CN102719500A - Method for producing gamma-amino butyric acid through continuous conversion of immobilized enzyme - Google Patents
Method for producing gamma-amino butyric acid through continuous conversion of immobilized enzyme Download PDFInfo
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Abstract
The invention discloses a method for producing gamma-amino butyric acid through continuous conversion of an immobilized enzyme. The method comprises the following steps of: inducing and expressing an established glutamic acid decarboxylase gene engineering bacterium BL21 (DE3)-pET35b-GAD; performing cell disruption; collecting supernatant; and researching optimal reaction conditions and immobilizing conditions of a glutamic acid decarboxylase in fusion expression with a cellulose binding domain (CBD). Cellulose is specific adsorbed by a CBD label, and a cellulose carrier is added into cell lysate, so that the specific adsorption of the glutamic acid decarboxylase is realized, and the immobilized glutamate decarboxylase is crosslinked by using a crosslinking agent. The conversion conditions are optimized and the optimal catalytic reaction conditions are as follows: the pH is 5.5; the reaction time is 2 hours; the concentration of a substrate L-sodium glutamate is 40g/ L; the concentration of the immobilized enzyme is 8mL/L; and the concentration of a coenzyme pyridoxal phosphate aldehyde is 0.1mM. Under the optimal reaction, continuous conversion is repeated for 10 times and the conversion rate of the substrate L-sodium glutamate is more than 93%.
Description
Technical field
The invention belongs to biotechnology and produce amino acid whose technical field, relate to the method for producing γ-An Jidingsuan through immobilized enzyme method.
Background technology
γ-An Jidingsuan is a kind of natural amino acid with very high physiologically active, is distributed widely in the plant-animal.Be to study comparatively deep a kind of important inhibitory nerve mediator at present, participate in multiple Metabolic activity.According to present research, find that the physiologically active of γ-An Jidingsuan mainly shows following several respects: (1) calm neural, anxiety; (2) bring high blood pressure down; (3) diseases such as treatment Parkinson's disease, epilepsy, senile dementia; (4) reduce blood ammonia, malicious to solve ammonia, thus promote liver function; (5) improve the brain vigor; (6) promote alcohol metabolism; (7) other are as preventing skin aging, eliminate body odor, improve lipid metabolism, preventing functions such as arteriosclerosis high-efficient fat reducing.γ-An Jidingsuan is existing in medicine at present to be used widely, and simultaneously as a kind of functional component with remarkable pharmacological action, it is also more and more paid close attention in Application in Food Industry.
Because γ-An Jidingsuan content in animal-plant material is very low, be difficult to directly from natural tissues, extract in a large number obtain, the preparation method of γ-An Jidingsuan mainly contains chemical synthesis and biological synthesis process at present.But the chemical synthesis yield is lower, and in production technique, uses dangerous solvents, or even noxious solvent, so the γ-An Jidingsuan of chemical synthesis preparation should not be used for the foodstuff additive use.
Biological synthesis process mainly is to utilize enzyme catalysis to transform, and is synthetic in the γ-An Jidingsuan branch road.Low, nontoxic, the safety and environmental protection of this method production cost belongs to low-carbon (LC), green production mode.With regard to the enzyme source, can be divided into two kinds of approach: plant tissue metabolism and microbial fermentation.Wherein, the plant tissue metabolism mainly utilizes endogenous enzyme to transform the preparation γ-An Jidingsuan, and being divided into plant tissue growth metabolism and three-dimensional plant tissue stress the metabolism dual mode.Microbe fermentation method mainly is to utilize the decarboxylation of L-Glutamic decarboxylase (GAD) biocatalysis L-glutamic acid to generate γ-An Jidingsuan.Up to now, coming to light has GAD mikrobe active and that be used for preparing γ-An Jidingsuan and mainly comprises intestinal bacteria E.coli, milk-acid bacteria, yeast, monascus ruber etc., and table 1 is the situation of present microorganisms producing γ-An Jidingsuan.
