CN105603016A - Method for preparing L-tert-leucine by coupling leucine dehydrogenase with glucose dehydrogenase - Google Patents
Method for preparing L-tert-leucine by coupling leucine dehydrogenase with glucose dehydrogenase Download PDFInfo
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Abstract
The invention discloses a method for preparing L-tert-leucine by coupling leucine dehydrogenase with glucose dehydrogenase, belonging to the technical field of enzyme engineering and chiral medical intermediate preparation. The method comprises the following steps: adding substrates trimethylpyruvic acid and glucose into a reaction system; adjusting the pH value of the system to 8.0-9.0; and adding a crude enzyme (containing leucine dehydrogenase, glucose dehydrogenase and coenzyme) with broken co-expressed strain and cultured by a culture medium added with substances such as niacin, and then starting reactions while controlling the temperature at 25-30 DEG C and pH value at 8.0-9.0 in the reaction process to generate L-tert-leucine. In the invention, a system in which leucine dehydrogenase is coupled with glucose dehydrogenase is adopted, and the L-tert-leucine is prepared by use of the coenzyme contained in the thalli; and the method has the characteristics of high concentration of single-batch reaction substrates, high reaction efficiency, no coenzyme addition and the like.
Description
Technical field
The present invention relates to a kind of method of utilizing leucine dehydrogenase coupling GDH to prepare S-Leucine, belong to enzyme workJourney and chiral medicinal intermediate preparing technical field.
Background technology
Optical voidness S-Leucine, contained tert-butyl group group not only has space steric effect and also has stronger hydrophobicity, therebyBe widely used as the synthetic template of asymmetry catalysis. It,, as the non-natural amino acid of one, is also usually applied to medicine simultaneouslyIn the middle of active peptide. Studies have reported that S-Leucine is for antineoplastic reagent, the medicines such as anti-AIDS protease inhibitors.Just because of the extensive use of S-Leucine, a large amount of synthetic methods are developed report. Wherein mainly comprise chemical resolution method,Chemistry dissymmetric synthesis, enzyme Split Method, and enzyme reductive amination synthetic method (Bommariusetal., 1995). Due in recent yearsThe enhancing of people's environmental consciousness, has severe reaction conditions, reaction process complexity, and the low chemical synthesis process of reaction efficiency is graduallyReplaced by biological synthesis process. Split in synthetic method at enzyme, some enzymes of mainly using comprise PA ase, lipase,(Abderhalden, Faust ， &Haase, 1934 such as protease; Agostaetal., 2006; Turner, Winterman, McCague,Parratt ， &Taylor, 1995). Because enzymatic resolution method exists complex operation step and theoretical yield only has 50%,The never report of correlation technique commercial Application.
Enzyme reduces amino synthetic method, has feature simple to operate and theoretical yield 100%. The enzyme of mainly using at present comprisesTwo kinds is respectively side chain aminopherase and leucine dehydrogenase. Hong etc. have reported and have utilized side chain transamination enzymatic synthesis L-Terleu, conversion ratio is greater than 90%, but the concentration of substrate of catalysis too low do not reach industrial requirements (Hong, Cha, Yun ， &Kim,2010). Another utilizes the synthetic S-Leucine of leucine dehydrogenase to be developed by Degussa (Germany) company in the nineties,And be applied to industrial production. The method is used enzyme mebrane reactor, leucine dehydrogenase and hydrogenlyase is fixed on enzyme membrane,Wherein hydrogenlyase can catalyzing N AD+Generate NADH, realize the regeneration of coenzyme, reduce coenzyme consumption (Liese,Seelbach ， &Wandrey, 2006). The synthetic S-Leucine of leucine dehydrogenase catalysis trimethyl pyruvic acid is considered to have mostThe synthesis path of industrial prospect.
Comprised Chinese patent 201110202325.4 about the patent report of leucine dehydrogenase synthesis path in the last few years,201210508084.0,201410695848.0,201310044292.4 etc. All there is following problem in these synthesis paths: 1. anti-In requisition for additional coenzyme, and coenzyme is expensive, less economical, if can not add coenzyme in synthetic engineering, and willCan significantly reduce the cost of industrial processes; 2. hydrogenlyase activity is low, needs to add large in asymmetric conversion processThe hydrogenlyase of amount, can give full play to leucine dehydrogenase and catalyze and synthesize effect, has increased the cost that enzyme drops into; 3. anti-Answer efficiency lower, just because of the lower hydrogenlyase of the activity of using in course of reaction, affect coenzyme transformation efficiency, increaseCompletely the needed reaction time of conversion of substrate, make reaction efficiency lower.
