CN1661026A - Method for preparing L-omithine through immobilized ectocellular enzyme method - Google Patents
Method for preparing L-omithine through immobilized ectocellular enzyme method Download PDFInfo
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Abstract
A process for preparing L-ornithin by imobilized cell enzyme method includes coating the fermented microbe cells by different immobilizing carrier, filling in column, making the L-arginine solution to flow through the column, collecting the flowing-out liquid, concentrating and crystallizing or purifying by cationic exchange column.
Description
One, technical field
The present invention relates to utilize fixation of microbial cell to prepare the method for L-ornithine, the present invention relates in particular and will contain the cell fixation of arginine deiminase and ornithine carbamyl transferase, the effect by the said fixing desmo enzyme is converted into the L-ornithine with substrate L-arginine.Belong to technical field of enzyme engineering.
Two, technical background
Proteinic main metabolites is a urea in the human body.Contain arginase in the liver, can produce L-ornithine and urea by hydrolysis L-arginine.The L-ornithine also is the arginic precursor of biosynthesizing L-, and the L-arginine can participate in proteinic composition.It is unusual that the L-ornithine plays a part in human body, and the formation of urea must have the participation of L-ornithine.The shortage of L-ornithine may be brought danger to life.The L-ornithine can be used for the treatment (GB1020492,1966) of liver poisoning.The L-ornithine excretes blood ammonia by urea cycle as toxinicide.The L-ornithine is combined its ability of separating the ammonia poison of back can be greatly improved (GB1083911,1967) with ATP.The hop that adds an amount of L-ornithine can be used for beauty treatment, chest enlarge, also can be used for preventing and treating benign tumour and malignant tumour (CA2404275,2001).If regularly take the L-arginine and the L-ornithine mixture of preparation by a certain percentage, have good fat-reducing effect (GB2199243,1988).The L-ornithine derivative can also be used for dysthymia disorders, treatment of diseases (GB1385562,1975) such as weak and nervous.The curative effect also very good (JP2188558,1990) of the alpha-difluoromethyl-treatment of L-ornithine psoriasis, prostatomegaly and tumour.
According to the existing literature report, L-ornithine production technique mainly contains following three kinds:
1 chemical method
Industrial the most frequently used production method is to adopt chemical method basic hydrolysis L-arginine to prepare the L-ornithine at present, and Shell Int.Rearsch adopted vinylformic acid and sodium cyanide as main raw material chemosynthesis DL-ornithine in 1966.(GB1020492，1966)。
2 fermentation methods
With some bacterium in the enterobacteria such as Escherichia coli, Aerobacter aerogenes, the mutant strain of Proteus rettgeri and Proteus mirabili carries out fermentative production L-ornithine, the L-ornithine is up to 15mg/mL (GB1098348,1968 in the fermented liquid; US3668072,1972), Nakazawa etc. adopt some kind of Corynebacterium genus as C.glutamicum AJ11589, C.glutamicum ATCC13032 and C.acetoacidophilum ATCC13870 fermentative production L-ornithine (JP57016696,1982).Shibuya etc. utilize the mutant strain fermentative production L-ornithine of Arthrobacter citreus 23-2A, and fermentation 72h productive rate is 35.2g/L.Micrococcusglutamicus ATCC No.13232 fermentative production L-ornithine such as Shigezo, fermentation 72h unit output is 26.2g/L, the real L-ornithine hydrochloride crystallization 18g/L (US2988489,1961) that gets.Brevibacteriumlactofermentum AJ-11678 (Yoshimura, et al., JP57166988,1982) also can be used for the fermentative production of L-ornithine.Reports such as Chen Ning utilized Corynebacterium glutamicum mutant fermentative production L-ornithine in 1999, output be 9.85g/L (the Tianjin Light Industry College journal, 2000,3:20-24).
3 enzyme process
1981, streptococcus faecium ATCC8043 such as Shen Shuying, Bacillus foecalis alkaligenes ATCC21400, streptococcus faecium S-43 free cell conversion of substrate L-arginine prepares the L-ornithine, and transformation efficiency is 95-100%, and extract yield is a 85-90% (Tianjin microorganism, 1981,3:28-38).Nineteen ninety-five, the arginase hydrolysis L-arginine that utilizations such as M Kyriakos are extracted from animal livers is used for the preparation (US5405761,1995) of multiple L-ornithine salt.