The research overview of table 1 microorganisms producing γ-An Jidingsuan
At present; Biological synthesis process is the main flow production technology of γ-An Jidingsuan, and the inside and outside biological synthesis process of comprehensive comparator is produced γ-An Jidingsuan, different strains and different production mode; The output difference of γ-An Jidingsuan is bigger; And fermentation period is all longer, how with fermented liquid separation, the purifying of product from complicated component, also becomes complicated, consuming time in the Production Flow Chart, as to increase a cost ring after the fermentation ends.In recent years, along with the development and application of enzyme immobilization technology at medicine industry, relevant immobilization GAD transforms the also existing report of research of γ-An Jidingsuan; Wu Guoqi etc. (analytical chemistry, 1999,27 (7): 759-763 adopts the biochemical resin of carboxymethylated allyl group VISOSE; (medicine biotechnology, 2005,12 (2): 101-105) adopt fixedly GAD of chitosan material such as Lin Shaoqin; People such as Chen Weiqing (Chinese biochemical drug magazine, 2007,28 (4): 224-227) adopt the immobilization of sodium-alginate and gelatin synergy; With sodium-alginate and gelatin is carrier, through calcium chloride sclerosis, glutaraldehyde cross-linking, intestinal bacteria GAD is carried out using embedding immobilization; And attempt adopting the packed bed reactor successive reaction to prepare γ-An Jidingsuan, but reaction efficiency is still waiting to improve raising.
Summary of the invention
The purpose of this invention is to provide a kind of new immobilized enzyme method and transform the green synthesis method of producing γ-An Jidingsuan continuously.
The inventive method relates to genetic engineering bacterium e. coli bl21 (DE3)-pET35b-GAD and BL21 (DE3)-pET21 (+)-GAD; Set out with substrate L-Sodium Glutamate or L-L-glutamic acid; The L-Glutamic decarboxylase of expressing through the perhaps immobilized genetic engineering bacterium of the L-Glutamic decarboxylase of expressing directly transforms L-Sodium Glutamate or L-L-glutamic acid, the preparation γ-An Jidingsuan.
Immobilized enzyme method of the present invention transforms the concrete grammar of producing γ-An Jidingsuan continuously:
With the recombinant expressed cellulose binding domain-L-Glutamic decarboxylase of immobilization (being called for short " immobilized enzyme ") fusion rotein is the enzyme source; Concentration is under the condition that exists of the Vitazechs of 0.05mM ~ 0.15mM, carries out enzymatic reaction, in the reaction process; In reaction system, add immobilized enzyme 4mL/L to 20mL/L; Substrate L-Sodium Glutamate or L-L-glutamic acid are 20g/L to 50g/L, 25 to 50 ℃ of temperature, and the pH value is 4 to 6; 1h to 4h produces γ-An Jidingsuan through the enzymatic conversion method reaction, and further separates acquisition γ-An Jidingsuan powder or crystal.
The preferred invert point of enzymatic reaction is 37 ℃; Preferred conversion pH value is 5.5; Preferred transformation time is 2h; The consumption of substrate L-Sodium Glutamate or L-L-glutamic acid preferably is respectively 40g/L; Coenzyme Vitazechs concentration is 0.1mM; The immobilized enzyme preferable amount is 8mL/L.
The immobilized enzyme source is reusable more than 15 times, and under The optimum reaction conditions and system, transforms continuously 10 times, and its catalytic substrate transformation efficiency all can reach more than 93%.
The preparation method of cellulose binding domain-L-Glutamic decarboxylase fusion rotein that described immobilization is recombinant expressed is:
At first; The present invention has made up engineering strain e. coli bl21 (the DE3)-pET35b-GAD of efficient amalgamation and expression cellulose binding domain-L-Glutamic decarboxylase voluntarily; Promptly the genome with e. coli bl21 is that template is carried out the pcr amplification glutamic acid decarboxylase gene; Make up pET21 (+)-GAD expression vector; And in e. coli bl21 (DE3), express, change the GAD gene of confirming over to pET35b subsequently, make up engineering strain e. coli bl21 (DE3)-pET35b-GAD; And in e. coli bl21 (DE3), express, wherein e. coli bl21 (DE3) and corresponding expression vector pET21a (+), pET35b are bibliographical information and the available expression system of commercialization;
Cell pyrolysis liquid with above-mentioned recombination engineering strain e. coli bl21 (DE3)-pET35b-GAD is initial enzyme source, through cellulose carrier absorption, accomplishes immobilization, obtains immobilized enzyme.
Described glutamic acid decarboxylase gene derives from recombinant bacterial strain BL21 (DE3)-pET21a (+)-GAD of structure, and this bacterial strain possesses can express the L-Glutamic decarboxylase fusion rotein, and can L-L-glutamic acid or its sodium salt be converted into γ-An Jidingsuan.