Summary of the invention
In order to overcome the problems referred to above, the present invention utilizes the broken crude enzyme liquid of coexpression leucine dehydrogenase and GDH bacterial strainAs catalyst, carry out catalysis trimethyl Pyruvate production S-Leucine. The relative formic acid of GDH that the present invention usesDehydrogenase has higher enzyme lives, thereby improves coenzyme utilization ratio. Adopt the reaction formation of crude enzyme liquid can ignore substrate forThe inhibition of somatic cells, improves concentration of substrate. By adding nicotinic acid in thalline incubation China and foreign countries, improve coenzyme concentration in born of the same parents, andIn realization response, zero coenzyme adds. Finally, realized and utilized leucine dehydrogenase coupling GDH efficiently to prepare uncle L-Leucine.
The object of this invention is to provide a kind of method of preparing S-Leucine, is to contain glucose, substrate trimethyl pyruvic acidIn reaction system, add the clasmatosis crude enzyme liquid of the recombinant bacterium of coexpression leucine dehydrogenase and GDH, stirring reaction.
In one embodiment of the invention, substrate trimethyl pyruvic acid concentration 0.5-1.2M in described method, concentration of glucoseBe the 1.0-1.3 of concentration of substrate doubly.
In one embodiment of the invention, the temperature of described reaction system is controlled at 20-35 DEG C, and pH is controlled at 7.5-9.0.Preferably temperature is 30 DEG C, and pH is 8.5.
In one embodiment of the invention, described pH controls by ammoniacal liquor.
In one embodiment of the invention, the time of described reaction is 1-2.5 hour. The preferred reaction time is 1.5h.
In one embodiment of the invention, the cell that in described reaction system, every L contains 20-60g (weight in wet base) recombinant bacterium is brokenBroken liquid, preferably 30g/L.
In one embodiment of the invention, the rotating speed of described stirring is 100-300r/min, is preferably 200r/min.
In one embodiment of the invention, described substrate trimethyl pyruvic acid concentration 1M, concentration of glucose is 1.2M, thickEnzyme liquid is that 30g/L (weight in wet base) cell obtains, and reaction temperature is 30 DEG C, and pH is 8.5, and the time is 1.5h.
In one embodiment of the invention, described leucine dehydrogenase and GDH derive from respectively BacillusCereus and Bacillussp.
In one embodiment of the invention, the preparation of described crude enzyme liquid: (1) builds coexpression amino acid sequence as SEQIDThe leucine dehydrogenase of NO.1 and amino acid sequence are as the recombinant bacterium of the GDH of SEQIDNO.2; (2) to recombinant bacteriumCultivate, inducible strain is expressed leucine dehydrogenase and GDH, obtains zymotic fluid; (3) by zymotic fluid centrifuging and takingCell, washing, then ultrasonication, obtains crude enzyme liquid.
In one embodiment of the invention, described step (1) specifically: de-at GDH GDH and leucineBetween hydrogen enzyme LDH, add SD-AS sequence to obtain gene GDH-SD-AS-LDH, be then connected on pET28a plasmid, thenBe transformed in e. coli bl21 host, obtain recombinant bacterium.
In one embodiment of the invention, described SD-AS sequence is GGAGATATACC (shown in SEDIDNO.3).
In one embodiment of the invention, the middle recombinant bacterium of described step (2) is cultivated in used culture medium and is added withPrecursor substance in coenzyme synthesis path is to improve endobacillary coenzyme concentration. In the time being added with precursor substance, for transforming productionWhen S-Leucine, cell addition can be reduced to 20-30g/L (weight in wet base); While not adding precursor substance, cell addition is carriedHigh to more than 40g/L, course of reaction does not need to add coenzyme, conversion ratio is high.
In one embodiment of the invention, described precursor substance is in nicotinic acid, niacinamide, L-Trp or L-Aspartic acidAny one or multiple; Preferably, described precursor substance is nicotinic acid.