Above-mentioned L-ornithine Production by Enzymes process all is to utilize free cell conversion of substrate L-arginine, so can contain a small amount of tropina and other impurity in the conversion fluid, is unfavorable for the separation and purification of product; And to remove thalline after transforming, reduce efficiency of pcr product, cause production cost to increase.Free cell transforms and is unfavorable for the industrialization continuous production, so the utilization ratio of enzyme is not high yet.
Up to the present, the relevant immobilized cell enzyme process method for preparing the L-ornithine is not appeared in the newspapers.
Three, summary of the invention
1. goal of the invention
The object of the present invention is to provide a kind of fixation of microbial cell enzyme process to prepare the method for L-ornithine.
2. technical scheme
(1) bacterial classification
In the present invention, the bacterial classification that is used for the production of L-ornithine has enterococcus faecalis (Enterococcusfaecalis), vibrio proteolyticus (Vibrio proteolyticus), Aeromonas media (Aeromonasmedia), Micrococcus sedentarius (Micrococcus sedentarius), Ge Shi Serratia (Serratiagrimesii) and Enterobacter sakazakii (Enterobacter sakazakii) etc.
(2) substratum and culture condition
1. seed culture
Seed culture medium (1000mL): glucose 10-30g, extractum carnis 5-10g, peptone 10-20g, potassium primary phosphate 1-5g, sal epsom 0.2-1.0g, the 10-100mL of Tomato juice, pH6.5-7.5,121 ℃ of sterilization 20min.
Liquid amount is the 50-100mL/250mL triangular flask during seed culture, and inoculum size is a test tube slant that covers with, and culture temperature is 30-37 ℃, and shaking speed is 100-200r/min.
2. fermentation culture
Fermention medium (1000mL): yeast extract paste 5-20g, glucose 10-20g, peptone 5-20g, corn steep liquor 5-10g, potassium primary phosphate 1.0-2.0g, sal epsom 0.5-1.0g, pH7.0-7.5.121 ℃ of sterilization 20min.
Liquid amount is the 50-150mL/250mL triangular flask during fermentation culture, and inoculum size is 2%-10%, and culture temperature is 25-37 ℃, and shaking speed is 100-200rpm.
(3) a kind of immobilized cell enzyme process prepares the method for L-ornithine, and its characterization step is as follows:
1. actication of culture
Learn from else's experience the bacterial classification of lyophil preservation with the dissolving of an amount of sterile liquid seed culture medium, insert constant temperature culture in the liquid seed culture medium then, get above-mentioned seed then and insert fresh seed culture medium and continue activation;
2. strain fermentation and somatic cells are collected
Above-mentioned bacterial classification is received in the fermention medium, cultivated and under aeration condition, carry out, can carry out pH regulator by adding acid to fermented liquid during the fermentation, be beneficial to thalli growth, improve thalline output; With the centrifugal collection thalline of of the right age fermented liquid;
3. cell fixation
Collected somatic cells and a certain amount of embedding medium are mixed, add curing molding in the forming agent, also can carry out intensive treatment, promptly get immobilized cell with reinforcer;
4. the arginic conversion of L-
With the mycetome cell colloid after said fixing processing dress post, the L-arginine solution gel column of flowing through is carried out bio-transformation, collect effusive conversion fluid, conversion fluid can repeat upper prop, is reformed completely into the L-ornithine up to substrate L-arginine;
5. the separation and purification of L-ornithine
After above-mentioned conversion fluid removed impurity with activated carbon decolorizing, be concentrated into suitable concn, crystallization, mother liquor can concentrate with conversion fluid merging next time, recrystallize, repeatedly repeat in the mother liquor of back the L-ornithine and can separate with cationic exchange coloum and obtain, crystal crude product can be made with extra care by recrystallization, and the L-ornithine that makes by ion exchange column needs further refining equally.The purified method can be recrystallization, or with ethanol sedimentation etc.
The bacterial classification of above-mentioned steps described in 1. comprises enterococcus faecalis (Enterococcus faecalis), vibrio proteolyticus (Vibrio proteolyticus), Aeromonas media (Aeromonas media), Micrococcus sedentarius (Micrococcus sedentarius), Ge Shi Serratia (Serratia grimesii) and Enterobacter sakazakii (Enterobacter sakazakii) etc.
