucose 1%, sucrose 0.5%, NaCl 0.5%, KH2PO40.04%, MgSO4•7H2O 0.03%, pH
7.2, seed liquor is inoculated in 300 L fermentation tanks according to the inoculum concentration of volume ratio 1%, 200 L of fermentation tank liquid amount, throughput
12 m3/ h, tank press 0.05 MPa, and 100 r/min of rotating speed, 35 DEG C are cultivated, the L- of stream plus final concentration of 60 g/L in fermentation process
Arginine, pH controls are 6.8 or so during fermentation, and the control of fermentation later stage pH is 7.2 or so, and fermentation 24 terminates when small, zymotic fluid warp
5.4 kg of wet cell with arginine deiminase activity, 230 L of fermented liquid supernatant are obtained after centrifugation, L- birds in fermented liquid supernatant
43.4 g/L of histidine content.
2nd, L-citrulline：The zymotic fluid of preparation L-arginine prepares substrate solution, and wherein L-arginine content is 250
G/L, pH 6.8, adds 1.0 g/L Tween-80s, adds 4 kg arginine deiminase wet thallus, cumulative volume 400 L, and 45
DEG C, when 80 r/min enzymatic reactions 28 are small, L-citrulline concentration is 248.9 g/L in sampling detection conversion fluid after reaction,
It is 99% to L-arginine molar yield, conversion fluid is centrifuged and removes thalline, supernatant is warming up to 80 DEG C of activated carbons and takes off after desalts
Color, plate compression, filtrate have been concentrated into a small amount of crystal and have separated out, and the absolute ethyl alcohol of 3 times of volumes, cooling knot are added into concentrate
Crystalline substance, filters, crystal is washed with 10 liters of ethanol, 84.6 kg of L-citrulline, yield 85%, specific rotation are obtained after dry= 25.9o
（c=8,6 mol/L hydrochloric acid）, crystalline mother solution recycling ethanol Posterior circle apply mechanically.
3rd, L-Orn is prepared：Fermented liquid supernatant containing L-Orn is after decolorization, upper cation exchange column, L- birds
Propylhomoserin is adsorbed, and is desorbed with 1 mol/L hydrochloric acid, and efflux is neutralized to pH 6.0, activated carbon decolorizing, vacuum concentration with sodium hydroxide
To there is crystal precipitation, the absolute ethyl alcohol of 3 times of volumes is added, crystallisation by cooling, filters, crystal is washed with a small amount of ethanol, obtained after dry
10.6 kg of L-ornithine hydrochloride, specific rotation= 23.7o（c=4,6 mol/L hydrochloric acid）.
All features disclosed in this specification, or disclosed all methods or during the step of, except mutually exclusive
Feature and/or step beyond, can combine in any way.This specification（Including claim, summary）Disclosed in
Any feature, unless specifically stated, can be replaced by other alternative features that are equivalent or have similar purpose.It is i.e. unless special
Do not describe, each feature is an example in a series of equivalent or similar characteristics.
The above is only the non-limiting embodiment of the present invention, substantial amounts of embodiment can also be derived, for ability
For the those of ordinary skill in domain, the invention design is not being departed from and on the premise of not making creative work, can be with
The embodiment of several modifications and improvements is made, these belong to protection scope of the present invention.
Claims (3)
1. a kind of bioanalysis prepares L-citrulline and the method for L-Orn, it is characterised in that：The method as follows into
OK：
（1）The cultivation and fermentation of strain：Enterococcus faecalis is cultivated in the medium, is fermented, culture medium includes following component：Corn
Starch 0.5-2.0g/ml, protein hydrolysate 0.5g/ml, beef extract 1.0-2.0 g/ml, maltose 0.5-1.0 g/ml, lactose
0.5-1.0 g/ml 、NaCl 0.5 g/ml、KH2PO4 0.04-0.5 g/ml、MgSO4•7H2O 0.02-0.05 g/ml, Portugal
1.0 g/ml of grape sugar, peptone 0.5-1.0 g/ml, 0.5 g/ml of sucrose, 1.0 g/ml of fructose, 3 g/ml of soya-bean cake hydrolyzate,
Yeast extract 0.5-1.0 g/ml, pH controls in culture medium are 7.2, and total carbon source mass concentration is 1-20g/L in the medium,
Total nitrogen source mass concentration be 1-20g/L, by the inoculum concentration kind of seed liquor 1-2% by volume in 300L fermentation tanks, is fermented canned
Liquid measure is 200L, and throughput 12m3/h, tank pressure is 0.05MPa, and rotating speed 100r/min, cultivation temperature is 35 DEG C, is fermented
Cheng Zhongliu adds L-arginine solution, during fermentation pH controls in 6.8-7.2, fermentation time for 15-24 it is small when, centrifuged after the completion of fermentation
Zymotic fluid, obtains the somatic cells with arginine depickling imines enzymatic activity and the fermented supernatant fluid containing L-Orn；
（2）Prepare L-citrulline：Substrate solution is prepared with the zymotic fluid of L-arginine, the pH value of substrate solution is 6.0-8.0,
Surfactant is added, somatic cells or enzyme extract with arginine deiminase activity are added, in 30-50 DEG C of condition
Lower carry out enzymatic reaction, when the reaction time is 16-30 small, after conversion reaction, is separated using the method for isoelectric point crystallizing
To product L-citrulline；
（3）Prepare L-Orn：By the fermented supernatant fluid containing L-Orn after decolorization, upper cation exchange column, L-
Ornithine is adsorbed, and the hydrochloric acid for being 1mol/L with concentration desorbs, and efflux is neutralized to pH value to 6.0 with sodium hydroxide, activated carbon
Decolourize, be concentrated in vacuo to crystal precipitation, add the absolute ethyl alcohol of 3 times of volumes, crystallisation by cooling, suction filtration, are washed with 10 liters of ethanol
Crystal, L-Orn is obtained after dry.
2. bioanalysis according to claim 1 prepares L-citrulline and the method for L-Orn, it is characterised in that：Described
Step（1）The concentration for the L-arginine solution that middle stream is added in zymotic fluid is 10-60g/L.
3. bioanalysis according to claim 1 prepares L-citrulline and the method for L-Orn, it is characterised in that：The side
Method step（2）The mass concentration of L-arginine in middle substrate solution is 150-250 g/L, and surfactant is：Tween 80, ten
Six alkyl trimethyl ammonium bromides, TritonX-100, its mass concentration are 0.1~1.0 g/L.
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