In one embodiment of the invention, the culture medium of described step (2) is LB culture medium (peptone 10g/L, fermentFemale extract 5g/L, NaCl10g/L, pH7.0, all the other are water, add kanamycin sulfate 50 μ g/mL before use); DescribedCultivation is to be cultured to thalline OD at 37 DEG C, 200r/min600Reach 0.6-2.0, then adding final concentration is the IPTG of 0.1mMIn cultivating 2-24h at 18 DEG C;
In one embodiment of the invention, the culture medium of described step (2) be self-induction culture medium (DEXTROSE ANHYDROUS 0.5g/L,Glycerine 5g/L, KH2PO46.8g/L，MgSO40.48g/L，Na2HPO47.1g/L，Na2SO40.71g/L，NH4Cl2.67G/L, albumen powder 10g/L, dusty yeast 5g/L; Inducer alpha-lactose 10g/L, pH7.60, with deionized water configuration, before useAdd kanamycin sulfate 50 μ g/mL), described cultivation is that incubation time is 24-48h under 30-37 DEG C, 200r/min.
In one embodiment of the invention, described method specifically: (1) is de-at GDH GDH and leucineBetween hydrogen enzyme LDH, add the SD-AS sequence of sequence as shown in SEQIDNO.3 to obtain gene GDH-SD-AS-LDH, soAfter be connected on pET28a plasmid, then be transformed in e. coli bl21 host, obtain recombinant bacterium; (2) recombinant bacterium is being containedHave in the culture medium of 1g/L nicotinic acid and cultivate, and inducible strain expresses leucine dehydrogenase and GDH, after cultivation, get thallineCell, washing; (3) in reaction system, add substrate trimethyl pyruvic acid concentration 1M, concentration of glucose is 1.2M, 30g/LThe crude enzyme liquid of cell, reaction temperature is 30 DEG C, with ammoniacal liquor control pH8.5, reaction 1.5h.
Beneficial effect
1, the recombinant bacterium of utilization of the present invention comprises leucine dehydrogenase, GDH and coenzyme, at catalysis trimethyl pyruvic acidIn the process of synthetic S-Leucine, effectively utilize the contained coenzyme of thalline self to realize coenzyme circular regeneration, anti-in catalysisIn the process of answering, do not need additionally to add coenzyme, realized zero interpolation of coenzyme, save the cost of coenzyme.
2, the present invention is in single batch of reaction, and concentration of substrate reaches 130g/L to carry out catalytic reaction and (be single batch the highest of reportConcentration of substrate), conversion ratio reaches more than 99%, and the reaction time shortens in 1.5h, and space-time yield reaches 2096gL-1d-1。
3, the present invention adopts the crude enzyme liquid of single recombinant bacterium to carry out catalytic reaction, has catalyst preparation process simple, and catalyst makesConsumption is few, and the feature that catalytic efficiency is high is applicable to suitability for industrialized production.
Brief description of the drawings
Fig. 1 is course of reaction schematic diagram of the present invention.
Detailed description of the invention
The testing conditions of substrate is: SB-AqColumn (4.6*150mm, 5um), and flow velocity 1ml/min, UV-detector 210nm,Mobile phase is 20mM potassium phosphate/acetonitrile solution (99.5:0.5v/v, pH2.0), sample size 5ul.
The testing conditions of product is: C18Column (4.6*250mm, 5um), utilize OPA derivatization, and flow velocity 1ml/min,UV-detector 338nm, mobile phase is 50mMNaAc/ acetonitrile solution (80:20v/v, pH7.2), sample size 10ul.
Embodiment 1: the Escherichia coli recombinant bacterial strain that builds coexpression leucine dehydrogenase and GDH
Utilize the gene order design primer in ncbi database, transfer respectively and derive from Bacilluscereus and BacillusspLeucine dehydrogenase and two kinds of enzyme genes of GDH, utilize restriction enzyme XhoI and NheI to genes of interest withExpression plasmid pET28a carries out double digestion, genes of interest obtain after being connected with carrier recombinant plasmid pET28a-LDH andPET28a-GDH. From the expression plasmid of two kinds of genes, utilize overlap extension pcr to exist again, between two genes, addEnter SD-AS sequence (GGAGATATACC). The primer that design comprises SD-AS sequence, obtains gene after two-wheeled PCRGDH-SD-AS-LDH, is connected to expression vector pET28a by this gene upper, proceeds in escherichia coli host BL21, obtainsRecombinant bacterium E.coliBL21/pET28a-G-SD-AS-L, thus realize two kinds of enzymes expressing in series on same plasmid.