Described cellulose carrier comprises cellulose microsphere, cellulose gel, absorbent cotton and filter paper.
Immobilized enzyme adopts cellulose binding domain-L-Glutamic decarboxylase fusion rotein specific adsorption fixed mode, or adopts the further crosslinked fixed mode of linking agent, and wherein linking agent is a LUTARALDEHYDE, and concentration is 0.1%.
In embodiments of the present invention, adopt pre-column derivatization HPLC method to measure the content of L-L-glutamic acid and γ-An Jidingsuan, derivatization reagent is the O-phthalic aldehyde reagent.The pre-column derivatization method is following: precision is measured 100 μ L sample solutions; (taking by weighing the 0.1g OPA dissolves with the 1mL acetonitrile to add 50 μ L derivatization reagents; Add 130 μ L beta-mercaptoethanols, be settled to 10mL with the 0.4mol/L borate buffer, pH10.2); Hybrid reaction 2min gets 20 μ L sample introduction analyses.Chromatographic column: C
18Chromatographic column (Inertsil ODS-SP5 μ, 4.6 * 250mm); Moving phase: 0-11min, B phase 8%-100%; Wherein the A phase (takes by weighing 3.2g crystallization sodium acetate, is settled to 400mL with water dissolution; Add 88 μ L triethylamines then, stirring down, the about 700 μ L of Dropwise 5 % acetic acid regulate pH to 7.20; Add the 2mL THF again, it is subsequent use to mix after-filtration), the B phase (takes by weighing 2.4g crystallization sodium acetate, is settled to 300mL with water dissolution; Dripping 2% acetic acid then transfers to 7.20 with pH to mix after-filtration subsequent use); Room temperature; Flow velocity is 1mL/min; Detect wavelength 340nm.
Advantage of the present invention and beneficial effect:
The present invention has made up the recombinant expression vector pET35b-GAD of amalgamation and expression L-Glutamic decarboxylase voluntarily and has been converted into E.coli BL21 (DE3); L-Glutamic decarboxylase to merging efficiently expresses, and obtains the cellulose binding domain-L-Glutamic decarboxylase (CBD-GAD) of high vigor.And discern cellulosic characteristic through the CBD biologic specificity dexterously, L-Glutamic decarboxylase has been carried out immobilization.Utilizing the CBD-GAD of immobilized efficient amalgamation and expression to be the enzyme source, is substrate with L-Sodium Glutamate or L-L-glutamic acid, can transform the preparation γ-An Jidingsuan continuously by enzyme process.Fermentation costs of the present invention is low; Immobilization process is simple and efficient; The enzymatic reaction catalytic efficiency (is high; Enzyme and product lock out operation are simple, and the green production process that therefore prepares γ-An Jidingsuan for immobilized enzyme method provides a new approach, have good industrial prospect in the field of industrialized production of γ-An Jidingsuan.
Description of drawings
Fig. 1 is that the pre-column derivatization HPLC of L-L-glutamic acid and γ-An Jidingsuan detects;
A: substrate L-L-glutamic acid; B: standard substance γ-An Jidingsuan; C: L-L-glutamic acid and γ-An Jidingsuan detect in the reaction solution.
Fig. 2 is that the concentration of linking agent is investigated.
Fig. 3 is the optimization of catalytic condition;
A: the relation of temperature of reaction and transformation efficiency; The relation of B:pH and transformation efficiency; C: the relation of reaction times and transformation efficiency; D: substrate L-Sodium Glutamate is to the relation of transformation efficiency; E: the relation of immobilized enzyme and transformation efficiency; F: the relation of Vitazechs and transformation efficiency.
Fig. 4 is the immobilized enzyme study on the stability.
Embodiment
The clone and the expression of embodiment 1 glutamic acid decarboxylase gene
The glutamic acid decarboxylase gene sequence of the e. coli bl21 of including according to ncbi database, design upstream primer GAD1:5 '-CGC GGA TCC ATG GAT AAG AAG CAA G-3 ' and downstream primer GAD2:5 ' CCG CTC GAG CGG TCA GGT ATG TTT AAA G-3 '.
Genome with e. coli bl21 is that template is carried out pcr amplification acquisition glutamic acid decarboxylase gene; After 1% agarose gel electrophoresis reclaims the purpose band; Connect pET21a (+) carrier; Make up pET21a (+)-GAD, and change BL21 (DE3) over to, obtain genetic engineering bacterium BL21 (DE3)-pET21a (+)-GAD.