The embedding medium of above-mentioned steps described in 3. comprises sodium alginate, carrageenin, polyvinyl alcohol, gelatin and chitin etc.; Forming agent comprises calcium chloride, Repone K, boric acid solution and sodium radio-phosphate,P-32 solution etc.; Reinforcer comprises glutaraldehyde etc.
The embedding medium sodium alginate of above-mentioned steps described in 3. and the concentration of carrageenin are 1-5%, and the amount of somatic cells is 1-20%, and the concentration of forming agent calcium chloride or Repone K is 1-5%, and be 1-3h set time, and solidification value is 20-40 ℃; With the embedding medium polyvinyl alcohol concentration is that 5-20% and carrageenan concentrations are the mixed solution of 0.3-1.2%, and the amount of somatic cells is 1-20%, and forming agent is Repone K-boric acid saturated solution of pH5-8, and solidification value is 10-50 ℃, and be 20-50h set time; Concentration with the embedding medium gelatin is 5-20%, and the amount of somatic cells is 1-20%, and solidification value is 4-10 ℃.The concentration of colloid reinforcer glutaraldehyde is 0.5-3%, and the intensive treatment time is 5-120min; Concentration with the embedding medium chitin is 1-10%, and the amount of somatic cells is 1-20%, and the pH of forming agent buffer solution of sodium phosphate is 4-9, and solidification value is 10-50 ℃, and be 12-48h set time.
The L-arginine concentration of above-mentioned steps described in 4. is 0.1-20%, and the speed of the enzymatic conversion post of flowing through is 10-1000mL/h, and invert point is 25-45 ℃.
3. beneficial effect
Adopt the method for immobilized cell, collection conversion fluid that can be easy carries out the product preparation, has simplified technological process, improved the utilization ratio of enzyme, substrate L-arginine transformation efficiency is more than 95%, and the product yield is higher than 80%, and helps the automatic control in the commercial process.
Four, embodiment
Embodiment 1 seed culture and fermentation culture
A kind of seed culture medium: glucose 15g, extractum carnis 7.5g, peptone 15g, potassium primary phosphate 3.5g, sal epsom 0.5g, the 100mL of Tomato juice, water 900mL, pH7.5 sterilized 20 minutes for 121 ℃.
A kind of fermention medium is as follows: glucose 2.0g, yeast extract paste 0.5g, peptone 0.7g, corn steep liquor 0.8g, potassium primary phosphate 0.1g, sal epsom 0.06g, L-arginine 0.3g, water 100mL, pH7.2.
Seed culture medium with an amount of sterilization dissolves freeze dried Aeromonas media bacterial classification, inserts then and is equipped with in the 250mL triangular flask of 50mL liquid seed culture medium, and in 37 ℃ of constant temperature culture, shaking speed is 200r/min.Get above-mentioned seed 10mL behind the 24hrs and insert fresh seed culture medium continuation activation.
Get the good bacterial classification of the above-mentioned activation of 4mL and insert in the 100mL fermention medium the following 37 ℃ of constant temperature culture 24h of 200rpm.
Embodiment 2 immobilized cell enzyme process prepare the method for L-ornithine
As fermentation culture 1000mL Aeromonas media as described in the embodiment 1, the centrifugal 20min of 6000rpm collects thalline, wet thallus 12.5g.Wash in the 1200mL fixing agent (3% sodium alginate soln) mixing.The sodium alginate soln that will contain thalline with syringe splashes into 3000mL forming agent (2%CaCl fast then
2Solution) in, remove forming agent behind the curing 1hr, and wash 3 times with 5000mL physiological saline.With the micelle of the gained diameter 5cm that packs into, in the glass column of high 50cm.1000mL 5%L-arginine solution is with the 20mL/h glue post of flowing through, transform fully after, collect effluent liquid and also be evaporated to 200mL, handle with the 2g activated carbon decolorizing.Destainer continues to be evaporated to 100mL, and add 200mL 95% ethanol and stir, crystallisation by cooling, vacuum filtration gets crystal dry weight 35.5g.[α]
D 20＝+11.0(C＝5.5，H
2O)。Crystalline mother solution is collected with next conversion fluid and is merged processing.