Embodiment 2:LB culture medium inducing expressing gene engineering strain
Utilize LB culture medium to carry out fermented and cultured to recombinant bacterium. LB medium component is peptone 10g/L, yeast extract 5g/L,NaCl10g/L, pH7.0, with deionized water preparation, adds kanamycin sulfate 50 μ g/mL before use. Condition of culture is initialTemperature is 37 DEG C, and shaking speed 200r/min, as thalline OD600Reach 0.6-2.0, adding final concentration is the IPTG of 0.1mM,Cultivate 17h at 18 DEG C.
Embodiment 3: the LB culture medium inducing expressing gene engineering strain of the materials such as additional nicotinic acid
In LB culture medium, add the nicotinic acid of 1g/L (or in the coenzyme synthesis path such as niacinamide, L-Trp, L-Aspartic acidPrecursor substance). LB medium component is peptone 10g/L, yeast extract 5g/L, and NaCl10g/L, pH7.0, to goIonized water preparation, adds kanamycin sulfate 50 μ g/mL before use. Condition of culture is that initial temperature is 37 DEG C, shaking speed200r/min, as thalline OD600Reach 0.6-2.0, adding final concentration is the IPTG of 0.1mM, cultivates 17h at 18 DEG C.
Embodiment 4: self-induction culture medium inducing expressing gene engineering strain
Utilize self-induction to cultivate recombinant bacterium is carried out to fermented and cultured. The composition of self-induction culture medium is DEXTROSE ANHYDROUS 0.5g/L, sweetOil 5g/L, KH2PO46.8g/L，MgSO40.48g/L，Na2HPO47.1g/L，Na2SO40.71g/L，NH4Cl2.67g/L，Albumen powder 10g/L, dusty yeast 5g/L, inducer alpha-lactose 10g/L, pH7.60, with deionized water configuration, adds before useKanamycin sulfate 50 μ g/mL. Condition of culture is, 37 DEG C of temperature, and rotating speed 200r/min, incubation time is 48h.
Embodiment 5: the self-induction culture medium inducing expressing gene engineering strain of the materials such as additional nicotinic acid
In self-induction culture medium, add nicotinic acid (or the coenzyme synthesis path such as niacinamide, L-Trp, L-Aspartic acid of 1g/LIn precursor substance, be applied to the synthetic S-Leucine of living things catalysis, effect is similar). The composition of self-induction culture medium is anhydrousGlucose 0.5g/L, glycerine 5g/L, KH2PO46.8g/L，MgSO40.48g/L，Na2HPO47.1g/L，Na2SO40.71g/L，NH4Cl2.67g/L, albumen powder 10g/L, dusty yeast 5g/L, inducer alpha-lactose 10g/L, pH7.60, joins with deionized waterPut, before use, add kanamycin sulfate 50 μ g/mL. Condition of culture is, 30 DEG C of temperature, rotating speed 200r/min, incubation timeFor 24h.
Embodiment 6: the preparation of biocatalyst
Zymotic fluid is carried out centrifugal, collect thalline, after physiological saline washing, recombinant bacterium is resuspended in to 0.1MpH7.2 by 1:1-1:10Tris-HCl buffer solution in, utilize ultrasonic disruption instrument to carry out fragmentation, obtain the broken crude enzyme liquid of bacterial strain, as biocatalyst.In this catalyst, comprise leucine dehydrogenase, GDH and coenzyme NAD (H) simultaneously.