Get a L-Glutamic decarboxylase engineering bacteria list colony inoculation in the LB substratum, 37 ℃ of overnight cultures are inoculated in saturated culture in the LB substratum that contains Amp (100 μ g/ml) with 1% to obtain saturated culture, and 37 ℃ are continued to cultivate OD
600Reach at 0.5 o'clock, add IPTG, induce 3h abduction delivering L-Glutamic decarboxylase for 37 ℃ to final concentration 0.1mmol/L.
After confirming that expression product possesses catalysis L-Sodium Glutamate and is converted into the γ-An Jidingsuan activity; GAD is connected to pET35b; Make up the pET35b-GAD carrier; And change BL21 (DE3) over to, and obtain genetic engineering bacterium BL21 (DE3)-pET35b-GAD, confirm behind the abduction delivering that expression product possesses the activity that catalysis L-Sodium Glutamate is converted into γ-An Jidingsuan.
The HPLC of embodiment 2 γ-An Jidingsuans detects
Adopt pre-column derivatization HPLC method to measure the content of L-L-glutamic acid and γ-An Jidingsuan, derivatization reagent is the O-phthalic aldehyde reagent.The pre-column derivatization method is following: precision is measured 100 μ L sample solutions, adds 50 μ L derivatization reagents, and hybrid reaction 2min gets 20 μ L sample introduction analyses.Chromatographic column: C
18Chromatographic column (Inertsil ODS-SP5 μ, 4.6 * 250mm); Moving phase: 0-11min, B phase 8%-100%; Room temperature; Flow velocity is 1mL/min; Detect wavelength 340nm.
Reaction solution centrifuging and taking supernatant carries out HPLC and analyzes after deriving as stated above, the result sees Fig. 1, with the comparison of γ-An Jidingsuan typical curve, calculates the molar yield of L-Sodium Glutamate in the substrate solution.
The immobilization of embodiment 3 L-Glutamic decarboxylases
Get 10mL cellulose microsphere (referring to Chinese patent, application number 200710057042.9) respectively, join in the cellular lysate liquid of genetic engineering bacterium BL21 (DE3)-pET35b-GAD that 50mL embodiment 1 obtains 4 ℃ of shaken overnight.After the sodium phosphate buffer washing, add the glutaraldehyde cross-linking of 0.01% to 1% different concns, with 1M Gly sealing 10h, the sodium phosphate buffer washing obtains immobilized L-Glutamic decarboxylase subsequently, i.e. immobilized enzyme, and 4 ℃ of preservations are subsequent use.Get the immobilized cellulose microsphere of 1mL different fixing condition respectively, add the L-Sodium Glutamate of same dosage, investigate immobilization efficiency, the result is as shown in Figure 2, and the optimum concn of linking agent LUTARALDEHYDE is 0.1%.
With immobilized enzyme in the foregoing description 3 is the enzyme source, and in every liter of reaction system, adding substrate L-L-glutamic acid respectively is 20 ~ 50g; Vitazechs is 0.05mM ~ 0.15mM; Invert point is 25 ~ 50 ℃; Transforming the pH value is 4 ~ 6; Transformation time is 1 ~ 4h, and immobilized enzyme is 4 ~ 20mL/L.Investigate of the influence of different conversion conditions, and adopt the content of the method detection γ-An Jidingsuan among the embodiment 2, calculate the transformation efficiency of L-Sodium Glutamate L-Sodium Glutamate transformation efficiency.
Fig. 3 A: the relation of investigating temperature of reaction and L-Sodium Glutamate transformation efficiency; Immobilized enzyme 4mL; Substrate L-Sodium Glutamate is 20g; Vitazechs is 0.1mM; Transforming the pH value is 5; Transformation time is 1h.
Fig. 3 B: the relation of investigating pH and L-Sodium Glutamate transformation efficiency; Immobilized enzyme 4mL; Substrate L-Sodium Glutamate consumption is 20g; Vitazechs is 0.1mM; Transformation time is 1h; Invert point is 37 ℃.
Fig. 3 C: the relation of investigating reaction times and L-Sodium Glutamate transformation efficiency; Immobilized enzyme 4mL; Substrate L-Sodium Glutamate consumption is 20g; Vitazechs is 0.1mM; Transforming the pH value is 5.5; Invert point is 37 ℃.