Embodiment 3 immobilized cell enzyme process prepare the method for L-ornithine
Cultural method fermentation culture enterococcus faecalis 1000mL shown in example 1, centrifugal collection thalline, centrifuge speed are 6000rpm, 20min.Claim thalline weight in wet base 10.2g, be suspended in the 50mL physiological saline 37 ℃ of water bath heat preservations.Get 10g polyvinyl alcohol (PVA), add 50mL distilled water immersion 1d, add the 0.5g carrageenin, in 110 ℃ of insulation 20min, PVA is fully dissolved in the rearmounted pressure kettle of mixing, take out and put 45 ℃ of water bath heat preservations.Then cell suspending liquid is mixed with the PVA-carrageenin mixed solution of equivalent, splash into needle tubing in the saturated boric acid solution of KCl-of pH6.4, leave standstill 30h under 4 ℃, make the immobilized cell micelle of enterococcus faecalis.With the gained micelle diameter 5cm that packs into, in the glass column of high 50cm.1000mL 5%L-arginine solution is with the 15mL/h glue post of flowing through, transform fully after, collect effluent liquid and also be evaporated to 200mL, handle with the 2g activated carbon decolorizing.Destainer continues to be evaporated to 100mL, and add 200mL 95% ethanol and stir, crystallisation by cooling, vacuum filtration gets crystal dry weight 32.5g.[α]
D 20＝+11.0(C＝5.5，H
2O)。
Embodiment 4 immobilized cell enzyme process prepare the method for L-ornithine
Method fermentation culture vibrio proteolyticus 100mL shown in embodiment 1 gets wet thallus 1.2g, washes in the 120mL fixing agent (3% carrageenan solutions) mixing.The carrageenan solutions that will contain thalline with syringe splashes in the 300mL forming agent (2%KCl solution) fast then, removes forming agent after solidifying 2hr, and washs 3 times with 500mL physiological saline.With the micelle of the gained diameter 3cm that packs into, in the glass column of high 20cm.The 5%L-arginine solution of 1000mL is with the 10mL/h glue post of flowing through, transform fully after, collect effluent liquid and also be evaporated to 200mL, handle with the 2g activated carbon decolorizing.Destainer continues to be evaporated to 100mL, and add 200mL 95% ethanol and stir, crystallisation by cooling, vacuum filtration gets crystal dry weight 34.2g.[α]
D 20＝+11.2(C＝5.5，H
2O)。
Embodiment 5 immobilized cell enzyme process prepare the method for L-ornithine
As fermentation culture 1000mL Micrococcus sedentarius as described in the embodiment 1, the centrifugal 20min of 6000rpm collects thalline, wet thallus 12g.Wash in the 2% fixing agent chitin solution of 1000mL mixing.Splash into then in the buffer solution of sodium phosphate of pH7.0 of 1000mL and solidify 24h.With the micelle of the gained diameter 5cm that packs into, in the glass column of high 50cm.1000mL 5%L-arginine solution is with the 20mL/h glue post of flowing through, transform fully after, collect effluent liquid and also be evaporated to 200mL, handle with the 2g activated carbon decolorizing.Destainer continues to be evaporated to 100mL, and add 200mL 95% ethanol and stir, crystallisation by cooling, vacuum filtration gets crystal dry weight 35.5g.[α]
D 20＝+11.0(C＝5.5，H
2O)。Crystalline mother solution is collected with next conversion fluid and is merged processing.
Embodiment 6 immobilized cell enzyme process prepare the method for L-ornithine
As fermentation culture 1000mL Ge Shi Serratia as described in the embodiment 1, the centrifugal 20min of 6000rpm collects thalline, wet thallus 10.5g.Wash in the 1000mL fixing agent (3% gelatin solution) mixing.Place 4 ℃ of low temperature to solidify then, solidify the micelle that blob of viscose is cut into behind the 2hr 5mm*5mm, and with 1% the glutaraldehyde reinforcement 1hr of 5000mL.With the micelle of the gained diameter 5cm that packs into, in the glass column of high 50cm.The 5%L-arginine solution of 1000mL is with the 20mL/h glue post of flowing through, transform fully after, collect effluent liquid and also be evaporated to 200mL, handle with the 2g activated carbon decolorizing.Destainer continues to be evaporated to 100mL, and add 200mL 95% ethanol and stir, crystallisation by cooling, vacuum filtration gets crystal dry weight 37.5g.[α]
D 20＝+10.8(C＝5.5，H
2O)。Crystalline mother solution is collected with next conversion fluid and is merged processing.