Embodiment 7: living things catalysis is synthetic
Configuration 100mL reaction system: add a certain amount of water and ammoniacal liquor in reactor, the substrate trimethyl of final concentration 0.5MPyruvic acid, utilizes ammoniacal liquor to regulate the pH of mixed liquor to reach 8.0 left and right, adds the glucose of 1.2 times of relative concentration of substrate, and 4g is (wetThe thalline (fragmentation becomes crude enzyme liquid) that heavily) prepared by embodiment 2, opens mechanical agitation simultaneously and starts reaction. In course of reaction, controlThe temperature of reaction system processed is at 35 DEG C, and pH value, in 7.5 left and right, is prepared S-Leucine. Sample in course of reaction is profit respectivelyWith ethanol and trichloroacetic acid precipitation albumen, then dilute certain multiple and carry out respectively the liquid phase detection of substrate and product afterwards. ReactionTime 1h, substrate conversion efficiency is greater than 99%. Spatial-temporal Transformation rate is 1039gL-1d-1。
Embodiment 8: living things catalysis is synthetic
Configuration 100mL reaction system: add a certain amount of water and ammoniacal liquor in reactor, the substrate trimethyl of final concentration 0.5MPyruvic acid, utilizes ammoniacal liquor to regulate the pH of mixed liquor to reach 8.0-9.0, adds the glucose of 1 times of relative concentration of substrate, and 6g is (wetThe thalline (fragmentation becomes crude enzyme liquid) that heavily) prepared by embodiment 4, opens mechanical agitation simultaneously and starts reaction. In course of reaction, controlThe temperature of reaction system processed is at 25 DEG C, and pH value, 9.0, is prepared S-Leucine. Sample in course of reaction utilizes respectively ethanolWith trichloroacetic acid precipitation albumen, then dilute certain multiple and carry out respectively the liquid phase detection of substrate and product afterwards. Reaction time 1h,Substrate conversion efficiency is greater than 99%. Spatial-temporal Transformation rate is 1043gL-1d-1。
Embodiment 9: living things catalysis is synthetic
Configuration 100mL reaction system: add a certain amount of water and ammoniacal liquor in reactor, the substrate trimethyl of final concentration 0.5MPyruvic acid, utilizes ammoniacal liquor to regulate the pH of mixed liquor to reach 8.0, adds the glucose of 1.3 times of relative concentration of substrate, 2g (weight in wet base)Thalline (fragmentation becomes crude enzyme liquid) prepared by embodiment 3, opens mechanical agitation simultaneously and starts reaction. In course of reaction, control anti-Answer the temperature of system at 30 DEG C, pH value, 8.0, is prepared S-Leucine. Sample in course of reaction utilizes respectively ethanol and threeMonoxone protein precipitation, then dilutes certain multiple and carries out respectively the liquid phase detection of substrate and product afterwards. Reaction time 2.5h,Substrate conversion efficiency is greater than 99%.
Embodiment 10: living things catalysis is synthetic
Configuration 100mL reaction system: add a certain amount of water and ammoniacal liquor in reactor, the substrate trimethyl third of final concentration 1MKetone acid, utilizes ammoniacal liquor to regulate the pH of mixed liquor to reach 8.5, adds the glucose of 1.2 times of relative concentration of substrate, 2g (weight in wet base)Thalline (fragmentation becomes crude enzyme liquid) prepared by embodiment 2, opens mechanical agitation simultaneously and starts reaction. In course of reaction, control anti-Answer the temperature of system at 25-30 DEG C, pH value, 8.0, is prepared S-Leucine. Sample in course of reaction utilizes respectively ethanolWith trichloroacetic acid precipitation albumen, then dilute certain multiple and carry out respectively the liquid phase detection of substrate and product afterwards. Reaction time1.5h, substrate conversion efficiency is greater than 99%. Production intensity can reach 84.25gL-1h-1, Spatial-temporal Transformation rate is 2076gL-1d-1。
Embodiment 11: living things catalysis is synthetic
Configuration 100mL reaction system: add a certain amount of water and ammoniacal liquor in reactor, the substrate trimethyl of final concentration 1.2MPyruvic acid, utilizes ammoniacal liquor to regulate the pH of mixed liquor to reach between 8.0-9.0, adds the glucose of 1.2 times of relative concentration of substrate,Thalline (fragmentation becomes crude enzyme liquid) prepared by 2g (weight in wet base) embodiment 5, opens mechanical agitation simultaneously and starts reaction. Course of reactionIn, control the temperature of reaction system at 28 DEG C, pH value, between 8.0-9.0, is prepared S-Leucine. Sample in course of reactionProduct utilize respectively ethanol and trichloroacetic acid precipitation albumen, then dilute certain multiple and carry out respectively the liquid phase inspection of substrate and product afterwardsSurvey. Reaction time 1h. Production intensity can reach 82.6gL-1h-1, Spatial-temporal Transformation rate 1982gL-1d-1。
Embodiment 11: living things catalysis is synthetic
Configuration 100mL reaction system: add a certain amount of water and ammoniacal liquor in reactor, the substrate trimethyl third of final concentration 1MKetone acid, utilizes ammoniacal liquor to regulate the pH of mixed liquor to reach 8.5 left and right, adds the glucose of 1.2 times of relative concentration of substrate, and 3g is (wetThe thalline (fragmentation becomes crude enzyme liquid) that heavily) prepared by embodiment 3, opens mechanical agitation simultaneously and starts reaction. In course of reaction, controlThe temperature of reaction system processed is at 30 DEG C, and pH value, in 8.5 left and right, is prepared S-Leucine. Sample in course of reaction is profit respectivelyWith ethanol and trichloroacetic acid precipitation albumen, then dilute certain multiple and carry out respectively the liquid phase detection of substrate and product afterwards. ReactionTime 1.5h, substrate conversion efficiency is greater than 99%, and production intensity can reach 87.3gL-1h-1, Spatial-temporal Transformation rate is 2096gL-1d-1。
Although the present invention is with preferred embodiment openly as above, it is not in order to limit the present invention, any person skilled in the art,Without departing from the spirit and scope of the present invention, all can do various changes and modification, therefore protection scope of the present invention should be withWhat claims defined is as the criterion.