Fig. 3 D: the relation of investigating substrate and L-Sodium Glutamate transformation efficiency; Immobilized enzyme 4mL; Vitazechs is 0.1mM; Invert point is 37 ℃; Transforming the pH value is 5.5; Transformation time is 2h.
Fig. 3 E: the relation of investigating immobilized enzyme and L-Sodium Glutamate transformation efficiency; Substrate L-Sodium Glutamate consumption is 40g; Vitazechs is 0.1mM; Transforming the pH value is 5.5; Transformation time is 2h; Invert point is 37 ℃.
Fig. 3 F: the relation of investigating Vitazechs and L-Sodium Glutamate transformation efficiency; Immobilized enzyme 8mL; Substrate L-Sodium Glutamate consumption is 40g; Invert point is 37 ℃; Transforming the pH value is 5.5; Transformation time is 2h.
Confirm optimum conversion condition: temperature of reaction is 37 ℃; Reaction system pH is 5.5; Reaction times is 2h; Substrate L-concentration of sodium glutamate is 40g/L; The immobilized enzyme consumption is 8mL/L; Coenzyme Vitazechs concentration is 0.1mM.
Under the righttest enzymatic reaction condition, the transformation efficiency of L-Sodium Glutamate is about 99.5%.
The study on the stability of embodiment 5 enzymes
With the top condition that embodiment 4 confirms, after the single catalyzed reaction finishes, filtering separation immobilized enzyme and product γ-An Jidingsuan, the immobilized enzyme of recovery is recycled and reused for catalyzed reaction next time.Use repeatedly 20 times, the repeated experiments result of immobilized enzyme shows that L-Sodium Glutamate transformation efficiency can reach more than 70%, and the transformation efficiency of preceding 15 substrate L-Sodium Glutamates is all more than 80%, and the transformation efficiency of preceding 10 substrate L-Sodium Glutamates is all at (Fig. 4) more than 93%.
With the top condition that embodiment 4 confirms, be the enzyme source with the immobilized enzyme, be substrate with the L-Sodium Glutamate; Enzyme process prepares γ-An Jidingsuan, adds the 8mL immobilized enzyme in the 1L system, adds Sodium Glutamate 40g; And coenzyme Vitazechs 0.1mM; 37 ° of C of controlled temperature, regulate pH5.5 with 3N hydrochloric acid, the reaction 2h that is hydrolyzed carries out successive reaction equally 10 times.Conversion fluid filters removes microballoon, and through the desalination of 001x7 cationic exchange resin adsorption, 0.5M ammoniacal liquor wash-out is collected elutriant; Vacuum concentration adds 95% ethanol of 3 times of volumes, 4 ° of C hold over night to thin viscous; Filter to collect crystal, and with 95% washing with alcohol of a small amount of precooling, the vacuum-drying of 60 ° of C; Get γ-An Jidingsuan white powder 195g, adopt HPLC to detect, product purity reaches 98.5%.
With the top condition that embodiment 4 confirms, be the enzyme source with the immobilized enzyme, be substrate with L-L-glutamic acid; Enzyme process prepares γ-An Jidingsuan, adds the 8mL immobilized enzyme in the 1L system, adds L-glutamic acid 40g; And coenzyme Vitazechs 0.1mM; 37 ° of C of controlled temperature, pH5.5, the reaction 2h that is hydrolyzed carries out successive reaction equally 10 times.Conversion fluid filters removes microballoon, and vacuum concentration adds 95% ethanol of 3 times of volumes to thin viscous; 4 ° of C hold over night filter to be collected crystal, and after 95% the washing with alcohol with a small amount of precooling; Vacuum drying oven inner drying in 60 ° of C; Get γ-An Jidingsuan white powder 224g, adopt HPLC to detect, product purity reaches 95.0%.
Claims (7)
1. one kind is adopted immobilized enzyme method to transform the method for producing γ-An Jidingsuan continuously, it is characterized in that this method comprises:
With the recombinant expressed cellulose binding domain of immobilization-L-Glutamic decarboxylase fusion rotein, abbreviation " immobilized enzyme " is the enzyme source; Concentration is under the condition that exists of the Vitazechs of 0.05mM ~ 0.15mM, carries out enzymatic reaction, in the reaction process; In reaction system, add immobilized enzyme 4mL/L to 20mL/L; Substrate L-Sodium Glutamate or L-L-glutamic acid are 20g/L to 50g/L, 25 to 50 ℃ of temperature, and the pH value is 4 to 6; 1h to 4h produces γ-An Jidingsuan through the enzymatic conversion method reaction, and further separates acquisition γ-An Jidingsuan powder or crystal.