Claims (10)
1. one kind prepares the method for L-ornithine with the immobilized cell enzyme process, and its preparation process is as follows:
1. actication of culture;
Learn from else's experience the bacterial classification of lyophil preservation with the dissolving of sterile liquid seed culture medium, insert then in the liquid seed culture medium, constant temperature culture is got above-mentioned seed then and is inserted fresh seed culture medium and continue activation;
2. strain fermentation and somatic cells are collected;
Above-mentioned bacterial classification is received in the fermention medium, under aeration condition, cultivated, then with the centrifugal collection thalline of of the right age fermented liquid;
3. cell fixation;
Collected somatic cells and embedding medium are mixed, add curing molding in the forming agent, also can add reinforcer and carry out intensive treatment, promptly get immobilized cell;
4. the L-arginine transforms;
With the colloid of mycetome cell after said fixing processing dress post, the L-arginine solution gel column of flowing through is carried out bio-transformation, collect effusive conversion fluid, conversion fluid can repeat upper prop, is reformed completely into the L-ornithine up to substrate L-arginine;
5. L-ornithine separation and purification;
After above-mentioned conversion fluid removed impurity with activated carbon decolorizing, be concentrated into crystallization, the L-ornithine adopts cationic exchange coloum to separate in the mother liquor; Crude product is made with extra care by recrystallization.
2. immobilized cell enzyme process according to claim 1 prepares the method for L-ornithine, it is characterized in that comprising enterococcus faecalis, vibrio proteolyticus, Aeromonas media, Micrococcus sedentarius, Ge Shi Serratia and Enterobacter sakazakii at the bacterial classification of step described in 1..
3. immobilized cell enzyme process according to claim 1 prepares the method for L-ornithine, it is characterized in that comprising sodium alginate, carrageenin, polyvinyl alcohol, gelatin and chitin at the embedding medium of step described in 3..
4. immobilized cell enzyme process according to claim 1 prepares the method for L-ornithine, it is characterized in that comprising calcium chloride, Repone K, boric acid solution and sodium radio-phosphate,P-32 solution at the forming agent of step described in 3..
5. immobilized cell enzyme process according to claim 1 prepares the method for L-ornithine, it is characterized in that at the reinforcer of step described in 3. be glutaraldehyde.
6. immobilized cell enzyme process according to claim 1 prepares the method for L-ornithine, it is characterized in that being 1-5% at the embedding medium sodium alginate of step described in 3. and the concentration of carrageenin, the amount of somatic cells is 1-20%, the concentration of forming agent calcium chloride or Repone K is 1-5%, be 1-3h set time, and solidification value is 20-40 ℃.
7. immobilized cell enzyme process according to claim 1 prepares the method for L-ornithine, it is characterized in that in the embedding medium polyvinyl alcohol concentration of step described in 3. be that 5-20% and carrageenan concentrations are the mixed solution of 0.3-1.2%, the amount of somatic cells is 1-20%, forming agent is Repone K-boric acid saturated solution of pH5-8, solidification value is 10-50 ℃, and be 20-50h set time.
8. immobilized cell enzyme process according to claim 1 prepares the method for L-ornithine, it is characterized in that the embedding medium gelatin concentration described in step 3. is 5-20%, the amount of somatic cells is 1-20%, solidification value is 4-10 ℃, the concentration of colloid reinforcer glutaraldehyde is 0.5-3%, and the intensive treatment time is 5-120min.
9. immobilized cell enzyme process according to claim 1 prepares the method for L-ornithine, it is characterized in that the embedding medium chitin concentration described in step 3. is 1-10%, the somatic cells amount is 1-20%, forming agent buffer solution of sodium phosphate pH is 4-9, solidification value is 10-50 ℃, be 12-48h set time.
10. immobilized cell enzyme process according to claim 1 prepares the method for L-ornithine, it is characterized in that described in step 4. described in the arginic concentration of L-be that the flow through speed of enzymatic conversion post of 0.1-20% is 10-1000mL/h, invert point is 25-45 ℃.