Claims (10)
1. a method of preparing S-Leucine, is characterized in that, described method is to contain glucose, substrate trimethyl acetoneIn acid reaction system, add the crude enzyme liquid of the recombinant bacterium of coexpression leucine dehydrogenase and GDH, stirring reaction.
2. method according to claim 1, is characterized in that, the preparation of described crude enzyme liquid: (1) builds coexpression amino acidSequence is if the leucine dehydrogenase of SEQIDNO.1 and amino acid sequence are as the restructuring of the GDH of SEQIDNO.2Bacterium; (2) recombinant bacterium is cultivated, and inducible strain is expressed leucine dehydrogenase and GDH; (3) will fermentLiquid centrifuging and taking cell, washing, then ultrasonication, obtains crude enzyme liquid.
3. method according to claim 1, is characterized in that, substrate trimethyl pyruvic acid concentration 0.5-1.2M in described method,Concentration of glucose is 1.0-1.3 times of concentration of substrate.
4. method according to claim 1, is characterized in that, the temperature of described reaction system is controlled at 20-35 DEG C, and pH controlsAt 7.5-9.0.
5. method according to claim 1, is characterized in that, the time of described reaction is 1-2.5h.
6. method according to claim 2, is characterized in that, in described step (2), recombinant bacterium is cultivated used trainingSupport in base and be added with the precursor substance in coenzyme synthesis path; Preferably, described precursor substance is nicotinic acid, niacinamide, L-look ammoniaAny one in acid or L-Aspartic acid or multiple; More preferably, described precursor substance is nicotinic acid.
7. method according to claim 2, is characterized in that, described step (1) is: at GDH GDH andBetween leucine dehydrogenase LDH, add the SD-AS sequence of sequence as shown in SEQIDNO.3 to obtain geneGDH-SD-AS-LDH, is then connected on pET28a plasmid, then is transformed in e. coli bl21 host, is recombinatedBacterium.
8. method according to claim 1, is characterized in that, described method specifically: (1) is at GDH GDHAnd between leucine dehydrogenase LDH, add the SD-AS sequence of sequence as shown in SEQIDNO.3 to obtain geneGDH-SD-AS-LDH, is then connected on pET28a plasmid, then is transformed in e. coli bl21 host, is recombinatedBacterium; (2) recombinant bacterium is cultivated in the culture medium that contains 1g/L nicotinic acid, and inducible strain is expressed leucine dehydrogenase and grapeGlucocorticoid dehydrogenase, gets somatic cells, washing after cultivation; (3) in reaction system, add substrate trimethyl pyruvic acid concentration 1M,Concentration of glucose is 1.2M, the crude enzyme liquid of 30g/L cell, and reaction temperature is 30 DEG C, with ammoniacal liquor control pH8.5, reaction 1.5h.
9. the S-Leucine obtaining according to the arbitrary described method of claim 1-8.
Described in claim 9 S-Leucine at chemical industry or prepare the application aspect medicine.
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| CN111676256A (en) * | 2020-06-30 | 2020-09-18 | 南通大学 | Biological preparation method of L-tert-leucine |
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| Title |
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| NONE: "Accession No：WP_013351020，MULTISPECIES：glucose-1-dehydrogenase [Bacillus subtilis group]", 《GENBANK DATABASE》 * |
| YAN,X.: "Accession No：AID61663，leucine dehydrogenase [Bacillus cereus]", 《GENBANK DATABASE》 * |
| 李静: "亮氨酸脱氢酶的基因发掘、催化性能及其应用研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
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| CN111676256A (en) * | 2020-06-30 | 2020-09-18 | 南通大学 | Biological preparation method of L-tert-leucine |
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