2. method according to claim 1 is characterized in that the preparation method of the recombinant expressed cellulose binding domain of described immobilization-L-Glutamic decarboxylase fusion rotein is:
At first; Make up engineering strain e. coli bl21 (the DE3)-pET35b-GAD of efficient amalgamation and expression cellulose binding domain-L-Glutamic decarboxylase; Promptly the genome with e. coli bl21 is that template is carried out the pcr amplification glutamic acid decarboxylase gene, makes up pET21 (+)-GAD expression vector, and in e. coli bl21 (DE3), expresses; Change the GAD gene of confirming over to pET35b subsequently; Obtain recombinant expression vector pET35b-GAD, make up engineering strain e. coli bl21 (DE3)-pET35b-GAD again, and in e. coli bl21 (DE3), express;
Cell pyrolysis liquid with above-mentioned recombination engineering strain e. coli bl21 (DE3)-pET35b-GAD is initial enzyme source, through cellulose carrier absorption, accomplishes immobilization, obtains immobilized enzyme.
3. method according to claim 2; It is characterized in that: described glutamic acid decarboxylase gene derives from recombinant bacterial strain BL21 (DE3)-pET21a (+)-GAD of structure; This bacterial strain possesses can express the L-Glutamic decarboxylase fusion rotein, and can L-L-glutamic acid or its sodium salt be converted into γ-An Jidingsuan.
4. according to claim 2 or 3 described methods, it is characterized in that: described cellulose carrier comprises cellulose microsphere, cellulose gel, absorbent cotton and filter paper.
5. according to claim 2 or 3 described methods; It is characterized in that: immobilized enzyme adopts cellulose binding domain-L-Glutamic decarboxylase fusion rotein specific adsorption fixed mode; Or adopt the further crosslinked fixed mode of linking agent, and wherein linking agent is a LUTARALDEHYDE, concentration is 0.1%.
6. method according to claim 1 is characterized in that: the preferred invert point of enzymatic reaction is 37 ℃; Preferred conversion pH value is 5.5; Preferred transformation time is 2h; The consumption of substrate L-Sodium Glutamate or L-L-glutamic acid preferably is respectively 40g/L; Coenzyme Vitazechs concentration is 0.1mM; The immobilized enzyme preferable amount is 8mL/L.
7. method according to claim 1 is characterized in that: the immobilized enzyme source is reusable more than 15 times, and under The optimum reaction conditions and system, transforms continuously 10 times, and its catalytic substrate transformation efficiency all can reach more than 93%.
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| CN114958878A (en) * | 2022-02-22 | 2022-08-30 | 山东蓝康生物科技有限公司 | Immobilized enzyme and application thereof in synthesizing NMN |
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| CN105936923A (en) * | 2016-01-25 | 2016-09-14 | 江苏纳克生物工程有限公司 | Method for producing gamma-aminobutyric acid by using recombinant Bacillus subtilis |
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| CN108642039A (en) * | 2018-06-29 | 2018-10-12 | 山东阳成生物科技有限公司 | A kind of immobilized glutamate decarboxylase and its method for producing γ-aminobutyric acid |
| CN109022412A (en) * | 2018-08-10 | 2018-12-18 | 浙江正硕生物科技有限公司 | A kind of method of fixed l-amino acid deaminase |
| CN111534551A (en) * | 2020-05-13 | 2020-08-14 | 福州三合元生物科技有限公司 | Process for preparing gamma-aminobutyric acid by immobilized enzyme |
| CN111534551B (en) * | 2020-05-13 | 2023-08-25 | 福州三合元生物科技有限公司 | Process for preparing gamma-aminobutyric acid by immobilized enzyme |
| CN114958878A (en) * | 2022-02-22 | 2022-08-30 | 山东蓝康生物科技有限公司 | Immobilized enzyme and application thereof in synthesizing NMN |
| CN114958878B (en) * | 2022-02-22 | 2023-10-13 | 山东蓝康药业有限公司 | Immobilized enzyme and application thereof in synthesis of NMN |
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