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| CN101851646A (en) * | 2009-03-31 | 2010-10-06 | 上海汉飞生化科技有限公司 | Method for producing L-ornithine hydrochloride through immobilized enzyme process |
| CN102250977A (en) * | 2011-06-28 | 2011-11-23 | 三峡大学 | Method for preparing L-isoleucine by using immobilized cells |
| CN102277395A (en) * | 2011-08-05 | 2011-12-14 | 上海汉飞生化科技有限公司 | Method for producing L-ornithine nitrate by enzymatic conversion |
| CN102286563A (en) * | 2011-07-19 | 2011-12-21 | 南开大学 | Method for preparing L-ornithine from immobilized enzyme |
| CN102373245A (en) * | 2010-08-18 | 2012-03-14 | 上海汉飞生化科技有限公司 | Preparation method for L-ornithine-alpha-ketoglutarate |
| CN103014086A (en) * | 2012-12-12 | 2013-04-03 | 武汉圣佳化工有限公司 | Method for continuously producing L-ornithine composite salt by enzyme immobilization |
| TWI426101B (en) * | 2011-01-24 | 2014-02-11 | Mackay Memorial Hospital | Compositions, sets and methods of making cell-containing hardened polymers |
| CN106631852A (en) * | 2016-09-30 | 2017-05-10 | 南京工业大学 | Method for extracting L-ornithine hydrochloride from L-ornithine fermentation broth |
| CN107630012A (en) * | 2017-10-18 | 2018-01-26 | 武夷学院 | A kind of embedding method of aerobic denitrifying bacteria |
| CN109071628A (en) * | 2016-03-29 | 2018-12-21 | 富士胶片株式会社 | Cell sheet embedding medium, composition and kit containing cell sheet |
| CN109486873A (en) * | 2018-07-13 | 2019-03-19 | 滨州职业学院 | A kind of immobilization Combined culture fermentation Unicell Oils and Fats technology |
| CN112195171A (en) * | 2019-07-08 | 2021-01-08 | 秦皇岛华恒生物工程有限公司 | Method for preparing beta-alanine by using immobilized enzyme |
Family Cites Families (1)
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| DE4020980C1 (en) * | 1990-07-02 | 1991-09-26 | Degussa Ag, 6000 Frankfurt, De |
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| CN101851646A (en) * | 2009-03-31 | 2010-10-06 | 上海汉飞生化科技有限公司 | Method for producing L-ornithine hydrochloride through immobilized enzyme process |
| CN102373245B (en) * | 2010-08-18 | 2013-08-07 | 上海汉飞生化科技有限公司 | Preparation method for L-ornithine-alpha-ketoglutarate |
| CN102373245A (en) * | 2010-08-18 | 2012-03-14 | 上海汉飞生化科技有限公司 | Preparation method for L-ornithine-alpha-ketoglutarate |
| TWI426101B (en) * | 2011-01-24 | 2014-02-11 | Mackay Memorial Hospital | Compositions, sets and methods of making cell-containing hardened polymers |
| CN102250977A (en) * | 2011-06-28 | 2011-11-23 | 三峡大学 | Method for preparing L-isoleucine by using immobilized cells |
| CN102286563A (en) * | 2011-07-19 | 2011-12-21 | 南开大学 | Method for preparing L-ornithine from immobilized enzyme |
| CN102286563B (en) * | 2011-07-19 | 2013-11-06 | 南开大学 | Method for preparing L-ornithine from immobilized enzyme |
| CN102277395A (en) * | 2011-08-05 | 2011-12-14 | 上海汉飞生化科技有限公司 | Method for producing L-ornithine nitrate by enzymatic conversion |
| CN103014086A (en) * | 2012-12-12 | 2013-04-03 | 武汉圣佳化工有限公司 | Method for continuously producing L-ornithine composite salt by enzyme immobilization |
| CN103014086B (en) * | 2012-12-12 | 2015-02-11 | 武汉圣佳化工有限公司 | Method for continuously producing L-ornithine composite salt by enzyme immobilization |
| CN109071628A (en) * | 2016-03-29 | 2018-12-21 | 富士胶片株式会社 | Cell sheet embedding medium, composition and kit containing cell sheet |
| CN106631852A (en) * | 2016-09-30 | 2017-05-10 | 南京工业大学 | Method for extracting L-ornithine hydrochloride from L-ornithine fermentation broth |
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| CN112195171A (en) * | 2019-07-08 | 2021-01-08 | 秦皇岛华恒生物工程有限公司 | Method for preparing beta-alanine by using immobilized enzyme |
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